Dr. Murphy is a founder, serves as CEO, and provides consulting in veterinary ophthalmology.
Dr. Murphy is a Board Certified Veterinary Ophthalmologist, and Professor of Comparative Ophthalmology in the Schools of Veterinary Medicine & Medicine, University of California-Davis. Dr. Murphy has advanced training and expertise in corneal diseases and surgery, comparative physiological optics, comparative ocular anatomy, as well as ocular diseases of laboratory and exotic animal species.
His laboratory has several active areas of research:
- Modulation of cell behaviors by biophysical attributes of the extracellular matrix: With many collaborators, Dr. Murphy is determining how biophysical attributes (nanoscale topography and compliance) of the extracellular matrix modulate fundamental cell behaviors such as orientation, adhesion, migration differentiation and proliferation. These studies have relevance to improved design of biomaterials as well as increasing our understanding of the microenvironment of cells in health and disease.
- Modulation of wound healing using engineering principals: A large interdisciplinary team is engaged in developing novel approaches to accelerate wound healing of the cornea and skin through funtionalzing wound beds rather than treating with standard soluble factors. The central concept is that improved wound healing outcomes can be achieved through "changing the wound" surface chemistry and biophysical attributes rather than treating the wound with soluble factors.
- Comparative ocular functional morphology: Dr. Murphy studies how various animals have evolved unique optical adaptations for optimizing their visual performance under challenging environmental conditions.
- Development of improved solutions for organ storage prior to transplantation: In collaboration with Professor Jon McAnulty, transplant surgeon at the School of Veterinary Medicine, Dr. Murphy has found that the addition of a unique combination of trophic factors can markedly extend the time that an organ can be stored and improve its performance once it is transplanted.
Standard services to industry provided by Dr. Murphy's laboratories include performance of in-vitro cytotoxicity assays using primary cultures from human ocular cell lines (cornea and trabecular meshwork) and conducting proof of principal studies using animal models.
Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo.
Exp Eye Res. 2020 Nov 18;:108362
Authors: Raghunathan V, Edwards SG, Leonard BC, Kim S, Evashenk AT, Song Y, Rewinski E, Price AM, Hoehn A, Chang C, Reilly CM, Muppala S, Murphy CJ, Thomasy SM
The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.
PMID: 33220237 [PubMed - as supplied by publisher]
Anionic nanoparticle-induced perturbation to phospholipid membranes affects ion channel function.
Proc Natl Acad Sci U S A. 2020 Oct 26;:
Authors: Foreman-Ortiz IU, Liang D, Laudadio ED, Calderin JD, Wu M, Keshri P, Zhang X, Schwartz MP, Hamers RJ, Rotello VM, Murphy CJ, Cui Q, Pedersen JA
Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.
PMID: 33106430 [PubMed - as supplied by publisher]
Engineered metal oxide nanomaterials inhibit corneal epithelial wound healing in vitro and in vivo.
NanoImpact. 2020 Jan;17:
Authors: Kim S, Gates B, Leonard BC, Gragg M, Pinkerton KE, Winkle LV, Murphy CJ, Pyrgiotakis G, Zhang Z, Demokritou P, Thomasy SM
Ocular exposure to metal oxide engineered nanomaterials (ENMs) is common as exemplified by zinc oxide (ZnO), a major constituent of sunscreens and cosmetics. The ocular surface that includes the transparent cornea and its protective tear film are common sites of exposure for metal ENMs. Despite the frequency of exposure of the ocular surface, there is a knowledge gap regarding the effects of metal oxide ENMs on the cornea in health and disease. Therefore, we studied the effects of metal oxide ENMs on the cornea in the presence or absence of injury. Cell viability of immortalized human corneal epithelial (hTCEpi) cells was assessed following treatment with 11 metal oxide ENMs with a concentration ranging from 0.5 to 250 μg/mL for 24 hours. An epithelial wound healing assay with a monolayer of hTCEpi cells was then performed using 11 metal oxide ENMs at select concentrations based on data from the viability assays. Subsequently, based on the in vitro results, in vivo testing of precorneal tear film (PTF) quantity and stability as well as a corneal epithelial wound healing were tested in the presence or absence ZnO or vanadium pentoxide (V2O5) at a concentration of 50 μg/mL. We found that WO3, ZnO, V2O5 and CuO ENMs significantly reduced hTCEpi cell viability in comparison to vehicle control or the other metal oxide ENMs tested. Furthermore, ZnO and V2O5 ENMs also significantly decreased hTCEpi cell migration. Although ZnO and V2O5 did not alter PTF parameters of rabbits in vivo, corneal epithelial wound healing was significantly delayed by topical ZnO while V2O5 did not alter wound healing. Finally, hyperspectral images confirmed penetration of ZnO and V2O5 through all corneal layers and into the iris stroma. Considering the marked epithelial toxicity and corneal penetration of ZnO, further investigations on the impact of this ENM on the eye are warranted.
PMID: 32154443 [PubMed]
Erratum: Author Correction: Identification of genes required for eye development by high-throughput screening of mouse knockouts.
Commun Biol. 2019;2:97
Authors: Moore BA, Leonard BC, Sebbag L, Edwards SG, Cooper A, Imai DM, Straiton E, Santos L, Reilly C, Griffey SM, Bower L, Clary D, Mason J, Roux MJ, Meziane H, Herault Y, International Mouse Phenotyping Consortium, McKerlie C, Flenniken AM, Nutter LMJ, Berberovic Z, Owen C, Newbigging S, Adissu H, Eskandarian M, Hsu CW, Kalaga S, Udensi U, Asomugha C, Bohat R, Gallegos JJ, Seavitt JR, Heaney JD, Beaudet AL, Dickinson ME, Justice MJ, Philip V, Kumar V, Svenson KL, Braun RE, Wells S, Cater H, Stewart M, Clementson-Mobbs S, Joynson R, Gao X, Suzuki T, Wakana S, Smedley D, Seong JK, Tocchini-Valentini G, Moore M, Fletcher C, Karp N, Ramirez-Solis R, White JK, de Angelis MH, Wurst W, Thomasy SM, Flicek P, Parkinson H, Brown SDM, Meehan TF, Nishina PM, Murray SA, Krebs MP, Mallon AM, Kent Lloyd KC, Murphy CJ, Moshiri A
[This corrects the article DOI: 10.1038/s42003-018-0226-0.].
PMID: 30854487 [PubMed - in process]
Presumed Photoreceptor Dysplasias In Peregrine Falcons ( Falco peregrinus) and Peregrine Falcon Hybrids.
J Wildl Dis. 2018 Oct 02;:
Authors: Moore BA, Murphy CJ, Marlar A, Dubielzig RR, Teixeira LBC, Ferrier WT, Hollingsworth SR
We describe a case series of photoreceptor dysplasia with secondary retinal degeneration in juvenile peregrine falcons. Six Peregrine Falcons ( Falco peregrinus) and three Peregrine Falcon × Prairie Falcon ( Falco mexicanus) hybrids had early-life visual deficits. Eight birds had visual defects shortly after hatching, and one bird had visual deficits first noticed at 5 mo of age. Complete ophthalmic examinations were performed in each animal. Eight of the animals had electroretinograms, and nine of the animals had their eyes examined histologically after euthanasia. Ophthalmic examinations did not reveal consistent and potentially blinding abnormalities, including an absence of ophthalmoscopic retinal lesions. Electroretinographic findings included subnormal amplitudes (with rod responses more abnormal than cone responses), with a negative b-wave amplitude occurring in one bird. Histologically, a reduction in the number of photoreceptors was present with numerous degenerative changes to the remaining photoreceptors, including frequent blunting and disorganization of photoreceptor outer segments, decreased numbers of cells in the inner nuclear layer, decreased numbers of ganglion cells, decreased thickness of the nerve fiber layer, and decreased myelinated axons within the optic nerve. Ultrastructurally, only minor cone outer segment changes and occasional phagocytic cells were seen. Results strongly suggested a primary retinopathy, characterized by photoreceptor dysplasia and secondary retinal degeneration with loss of cellular elements throughout the retina. The presence of a similar spectrum of findings in related individuals, the early age of onset, and the relative lack of other environmental, ocular, or systemic abnormalities suggested possible heritability.
PMID: 30277829 [PubMed - as supplied by publisher]
Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance.
Exp Eye Res. 2018 Sep 04;:
Authors: Miyagi H, Jalilian I, Murphy CJ, Thomasy SM
Corneal wound healing is a complex process that consists of cellular integration of multiple soluble biochemical cues and cellular responses to biophysical attributes associated with the matrix of the wound space. Upon corneal stromal wounding, the transformation of corneal fibroblasts to myofibroblasts is promoted by transforming growth factor-β (TGFβ). This process is critical for wound healing; however, excessive persistence of myofibroblasts in the wound space has been associated with corneal fibrosis resulting in severe vision loss. The objective of this study was to determine the effect of hepatocyte growth factor (HGF), which can modulate TGFβ signaling, on corneal myofibroblast transformation by analyzing the expression of α-smooth muscle actin (αSMA) as a marker of myofibroblast phenotype particularly as it relates to biomechanical cues. Human corneal fibroblasts were cultured on tissue culture plastic (>1 GPa) or hydrogel substrates mimicking human normal or wounded corneal stiffness (25 and 75 kPa) in media containing TGFβ1 ± HGF. The expression of αSMA was analyzed by quantitative PCR, Western blot and immunocytochemistry. Cellular stiffness, which is correlated with cellular phenotype, was measured by atomic force microscopy (AFM). In primary human corneal fibroblasts, the mRNA expression of αSMA showed a clear dose response to TGFβ1. The expression was significantly suppressed when cells were incubated with 20 ng/ml HGF in the presence of 2 ng/ml of TGFβ1. The protein expression of αSMA induced by 5 ng/ml TGFβ1 was also decreased by 20 ng/ml of HGF. Cells cultured on hydrogels mimicking human normal (25 kPa) and fibrotic (75 kPa) cornea also showed an inhibitory effect of HGF on αSMA expression in the presence or absence of TGFβ1. Cellular stiffness was decreased by HGF in the presence of TGFβ1 as measured by AFM. In this study, we have demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGFβ1 in human corneal stromal cells. These data suggest that HGF holds the potential as a therapeutic agent to improve wound healing outcomes by minimizing corneal fibrosis.
PMID: 30193807 [PubMed - as supplied by publisher]
Effects of 5% sodium chloride ophthalmic ointment on thickness and morphology of the normal canine cornea.
Vet Ophthalmol. 2018 Jun 21;:
Authors: Samuel M, Thomasy SM, Calderon AS, Kass PH, Collins K, Murphy CJ
OBJECTIVE: To determine the effect of 5% sodium chloride ophthalmic ointment (5% NaCl) on thickness and morphology of the normal canine cornea using ultrasonic pachymetry (USP), in vivo confocal microscopy (IVCM), and Fourier-domain optical coherence tomography (FD-OCT).
METHODS: Five healthy laboratory Beagles received ophthalmic examinations including USP, IVCM, and FD-OCT prior to and at fixed intervals following treatment. The right and left eyes were treated with 5% NaCl and artificial tears ophthalmic ointment (AT), respectively, every 2 hours for 4 treatments/d (days 2-9), and then hourly for 7 treatments/d (day 10). Treatment groups were statistically compared using mixed-effects linear regression.
RESULTS: Treatment with 5% NaCl resulted in a 12 μm decrease in corneal thickness from baseline (P < .001), while there was no significant difference in corneal thickness between values obtained at baseline and following treatment with AT (P = .82). Epithelial cell density significantly increased from baseline (530 ± 52 cells/mm2 ) to 577 ± 43 and 567 ± 15 cells/mm2 with 5% NaCl and AT, respectively (P = .003 and .005, respectively). However, keratocyte cell density in the anterior and posterior stroma and endothelial cell density did not significantly differ following treatment with 5% NaCl or AT ointment (P > .05).
CONCLUSIONS: Short-term topical treatment with 5% NaCl decreased corneal thickness in normal dogs with no observable changes in corneal morphology or signs of ocular toxicity.
PMID: 29927055 [PubMed - as supplied by publisher]
A Population Study of Common Ocular Abnormalities in C57BL/6N rd8 Mice.
Invest Ophthalmol Vis Sci. 2018 May 01;59(6):2252-2261
Authors: Moore BA, Roux MJ, Sebbag L, Cooper A, Edwards SG, Leonard BC, Imai DM, Griffey S, Bower L, Clary D, Lloyd KCK, Hérault Y, Thomasy SM, Murphy CJ, Moshiri A
Purpose: The purpose of this study was to quantify the frequency and severity of ocular abnormalities affecting wild-type C57BL/6N mice, the most common strain used worldwide for the creation of single-gene knockouts.
Methods: A total of 2773 animals (5546 eyes) were examined at one colony at UC Davis and in three more colonies at the Institut Clinique de la Souris in Strasbourg, France. Mice were examined at 15 to 16 weeks postnatal age by performing anterior segment biomicroscopy, posterior segment examination by indirect ophthalmoscopy, intraocular pressure measurement, and optical coherence tomography of anterior and posterior segment structures.
Results: Common ocular findings in the C57BL/6N strain included corneal deposits (3%), increased optical density of the anterior lens capsule (67%), punctate nuclear cataracts (98%), vitreous crystalline deposits (61%), hyaloid vascular remnant (6%), and retinal dysplasia attributed to the rd8 mutation (58%). Interestingly, retinal dysplasia was more common in male mice in all four breeding colonies evaluated in this study. The thickness of ocular tissues and compartments were measured by spectral-domain optical coherence tomography, including the central cornea, anterior chamber, vitreous, and retinal layers. Intraocular pressure was measured by rebound tonometry.
Conclusions: Ocular abnormalities are common in anterior and posterior segments of the C57BL/6N mouse, the most common background on which single-gene knockout mice have been made. It is important that vision scientists understand the extent and variability of ocular findings associated with this particular genetic background of mice.
PMID: 29847629 [PubMed - in process]
Glaucomatous cell derived matrices differentially modulate non-glaucomatous trabecular meshwork cellular behavior.
Acta Biomater. 2018 Mar 07;:
Authors: Raghunathan V, Benoit J, Kasetti R, Zode G, Salemi M, Phinney BS, Keller KE, Staverosky JA, Murphy CJ, Acott T, Vranka J
Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment.
STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.
PMID: 29524673 [PubMed - as supplied by publisher]
Whorl pattern keratopathies in veterinary and human patients.
Vet Ophthalmol. 2018 Feb 06;:
Authors: Kim S, Thomasy SM, Ramsey D, Zhao M, Mannis MJ, Murphy CJ
The course travelled by corneal epithelial cells from their stem cell niche at the limbus toward the vertex of the cornea is normally not evident due to their transparency, but in certain conditions, the epithelial cells can be rendered visible to the clinician. In such cases, the pathway taken by epithelial cells can manifest as a whorl pattern described using a variety of terms including hurricane keratitis/keratopathy, vortex keratopathy, whorl keratopathy, cornea verticillata, and at times, named after causative agents as exemplified by amiodarone keratopathy. Here, we briefly discuss the terminology used and the spectrum of conditions that can result in keratopathies with whorl patterns in human patients. We review the manifestations of such patterns in veterinary patients and discuss the state of understanding of the underlying forces that create the whorl distribution of epithelial cells on the ocular surface.
PMID: 29411482 [PubMed - as supplied by publisher]
Ocular anatomy of the black pacu (Colossoma macropomum): gross, histologic, and diagnostic imaging.
Vet Ophthalmol. 2018 Jan 30;:
Authors: Gustavsen KA, Paul-Murphy JR, Weber ES, Zwingenberger AL, Dunker FH, Dubielzig RR, Reilly CM, Murphy CJ
OBJECTIVE: To describe the ocular anatomy of the black pacu (Colossoma macropomum), a freshwater teleost fish of the Amazon River basin, including an unusual choroid laden with adipose tissue.
PROCEDURES: Three adult black pacu were anesthetized and examined clinically and with ocular ultrasonography, then euthanized. Three fish were euthanized and their heads imaged immediately postmortem using computed tomography. One fish was euthanized and its exenterated eyes imaged by high-resolution magnetic resonance imaging. The exenterated eyes of all seven fish were fixed in formalin; eyes from three fish were examined grossly and histologically. Additionally, archived histologic sections from two smaller black pacu specimens were examined.
RESULTS: Findings were consistent among the ocular imaging modalities used. Intrinsic to the sclera were circumferential ossicles and scleral cartilage. The lens was spherical and protruded through the ovoid pupil with an aphakic space inferiorly when the accommodative mechanism was relaxed under anesthesia. Both a small falciform process and epiretinal vasculature were present in the posterior segment. The retina was cone-rich, and processes of the retinal pigment epithelium enveloped the photoreceptor outer segments. Remarkably, the choroid occupied one-third of the anteroposterior length of the globe; histology confirmed that the bulk of the choroid was composed of adipose tissue.
CONCLUSIONS: The eye of the pacu overall is typical of teleosts but has the notable and consistent finding of a substantive store of choroidal fat of unknown function.
PMID: 29380518 [PubMed - as supplied by publisher]
Identification of genes required for eye development by high-throughput screening of mouse knockouts.
Commun Biol. 2018;1:236
Authors: Moore BA, Leonard BC, Sebbag L, Edwards SG, Cooper A, Imai DM, Straiton E, Santos L, Reilly C, Griffey SM, Bower L, Clary D, Mason J, Roux MJ, Meziane H, Herault Y, International Mouse Phenotyping Consortium, McKerlie C, Flenniken AM, Nutter LMJ, Berberovic Z, Owen C, Newbigging S, Adissu H, Eskandarian M, Hsu CW, Kalaga S, Udensi U, Asomugha C, Bohat R, Gallegos JJ, Seavitt JR, Heaney JD, Beaudet AL, Dickinson ME, Justice MJ, Philip V, Kumar V, Svenson KL, Braun RE, Wells S, Cater H, Stewart M, Clementson-Mobbs S, Joynson R, Gao X, Suzuki T, Wakana S, Smedley D, Seong JK, Tocchini-Valentini G, Moore M, Fletcher C, Karp N, Ramirez-Solis R, White JK, de Angelis MH, Wurst W, Thomasy SM, Flicek P, Parkinson H, Brown SDM, Meehan TF, Nishina PM, Murray SA, Krebs MP, Mallon AM, Lloyd KCK, Murphy CJ, Moshiri A
Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
PMID: 30588515 [PubMed]
The SPOTS System: An Ocular Scoring System Optimized for Use in Modern Preclinical Drug Development and Toxicology.
J Ocul Pharmacol Ther. 2017 Dec;33(10):718-734
Authors: Eaton JS, Miller PE, Bentley E, Thomasy SM, Murphy CJ
PURPOSE: To present a semiquantitative ocular scoring system comprising elements and criteria that address many of the limitations associated with systems commonly used in preclinical studies, providing enhanced cross-species applicability and predictive value in modern ocular drug and device development.
METHODS: Revisions to the ocular scoring systems of McDonald-Shadduck and Hackett-McDonald were conducted by board-certified veterinary ophthalmologists at Ocular Services On Demand (OSOD) over the execution of hundreds of in vivo preclinical ocular drug and device development studies and general toxicological investigations. This semiquantitative preclinical ocular toxicology scoring (SPOTS) system was driven by limitations of previously published systems identified by our group's recent review of slit lamp-based scoring systems in clinical ophthalmology, toxicology, and vision science.
RESULTS: The SPOTS system provides scoring criteria for the anterior segment, posterior segment, and characterization of intravitreal test articles. Key elements include: standardized slit lamp settings; expansion of criteria to enhance applicability to nonrabbit species; refinement and disambiguation of scoring criteria for corneal opacity, fluorescein staining severity, and aqueous flare; introduction of novel criteria for scoring of aqueous and anterior vitreous cell; and introduction of criteria for findings observed with drugs/devices targeting the posterior segment. A modified Standardization of Uveitis Nomenclature (SUN) system is also introduced to facilitate accurate use of SUN's criteria in laboratory species.
CONCLUSIONS: The SPOTS systems provide criteria that stand to enhance the applicability of semiquantitative scoring criteria to the full range of laboratory species, in the context of modern approaches to ocular therapeutics and drug delivery and drug and device development.
PMID: 29239680 [PubMed - in process]
Slit Lamp-Based Ocular Scoring Systems in Toxicology and Drug Development: A Literature Survey.
J Ocul Pharmacol Ther. 2017 Nov 07;:
Authors: Eaton JS, Miller PE, Bentley E, Thomasy SM, Murphy CJ
PURPOSE: To present a survey of the features of published slit lamp-based scoring systems and their applicability in the context of modern ocular toxicology and drug development.
METHODS: References describing original or modified slit lamp-based scoring systems for human or veterinary clinical patients or in investigative or toxicologic research were collected following a comprehensive literature review using textbooks and online publication searches. Each system's indications and features were compiled to facilitate comparison.
RESULTS: Literature review identified 138 original or modified scoring systems. Most (48%) were published for evaluation of the ocular surface, 34% for the general anterior segment, and 18% for the lens. Most systems were described for assessment of human patients (50%) and small albino laboratory species such as rabbits (19%), rats (12%), and mice (8%). Systems described for pigmented laboratory species and for larger species such as dogs, cats, pigs, and nonhuman primates (NHPs) were comparatively underrepresented. No systems described a lens scoring scheme specific to the dog, cat, pig, or NHP. Scoring schemes for aqueous and vitreous cells were infrequently described for laboratory species.
CONCLUSIONS: Many slit lamp-based scoring systems have been published, but the features of each differ and complicate translation of findings between different species. Use and interpretation of any scoring system in toxicology and drug development must be done with awareness of the limitations of the system being used.
PMID: 29111862 [PubMed - as supplied by publisher]
Biomechanical, ultrastructural, and electrophysiological characterization of the non-human primate experimental glaucoma model.
Sci Rep. 2017 Oct 30;7(1):14329
Authors: Raghunathan V, Eaton JS, Christian BJ, Morgan JT, Ver Hoeve JN, Yang CC, Gong H, Rasmussen CA, Miller PE, Russell P, Nork TM, Murphy CJ
Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We studied NHPs with ExGl of several years duration. As expected, ExGl eyes exhibited selective reductions of the retinal nerve fiber layer that correlate with electrophysiologic measures documenting a link between morphologic and elctrophysiologic endpoints. Softening of unlasered TM in ExGl eyes compared to untreated controls was observed. The degree of TM softening was consistent, regardless of pre-mortem clinical findings including severity of IOP elevation, retinal nerve fiber layer thinning, or electrodiagnostic findings. Importantly, this softening is contrary to TM stiffening reported in glaucomatous human eyes. Furthermore, microscopic analysis of unlasered TM from eyes with ExGl demonstrated TM thinning with collapse of Schlemm's canal; and proteomic analysis confirmed downregulation of metabolic and structural proteins. These data demonstrate unexpected and compensatory changes involving the TM in the NHP model of ExGl. The data suggest that compensatory mechanisms exist in normal animals and respond to elevated IOP through softening of the meshwork to increase outflow.
PMID: 29085025 [PubMed - in process]
Phenotypic Characterization of Corneal Endothelial Dystrophy in German Shorthaired and Wirehaired Pointers Using In Vivo Advanced Corneal Imaging and Histopathology.
Cornea. 2017 Oct 25;:
Authors: Shull OR, Reilly CM, Davis LB, Murphy CJ, Thomasy SM
PURPOSE: To evaluate corneal morphology using ultrasonic pachymetry (USP), Fourier-domain optical coherence tomography (FD-OCT), and in vivo confocal microscopy (IVCM) in 2 related canine breeds-German shorthaired pointers (GSHPs) and German wirehaired pointers (GWHPs)-with and without corneal endothelial dystrophy (CED). This condition is characterized by premature endothelial cell degeneration leading to concomitant corneal edema and is similar to Fuchs endothelial corneal dystrophy.
METHODS: Corneas of 10 CED-affected (4 GSHP and 6 GWHP) and 19 unaffected, age-matched (15 GSHP and 4 GWHP) dogs were examined using USP, FD-OCT, and IVCM. A 2-sample t test or Mann-Whitney rank-sum test was used to statistically compare parameters between both groups. Data are presented as mean ± SD or median (range).
RESULTS: Central corneal thickness determined using USP was significantly greater in CED-affected than in unaffected dogs at 1179 (953-1959) and 646 (497-737) μm, respectively (P < 0.001). Central epithelial thickness was found to be significantly decreased in CED-affected versus unaffected dogs at 47 ± 7.1 and 55 ± 7.1 μm, respectively (P = 0.011), using FD-OCT. With IVCM, corneal endothelial density was significantly less (P < 0.001) in 5 dogs with CED versus 19 unaffected controls at 499 ± 315 versus 1805 ± 298 cells/mm, respectively. CED-affected dogs exhibited endothelial pleomorphism and polymegethism, whereas CED-unaffected dogs had regular hexagonal arrangement of cells.
CONCLUSIONS: GSHPs and GWHPs with CED exhibit marked differences in corneal morphology when compared with age-matched control dogs. These 2 CED-affected breeds represent spontaneous, large animal models for human Fuchs endothelial corneal dystrophy.
PMID: 29077583 [PubMed - as supplied by publisher]
The role of hepatocyte growth factor in corneal wound healing.
Exp Eye Res. 2017 Oct 10;166:49-55
Authors: Miyagi H, Thomasy SM, Russell P, Murphy CJ
Hepatocyte growth factor (HGF) is a glycoprotein produced by mesenchymal cells and operates as a key molecule for tissue generation and renewal. During corneal injury, HGF is primarily secreted by stromal fibroblasts and promotes epithelial wound healing in a paracrine manner. While this mesenchymal-epithelial interaction is well characterized in various organs and the cornea, the role of HGF in corneal stromal and endothelial wound healing is understudied. In addition, HGF has been shown to play an anti-fibrotic role by inhibiting myofibroblast generation and subsequent production of a disorganized extracellular matrix and tissue fibrosis. Therefore, HGF represents a potential therapeutic tool in numerous organs in which myofibroblasts are responsible for tissue scarring. Corneal fibrosis can be a devastating sequela of injury and can result in corneal opacification and retrocorneal membrane formation leading to severe vision loss. In this article, we concisely review the available literature regarding the role of HGF in corneal wound healing. We highlight the influence of HGF on cellular behaviors in each corneal layer. Additionally, we suggest the possibility that HGF may represent a therapeutic tool for interrupting dysregulated corneal repair processes to improve patient outcomes.
PMID: 29024692 [PubMed - as supplied by publisher]
Genetic analysis of optic nerve head coloboma in the Nova Scotia Duck Tolling Retriever identifies discordance with the NHEJ1 intronic deletion (collie eye anomaly mutation).
Vet Ophthalmol. 2017 Jul 12;:
Authors: Brown EA, Thomasy SM, Murphy CJ, Bannasch DL
Collie eye anomaly (CEA) encompasses a spectrum of different ophthalmic phenotypes from clinically inconsequential choroidal hypoplasia to blindness from coloboma of the optic nerve head (ONH). A previous study found a 7.8-kb deletion in intron 4 of the NHEJ1 gene to be associated with CEA. A genetic test based on this association is recommended for many breeds, including the Nova Scotia Duck Tolling Retriever (NSDTR). Collection of ONH coloboma-affected NSDTR showed lack of concordance of the NHEJ1 intronic deletion with ONH coloboma. Using genomewide single nucleotide polymorphism (SNP) genotyping in 7 ONH coloboma-affected NSDTR cases and 47 unaffected NSDTR controls with no ophthalmic signs, one SNP, located on chromosome 7, demonstrated genomewide significance. However, high genomic inflation may have confounded the results. Therefore, the genomewide association study was repeated using EMMAX to control for population structure in the cohort of 7 cases and 47 controls. However, no regions of the genome were significantly associated with ONH coloboma. These results failed to document significant association with the CEA locus. Due to the complex genetic etiology of ONH coloboma, the NHEJ1 intronic deletion test results should be carefully considered when making breeding decisions. If the goal is to select for visually competent dogs, our data suggest that eye examinations of puppies would be more effective as a guide in selection of breeding pairs than relying solely on currently available genetic tests.
PMID: 28702949 [PubMed - as supplied by publisher]
Tissue and cellular biomechanics during corneal wound injury and repair.
Acta Biomater. 2017 May 27;:
Authors: Raghunathan VK, Thomasy SM, Strøm P, Yañez-Soto B, Garland SP, Sermeno J, Reilly CM, Murphy CJ
Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical cues, as well as cellular responses to the intrinsic chemistry and biophysical attributes associated with the matrix of the wound space. Here, we document how the biomechanics of the corneal stroma are altered through the course of wound repair following keratoablative procedures in rabbits. Further we documented the influence that substrate stiffness has on stromal cell mechanics. Following corneal epithelial debridement, New Zealand white rabbits underwent phototherapeutic keratectomy (PTK) on the right eye (OD). Wound healing was monitored using advanced imaging modalities. Rabbits were euthanized and corneas were harvested at various time points following PTK. Tissues were characterized for biomechanics with atomic force microscopy and with histology to assess inflammation and fibrosis. Factor analysis was performed to determine any discernable patterns in wound healing parameters. The matrix associated with the wound space was stiffest at 7 days post PTK. The greatest number of inflammatory cells were observed 3 days after wounding. The highest number of myofibroblasts and the greatest degree of fibrosis occurred 21 days after wounding. While all clinical parameters returned to normal values 400 days after wounding, the elastic modulus remained greater than pre-surgical values. Factor analysis demonstrated dynamic remodeling of stroma occurs between days 10 and 42 during corneal stromal wound repair. Elastic modulus of the anterior corneal stroma is dramatically altered following PTK and its changes coincide initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Factor analysis demonstrates strongest correlation between elastic modulus, myofibroblasts, fibrosis and stromal haze thickness, and between edema and central corneal thickness.
STATEMENT OF SIGNIFICANCE: Tissue biomechanics during the course of corneal wound healing is documented for the first time through atomic force microscopy, and is correlated with advanced clinical imaging and immunohistochemistry. Parameters obtained from the study are applied in a multivariate statistical model to cluster the data for better classification and monitor the wound repair process. Elastic modulus of the anterior corneal stroma is dramatically altered following wounding and correlates initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Importantly, the occurrence of myofibroblasts is preceded by changes in tissue mechanics, which is important to consider in light of crosslinking procedures applied to treat corneal diseases.
PMID: 28559158 [PubMed - as supplied by publisher]
Cascading Effects of Nanoparticle Coatings: Surface Functionalization Dictates the Assemblage of Complexed Proteins and Subsequent Interaction with Model Cell Membranes.
ACS Nano. 2017 May 22;:
Authors: Melby ES, Lohse SE, Park JE, Vartanian AM, Putans RA, Abbott HB, Hamers RJ, Murphy CJ, Pedersen JA
Interactions of functionalized nanomaterials with biological membranes are expected to be governed by not only nanoparticle physiochemical properties but also coatings or "coronas" of biomacromolecules acquired after immersion in biological fluids. Here we prepared a library of 4-5 nm gold nanoparticles (AuNPs) coated with either ω-functionalized thiols or polyelectrolyte wrappings to examine the influence of surface functional groups on the assemblage of proteins complexing the nanoparticles and its subsequent impact on attachment to model biological membranes. We find that the initial nanoparticle surface coating has a cascading effect on interactions with model cell membranes by determining the assemblage of complexing proteins, which in turn influences subsequent interaction with model biological membranes. Each type of functionalized AuNP investigated formed complexes with a unique ensemble of serum proteins that depended on the initial surface coating of the nanoparticles. Formation of protein-nanoparticle complexes altered the electrokinetic, hydrodynamic, and plasmonic properties of the AuNPs. Complexation of the nanoparticles with proteins reduced the attachment of cationic AuNPs and promoted attachment of anionic AuNPs to supported lipid bilayers; this trend is observed with both lipid bilayers comprising 100% zwitterionic phospholipids and those incorporating anionic phosphatidylinositol. Complexation with serum proteins led to attachment of otherwise noninteracting oligo(ethylene glycol)-functionalized AuNPs to bilayers containing phosphatidylinositol. These results demonstrate the importance of considering both facets of the nano-bio interface: functional groups displayed on the nanoparticle surface and proteins complexing the nanoparticles influence interaction with biological membranes as does the molecular makeup of the membranes themselves.
PMID: 28482159 [PubMed - as supplied by publisher]
Growth-Based Bacterial Viability Assay for Interference-Free and High-Throughput Toxicity Screening of Nanomaterials.
Anal Chem. 2017 Feb 07;89(3):2057-2064
Authors: Qiu TA, Nguyen TH, Hudson-Smith NV, Clement PL, Forester DC, Frew H, Hang MN, Murphy CJ, Hamers RJ, Feng ZV, Haynes CL
Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates. In this assay, bacteria were exposed to various materials and then diluted by a large factor into fresh growth medium. The large dilution ensured minimal optical interference from the nanomaterial when reading optical density, and the residue left from the exposure mixture after dilution was confirmed not to impact the bacterial growth profile. The fractions of viable cells after exposure were allowed to grow in fresh medium to generate measurable growth curves. Bacterial viability was then quantitatively correlated to the delay of bacterial growth compared to a reference regarded as 100% viable cells; data analysis was inspired by that in quantitative polymerase chain reactions, where the delay in the amplification curve is correlated to the starting amount of the template nucleic acid. Fast and robust data analysis was achieved by developing computer algorithms carried out using R. This method was tested on four bacterial strains, including both Gram-negative and Gram-positive bacteria, showing great potential for application to all culturable bacterial strains. With the increasing diversity of engineered nanomaterials being considered for large-scale use, this high-throughput screening method will facilitate rapid screening of nanomaterial toxicity and thus inform the risk assessment of nanoparticles in a timely fashion.
PMID: 28208291 [PubMed - in process]
Biomechanical relationships between the corneal endothelium and Descemet's membrane.
Exp Eye Res. 2016 Nov;152:57-70
Authors: Ali M, Raghunathan V, Li JY, Murphy CJ, Thomasy SM
The posterior face of the cornea consists of the corneal endothelium, a monolayer of cuboidal cells that secrete and attach to Descemet's membrane, an exaggerated basement membrane. Dysfunction of the endothelium compromises the barrier and pump functions of this layer that maintain corneal deturgesence. A large number of corneal endothelial dystrophies feature irregularities in Descemet's membrane, suggesting that cells create and respond to the biophysical signals offered by their underlying matrix. This review provides an overview of the bidirectional relationship between Descemet's membrane and the corneal endothelium. Several experimental methods have characterized a richly topographic and compliant biophysical microenvironment presented by the posterior surface of Descemet's membrane, as well as the ultrastructure and composition of the membrane as it builds during a lifetime. We highlight the signaling pathways involved in the mechanotransduction of biophysical cues that influence cell behavior. We present the specific example of Fuchs' corneal endothelial dystrophy as a condition in which a dysregulated Descemet's membrane may influence the progression of disease. Finally, we discuss some disease models and regenerative strategies that may facilitate improved treatments for corneal dystrophies.
PMID: 27639516 [PubMed - in process]
Superficial Keratectomy and Conjunctival Advancement Hood Flap (SKCAHF) for the Management of Bullous Keratopathy: Validation in Dogs With Spontaneous Disease.
Cornea. 2016 Oct;35(10):1295-304
Authors: Horikawa T, Thomasy SM, Stanley AA, Calderon AS, Li J, Linton LL, Murphy CJ
PURPOSE: To evaluate the efficacy of superficial keratectomy and conjunctival advancement hood flap (SKCAHF) for the treatment of bullous keratopathy in canine patients.
METHODS: Nine dogs (12 eyes) diagnosed with progressive corneal edema underwent superficial keratectomy followed by placement of conjunctival advancement hood flaps. The canine patients were examined pre- and postoperatively using in vivo confocal microscopy, ultrasonic pachymetry (USP), and Fourier-domain optical coherence tomography (FD-OCT). All owners responded to a survey regarding treatment outcomes.
RESULTS: Mean central corneal thickness (CCT) as measured by FD-OCT was 1163 ± 290 μm preoperatively and significantly decreased postoperatively to 795 ± 197 μm (P = 0.001), 869 ± 190 μm (P = 0.005), and 969 ± 162 μm (P = 0.033) at median postoperative evaluations occurring at 2.2, 6.8, and 12.3 months, respectively. Owners reported significant improvement (P < 0.05) in vision and corneal cloudiness at 6.8 and 12.3 months postoperatively. The percentage of cornea covered by the conjunctival flap was correlated (P = 0.0159) with a reduction in CCT by USP at 12.3 months postoperatively. All canine patients were comfortable pre- and postoperatively.
CONCLUSIONS: SKCAHF results in a reduction of corneal thickness in canine patients with bullous keratopathy. The increase in corneal thickness over time, after performing SKCAHF, is likely because of progressive endothelial decompensation. This surgery is a potentially effective intervention for progressive corneal edema in dogs that may have value in treatment of human patients with bullous keratopathy.
PMID: 27538190 [PubMed - in process]
Intravitreal Administration of Human Bone Marrow CD34+ Stem Cells in a Murine Model of Retinal Degeneration.
Invest Ophthalmol Vis Sci. 2016 Aug 01;57(10):4125-35
Authors: Moisseiev E, Smit-McBride Z, Oltjen S, Zhang P, Zawadzki RJ, Motta M, Murphy CJ, Cary W, Annett G, Nolta JA, Park SS
PURPOSE: Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model.
METHODS: C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina.
RESULTS: In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis.
CONCLUSIONS: Intravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration.
PMID: 27537262 [PubMed - in process]
Presumptive keratoglobus in a great horned owl (Bubo virginianus).
Vet Ophthalmol. 2016 Jul 31;:
Authors: Lau RK, Moresco A, Woods SJ, Reilly CM, Hawkins MG, Murphy CJ, Hollingsworth SR, Hacker D, Freeman KS
A juvenile to young adult, male, great horned owl (Bubo virginianus,GHOW) was presented to the wildlife rehabilitation hospital at Lindsay Wildlife Museum (WRHLWM) due to trauma to the right patagium from barbed wire entanglement. On presentation, both corneas were irregular, dry, and no movement of the third eyelid was noted. A severe corneal enlargement/globoid appearance was the predominant ophthalmic feature. The fundus was normal in both eyes (OU). Over the course of several days, both corneas developed edema combined with further dessication at the ocular surface associated with diffuse dorsal fluorescein stain uptake. Repeated ophthalmic examinations found normal intraocular pressures and an inability to move the third eyelid over the enlarged corneas. The bird was deemed nonreleasable due to severe wing damage and poor prognosis associated with eye abnormalities and was humanely euthanized. Postmortem CT, enucleation, and histopathology were performed to evaluate the ocular anatomical abnormality and confirm the suspected diagnosis of keratoglobus. This GHOW represents the first reported case of presumptive keratoglobus in a raptor.
PMID: 27477943 [PubMed - as supplied by publisher]
In Vivo Imaging of Corneal Endothelial Dystrophy in Boston Terriers: A Spontaneous, Canine Model for Fuchs' Endothelial Corneal Dystrophy.
Invest Ophthalmol Vis Sci. 2016 Jul 01;57(9):OCT495-503
Authors: Thomasy SM, Cortes DE, Hoehn AL, Calderon AC, Li JY, Murphy CJ
PURPOSE: Boston Terriers (BTs) have a greater prevalence of corneal endothelial dystrophy (CED), in comparison to other canine breeds. Similar to Fuchs' endothelial corneal dystrophy (FECD), this condition is characterized by endothelial cell degeneration with secondary corneal edema. This study assessed corneal morphology using in vivo confocal microscopy (IVCM) and Fourier-domain optical coherence tomography (FD-OCT) in BTs with and without CED.
METHODS: The corneas of 16 BTs with CED and 15 unaffected, age-matched BTs underwent clinical evaluation and were imaged using IVCM and FD-OCT. A two-sample t-test or Mann-Whitney rank sum test were used to statistically compare parameters between groups. Data are presented as mean ± SD or median (range).
RESULTS: Mean age did not significantly differ between affected and unaffected dogs at 10.0 ± 2.0 and 10.6 ± 2.4 years, respectively (P = 0.437). Females (69%) were overrepresented among the CED-affected dogs. In CED patients, IVCM demonstrated endothelial polymegathism and pleomorphism. Corneal endothelial density was significantly less (P < 0.001) in dogs with CED (1026 ± 260 cells/mm2) versus age-matched controls (2297 ± 372 cells/mm2). Fourier-domain OCT demonstrated a significant increase (P < 0.01) in central corneal and endothelium-Descemet's complex thickness in dogs with CED versus age-matched controls at 1019 (485-1550) or 536 (464-650) μm and 32 (22-56) or 25 (15-34) μm, respectively.
CONCLUSIONS: Corneal endothelial dystrophy in BTs is a bilateral, adult-onset condition that shares many similarities with FECD. Thus, CED could serve as a spontaneous disease model to study the pathogenesis of and develop novel treatments for FECD.
PMID: 27454658 [PubMed - in process]
Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.
Environ Sci Technol. 2016 Jun 21;50(12):6124-45
Authors: Holden PA, Gardea-Torresdey JL, Klaessig F, Turco RF, Mortimer M, Hund-Rinke K, Cohen Hubal EA, Avery D, Barceló D, Behra R, Cohen Y, Deydier-Stephan L, Ferguson PL, Fernandes TF, Herr Harthorn B, Henderson WM, Hoke RA, Hristozov D, Johnston JM, Kane AB, Kapustka L, Keller AA, Lenihan HS, Lovell W, Murphy CJ, Nisbet RM, Petersen EJ, Salinas ER, Scheringer M, Sharma M, Speed DE, Sultan Y, Westerhoff P, White JC, Wiesner MR, Wong EM, Xing B, Steele Horan M, Godwin HA, Nel AE
Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.
PMID: 27177237 [PubMed - in process]
Biological Responses to Engineered Nanomaterials: Needs for the Next Decade.
ACS Cent Sci. 2015 Jun 24;1(3):117-23
Authors: Murphy CJ, Vartanian AM, Geiger FM, Hamers RJ, Pedersen J, Cui Q, Haynes CL, Carlson EE, Hernandez R, Klaper RD, Orr G, Rosenzweig Z
The interaction of nanomaterials with biomolecules, cells, and organisms is an enormously vital area of current research, with applications in nanoenabled diagnostics, imaging agents, therapeutics, and contaminant removal technologies. Yet the potential for adverse biological and environmental impacts of nanomaterial exposure is considerable and needs to be addressed to ensure sustainable development of nanomaterials. In this Outlook four research needs for the next decade are outlined: (i) measurement of the chemical nature of nanomaterials in dynamic, complex aqueous environments; (ii) real-time measurements of nanomaterial-biological interactions with chemical specificity; (iii) delineation of molecular modes of action for nanomaterial effects on living systems as functions of nanomaterial properties; and (iv) an integrated systems approach that includes computation and simulation across orders of magnitude in time and space.
PMID: 27162961 [PubMed]
Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells.
Exp Eye Res. 2015 Jun;135:127-33
Authors: Morgan JT, Kwon HS, Wood JA, Borjesson DL, Tomarev SI, Murphy CJ, Russell P
Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.
PMID: 25720657 [PubMed - in process]
Effects of charge and surface ligand properties of nanoparticles on oxidative stress and gene expression within the gut of Daphnia magna.
Aquat Toxicol. 2015 May;162:1-9
Authors: Dominguez GA, Lohse SE, Torelli MD, Murphy CJ, Hamers RJ, Orr G, Klaper RD
Concern has been raised regarding the current and future release of engineered nanomaterials into aquatic environments from industry and other sources. However, not all nanomaterials may cause an environmental impact and identifying which nanomaterials may be of greatest concern has been difficult. It is thought that the surface groups of a functionalized nanoparticles (NPs) may play a significant role in determining their interactions with aquatic organisms, but the way in which surface properties of NPs impact their toxicity in whole organisms has been minimally explored. A major point of interaction of NPs with aquatic organisms is in the gastrointestinal tract as they ingest particulates from the water column or from the sediment. The main goal of this study was to use model gold NP (AuNPs) to evaluate the potential effects of the different surfaces groups on NPs on the gut of an aquatic model organism, Daphnia magna. In this study, we exposed daphnids to a range of AuNPs concentrations and assessed the impact of AuNP exposure in the daphnid gut by measuring reactive oxygen species (ROS) production and expression of genes associated with oxidative stress and general cellular stress: glutathione S-transferase (gst), catalase (cat), heat shock protein 70 (hsp70), and metallothionein1 (mt1). We found ROS formation and gene expression were impacted by both charge and the specific surface ligand used. We detected some degree of ROS production in all NP exposures, but positively charged AuNPs induced a greater ROS response. Similarly, we observed that, compared to controls, both positively charged AuNPs and only one negatively AuNP impacted expression of genes associated with cellular stress. Finally, ligand-AuNP exposures showed a different toxicity and gene expression profile than the ligand alone, indicating a NP specific effect.
PMID: 25734859 [PubMed - in process]
The intrinsic stiffness of human trabecular meshwork cells increases with senescence.
Oncotarget. 2015 Apr 12;
Authors: Morgan JT, Raghunathan VK, Chang YR, Murphy CJ, Russell P
Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases.
PMID: 25915531 [PubMed - as supplied by publisher]
Quantitative determination of ligand densities on nanomaterials by X-ray photoelectron spectroscopy.
ACS Appl Mater Interfaces. 2015 Jan 28;7(3):1720-5
Authors: Torelli MD, Putans RA, Tan Y, Lohse SE, Murphy CJ, Hamers RJ
X-ray photoelectron spectroscopy (XPS) is a nearly universal method for quantitative characterization of both organic and inorganic layers on surfaces. When applied to nanoparticles, the analysis is complicated by the strong curvature of the surface and by the fact that the electron attenuation length can be comparable to the diameter of the nanoparticles, making it necessary to explicitly include the shape of the nanoparticle to achieve quantitative analysis. We describe a combined experimental and computational analysis of XPS data for molecular ligands on gold nanoparticles. The analysis includes scattering in both Au core and organic shells and is valid even for nanoparticles having diameters comparable to the electron attenuation length (EAL). To test this model, we show experimentally how varying particle diameter from 1.3 to 6.3 nm leads to a change in the measured AC/AAu peak area ratio, changing by a factor of 15. By analyzing the data in a simple computational model, we demonstrate that ligand densities can be obtained, and, moreover, that the actual ligand densities for these nanoparticles are a constant value of 3.9 ± 0.2 molecules nm(-2). This model can be easily extended to a wide range of core-shell nanoparticles, providing a simple pathway to extend XPS quantitative analysis to a broader range of nanomaterials.
PMID: 25514372 [PubMed]
Gross, histologic, and micro-computed tomographic anatomy of the lacrimal system of snakes.
Vet Ophthalmol. 2015 Jan;18 Suppl 1:15-22
Authors: Souza NM, Maggs DJ, Park SA, Puchalski SM, Reilly CM, Paul-Murphy J, Murphy CJ
OBJECTIVE: To describe the lacrimal system of snakes using contrast micro-computed tomography (micro-CT) with 3-dimensional reconstruction, fluorescein passage ('Jones') testing, histology, and gross dissection.
ANIMALS STUDIED: One royal python and 19 snake cadavers representing 10 species.
PROCEDURES: Direct observation following injection of fluorescein into the subspectacular space, micro-CT following injection of three contrast agents into the subspectacular space, gross dissection following injection of latex into the subspectacular space, and histopathology.
RESULTS: Injection of fluorescein confirmed patency, but not course of the lacrimal duct. Barium enabled clear visualization of the lacrimal duct, whereas two iodinated contrast agents proved inadequate. Collectively, micro-CT, anatomic dissections, and histology suggest tears are produced by a single, large, serous, retrobulbar gland, released into the subspectacular space via several ductules, and drained through a single punctum originating in the ventronasal subspectacular space, and the lacrimal duct, which takes one of three routes of variable tortuosity before opening into the oral cavity in close association with the opening of the duct of the vomeronasal organ.
CONCLUSIONS: The ophidian lacrimal duct has a generally tortuous course, and the details of its anatomy are species-variable. The tortuous course of the duct likely predisposes snakes to duct occlusion and must be considered when planning medical and surgical interventions in snakes with pseudobuphthalmos and subspectacular abscessation.
PMID: 24862081 [PubMed - in process]
Spectral domain optical coherence tomography imaging of spectacular ecdysis in the royal python (Python regius).
Vet Ophthalmol. 2015 Jan;18 Suppl 1:1-7
Authors: Tusler CA, Maggs DJ, Kass PH, Paul-Murphy JR, Schwab IR, Murphy CJ
OBJECTIVE: To describe using spectral domain optical coherence tomography (SD-OCT), digital slit-lamp biomicroscopy, and external photography, changes in the ophidian cuticle, spectacle, and cornea during ecdysis.
ANIMALS STUDIED: Four normal royal pythons (Python regius).
PROCEDURES: Snakes were assessed once daily throughout a complete shed cycle using nasal, axial, and temporal SD-OCT images, digital slit-lamp biomicroscopy, and external photography.
RESULTS: Spectral domain optical coherence tomography (SD-OCT) images reliably showed the spectacular cuticle and stroma, subcuticular space (SCS), cornea, anterior chamber, iris, and Schlemm's canal. When visible, the subspectacular space (SSS) was more distended peripherally than axially. Ocular surface changes throughout ecdysis were relatively conserved among snakes at all three regions imaged. From baseline (7 days following completion of a full cycle), the spectacle gradually thickened before separating into superficial cuticular and deep, hyper-reflective stromal components, thereby creating the SCS. During spectacular separation, the stroma regained original reflectivity, and multiple hyper-reflective foci (likely fragments from the cuticular-stromal interface) were noted within the SCS. The cornea was relatively unchanged in character or thickness throughout all stages of ecdysis. Slit-lamp images did not permit observation of these changes.
CONCLUSIONS: Spectral domain optical coherence tomography (SD-OCT) provided excellent high-resolution images of the snake anterior segment, and especially the cuticle, spectacle, and cornea of manually restrained normal snakes at all stages of ecdysis and warrants investigation in snakes with anterior segment disease. The peripheral spectacle may be the preferred entry point for diagnostic or therapeutic injections into the SSS and for initiating spectacular surgery.
PMID: 24824651 [PubMed - in process]
The effect of needle gauge, needle type, and needle orientation on the volume of a drop.
Vet Ophthalmol. 2015 Jan 27;
Authors: Tripp GK, Good KL, Motta MJ, Kass PH, Murphy CJ
OBJECTIVE: The purpose of this study was to determine impact of needle gauge, type, and orientation on average volume of drop dispensed.
PROCEDURE: Five needle gauges (22G, 23G, 25G, 27G, and 30G) were examined. For each gauge, volume of drop delivered was determined for standard sharp beveled tip, blunt tip, and after breaking off of the sharp needle from the hub. Vertical and horizontal orientation of the needle was tested for effect on drop volume for 22-G and 30-G sharp beveled needles.
RESULTS: Mean drop volume was affected by needle gauge, needle orientation, and whether the needle had been broken off from its hub. Mean drop volume scaled directly with needle diameter with drop volumes of 25.0 μL (±20.2) and 83.9 μL (±16.5) being found for 30-gauge and 22-gauge needles, respectively. Intermediate gauges (27, 25, 23G) yielded intermediate drop volumes. Blunt needles tended to produce larger drop volumes compared to sharp beveled needles, but these differences did not reach statistical significance. Breaking off of the needle from the hub produced substantially larger drop volumes with little difference being found between needle gauges. Average volumes of 1 drop from a 22-G vertical, 22-G horizontal, 30-G vertical, and 30-G horizontal sharp beveled needle were 20.2, 9.1, 10.1, and 3.3 μL, respectively.
CONCLUSIONS: These findings have relevance for controlled delivery of topical ophthalmic medications to patients.
PMID: 25643934 [PubMed - as supplied by publisher]
KCNJ15/Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis.
Nat Commun. 2015;6:8532
Authors: Nakajima K, Zhu K, Sun YH, Hegyi B, Zeng Q, Murphy CJ, Small JV, Chen-Izu Y, Izumiya Y, Penninger JM, Zhao M
Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K(+) channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields.
PMID: 26449415 [PubMed - in process]
The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.
Exp Cell Res. 2014 Oct 15;328(1):164-71
Authors: Murphy KC, Morgan JT, Wood JA, Sadeli A, Murphy CJ, Russell P
The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.
PMID: 24992043 [PubMed - indexed for MEDLINE]
Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes.
ACS Appl Mater Interfaces. 2014 Sep 23;
Authors: You J, Raghunathan VK, Son KJ, Patel D, Haque A, Murphy CJ, Revzin A
Hepatocytes, the main epithelial cell type in the liver, perform most of the biochemical functions of the liver. Thus, maintenance of a primary hepatocyte phenotype is crucial for investigations of in vitro drug metabolism, toxicity, and development of bioartificial liver constructs. Here, we report the impact of topographic cues alone and in combination with soluble signals provided by encapsulated feeder cells on maintenance of the primary hepatocyte phenotype. Topographic features were 300 nm deep with pitches of either 400, 1400, or 4000 nm. Hepatocyte cell attachment, morphology and function were markedly better on 400 nm pitch patterns compared with larger scale topographies or planar substrates. Interestingly, topographic features having biomimetic size scale dramatically increased cell adhesion whether or not substrates had been precoated with collagen I. Albumin production in primary hepatocytes cultured on 400 nm pitch substrates without collagen I was maintained over 10 days and was considerably higher compared to albumin synthesis on collagen-coated flat substrates. In order to investigate the potential interaction of soluble cytoactive factors supplied by feeder cells with topographic cues in determining cell phenotype, bioactive heparin-containing hydrogel microstructures were molded (100 μm spacing, 100 μm width) over the surface of the topographically patterned substrates. These hydrogel microstructures either carried encapsulated fibroblasts or were free of cells. Hepatocytes cultured on nanopatterned substrates next to fibroblast carrying hydrogel microstructures were significantly more functional than hepatocytes cultured on nanopatterned surfaces without hydrogels or stromal cells significantly elevated albumin expression and cell junction formation compared to cells provided with topographic cues only. The simultaneous presentation of topographic biomechanical cues along with soluble signaling molecules provided by encapsulated fibroblasts cells resulted in optimal functionality of cultured hepatocytes. The provision of both topographic and soluble signaling cues could enhance our ability to create liver surrogates and inform the development of engineered liver constructs.
PMID: 25247391 [PubMed - as supplied by publisher]
Automated AFM force curve analysis for determining elastic modulus of biomaterials and biological samples.
J Mech Behav Biomed Mater. 2014 Sep;37:209-18
Authors: Chang YR, Raghunathan VK, Garland SP, Morgan JT, Russell P, Murphy CJ
The analysis of atomic force microscopy (AFM) force data requires the selection of a contact point (CP) and is often time consuming and subjective due to influence from intermolecular forces and low signal-to-noise ratios (SNR). In this report, we present an automated algorithm for the selection of CPs in AFM force data and the evaluation of elastic moduli. We propose that CP may be algorithmically easier to detect by identifying a linear elastic indentation region of data (high SNR) rather than the contact point itself (low SNR). Utilizing Hertzian mechanics, the data are fitted for the CP. We first detail the algorithm and then evaluate it on sample polymeric and biological materials. As a demonstration of automation, 64 × 64 force maps were analyzed to yield spatially varying topographical and mechanical information of cells. Finally, we compared manually selected CPs to automatically identified CPs and demonstrated that our automated approach is both accurate (< 10nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show that the algorithm is useful for analysis of both biomaterials and biological samples.
PMID: 24951927 [PubMed - indexed for MEDLINE]
Reduction in wound bioburden using a silver-loaded dissolvable microfilm construct.
Adv Healthc Mater. 2014 Jun;3(6):916-28
Authors: Herron M, Agarwal A, Kierski PR, Calderon DF, Teixeira LB, Schurr MJ, Murphy CJ, Czuprynski CJ, McAnulty JF, Abbott NL
Silver is a widely used antimicrobial agent, yet, when impregnated in macroscopic dressings, it stains wounds, can lead to tissue toxicity, and can inhibit healing. Recently, polymeric nanofilms containing silver nanoparticles were reported to exhibit antimicrobial activity at loadings and release rates of silver that are 100× lower than conventional dressings. Here, fabrication of composite microfilm constructs that provide a facile way to transfer the silver-loaded polymeric nanofilms onto wounds in vivo is reported. The construct is fabricated from a silver nanoparticle-loaded polymeric nanofilm that is laminated with a micrometer-thick-soluble film of polyvinylalcohol (PVA). When placed on a moist wound, the PVA dissolves, leaving the silver-loaded nanofilm immobilized on the wound-bed. In vitro, the immobilized nanofilms release <1 μg cm(-2) d(-1) of silver over 30 d from skin dermis and they kill 5 log10 CFUs of Staphylococcus aureus in 24 h. In mice, wounds inoculated with 10(5) CFU S. aureus presented up to 3 log10 less bacterial burden when treated with silver/nanofilms for 3 d, as compared to unmodified wounds. In uncontaminated wounds, silver/nanofilms allow normal and complete wound closure by re-epithelialization. Dissolvable microfilm constructs may overcome key limitations associated with current uses of silver in wound healing.
PMID: 24523027 [PubMed - indexed for MEDLINE]
Epidermal growth factor-functionalized polymeric multilayer films: interplay between spatial location and bioavailability of EGF.
J Invest Dermatol. 2014 Jun;134(6):1757-60
Authors: Gorouhi F, Shah NM, Raghunathan VK, Mohabbati Y, Abbott NL, Isseroff RR, Murphy CJ
PMID: 24390131 [PubMed - indexed for MEDLINE]
Raman spectroscopy enables noninvasive biochemical characterization and identification of the stage of healing of a wound.
Anal Chem. 2014 Apr 15;86(8):3764-72
Authors: Jain R, Calderon D, Kierski PR, Schurr MJ, Czuprynski CJ, Murphy CJ, McAnulty JF, Abbott NL
Accurate and rapid assessment of the healing status of a wound in a simple and noninvasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of nonhealing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret, and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spectral analysis as a simple, noninvasive method to biochemically characterize healing wounds in mice and to accurately identify different phases of healing of wounds at different time-points. Raman spectra were collected from "splinted" full thickness dermal wounds in mice at 4 time-points (0, 1, 5, and 7 days) corresponding to different phases of wound healing, as verified by histopathology. Spectra were deconvolved using multivariate factor analysis (MFA) into 3 "factor score spectra" (that act as spectral signatures for different stages of healing) that were successfully correlated with spectra of prominent pure wound bed constituents (i.e., collagen, lipids, fibrin, fibronectin, etc.) using non-negative least squares (NNLS) fitting. We show that the factor loadings (weights) of spectra that belonged to wounds at different time-points provide a quantitative measure of wound healing progress in terms of key parameters such as inflammation and granulation. Wounds at similar stages of healing were characterized by clusters of loading values and slowly healing wounds among them were successfully identified as "outliers". Overall, our results demonstrate that Raman spectroscopy can be used as a noninvasive technique to provide insight into the status of normally healing and slow-to-heal wounds and that it may find use as a complementary tool for real-time, in situ biochemical characterization in wound healing studies and clinical diagnosis.
PMID: 24559115 [PubMed - in process]
Anchoring a cytoactive factor in a wound bed promotes healing.
J Tissue Eng Regen Med. 2014 Mar 27;
Authors: Chattopadhyay S, Guthrie KM, Teixeira L, Murphy CJ, Dubielzig RR, McAnulty JF, Raines RT
Wound healing is a complex process that requires the intervention of cytoactive factors. The one-time application of soluble factors to a wound bed does not maintain a steady, sufficient concentration. Here we investigated the benefits of anchoring a factor in a wound bed via a tether to endogenous collagen. We used a collagen-mimetic peptide (CMP) as a pylon. The CMP binds to damaged but not intact collagen and thus localizes a pendant cytoactive factor in the regions of a wound bed that require intervention. As a model factor, we chose substance P, a peptide of the tachykinin family that promotes wound healing. Using splinted wounds in db/db mice, we found that the one-time application of a CMP-substance P conjugate enhances wound healing compared to unconjugated substance P and other controls. Specifically, all 16 wounds treated with the conjugate closed more thoroughly and, did so with extensive re-epithelialization and mitigated inflammatory activity. These data validate a simple and general strategy for re-engineering wound beds by the integration of beneficial cytoactive factors. Copyright © 2014 John Wiley & Sons, Ltd.
PMID: 24677775 [PubMed - as supplied by publisher]
A cell culture substrate with biologically relevant size-scale topography and compliance of the basement membrane.
Langmuir. 2014 Mar 4;30(8):2101-8
Authors: Garland SP, McKee CT, Chang YR, Raghunathan VK, Russell P, Murphy CJ
A growing body of literature broadly documents that a wide array of fundamental cell behaviors are modulated by the physical attributes of the cellular microenvironment, yet in vitro assays are typically carried out using tissue culture plastic or glass substrates that lack the 3-dimensional topography present in vivo and have stiffness values that far exceed that of cellular and stromal microenvironments. This work presents a method for the fabrication of thin hydrogel films that can replicate arbitrary topographies with a resolution of 400 nm that possess an elastic modulus of approximately 250 kPa. Material characterization including swelling behavior and mechanics were performed and reported. Cells cultured on these surfaces patterned with anisotropic ridges and grooves react to the biophysical cues present and show an alignment response.
PMID: 24524303 [PubMed - indexed for MEDLINE]
Elastic modulus and collagen organization of the rabbit cornea: epithelium to endothelium.
Acta Biomater. 2014 Feb;10(2):785-91
Authors: Thomasy SM, Raghunathan VK, Winkler M, Reilly CM, Sadeli AR, Russell P, Jester JV, Murphy CJ
The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet's membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.
PMID: 24084333 [PubMed - indexed for MEDLINE]
Photopatternable and photoactive hydrogel for on-demand generation of hydrogen peroxide in cell culture.
Biomaterials. 2014 Feb;35(5):1762-70
Authors: Garland SP, Wang RY, Raghunathan VK, Lam KS, Murphy CJ, Russell P, Sun G, Pan T
Oxidative stress, largely mediated by reactive oxygen species (ROS), is a nearly ubiquitous component in complex biological processes such as aging and disease. Optimal in vitro methods used in elucidating disease mechanisms would deliver of low levels of hydrogen peroxide, emulating the in vivo pathological state, but current methods are limited by kinetic stability or accurate measurement of the dose administered. Here we present an in vitro platform that exploits anthraquinone catalysts for the photocatalytic production of hydrogen peroxide. This system can be dynamically tuned to provide constant generation of hydrogen peroxide at a desired physiologic rate over at least 14 days and is described using a kinetic model. Material characterization and stability is discussed along with a proof-of-concept in vitro study that assessed the viability of cells as they were oxidatively challenged over 24 h at different ROS generation rates.
PMID: 24290809 [PubMed - indexed for MEDLINE]
Robust and artifact-free mounting of tissue samples for atomic force microscopy.
Biotechniques. 2014 Jan;56(1):40-2
Authors: Morgan JT, Raghunathan VK, Thomasy SM, Murphy CJ, Russell P
Immobilization of tissue-samples for atomic for microscopy (AFM) is typically done using either semi-dry tissue or by gluing the tissue sample down, both of which can introduce artifacts. Here, we describe the design of a Soft- Clamping Immobilizing Retainer of Tissue (SCIRT) for consistent and nondestructive immobilization of tissues for AFM analysis. We compare the performance of our SCIRT method with glue-immobilization for two difficult to handle tissue types: human trabecular meshwork (HTM) and rabbit cornea (RC). Our results demonstrate that the SCIRT method has several advantages, including: (i) allowing for small sample sizes, (ii) enabling continuous hydration, (iii) eliminating contact with glue or associated solvents, (iv) permitting sample recovery following measurement, and (v) ease of use. In conclusion, the SCIRT method is a simple and effective means of immobilizing soft, hydrated tissue samples consistently and without artifacts.
PMID: 24447138 [PubMed - indexed for MEDLINE]
Involvement of YAP, TAZ and HSP90 in contact guidance and intercellular junction formation in corneal epithelial cells.
PLoS One. 2014;9(10):e109811
Authors: Raghunathan VK, Dreier B, Morgan JT, Tuyen BC, Rose BW, Reilly CM, Russell P, Murphy CJ
The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.
PMID: 25290150 [PubMed - in process]
Full-thickness splinted skin wound healing models in db/db and heterozygous mice: implications for wound healing impairment.
Wound Repair Regen. 2014 May-Jun;22(3):368-80
Authors: Park SA, Teixeira LB, Raghunathan VK, Covert J, Dubielzig RR, Isseroff RR, Schurr M, Abbott NL, McAnulty J, Murphy CJ
The excisional dorsal full-thickness skin wound model with or without splinting is widely utilized in wound healing studies using diabetic or normal mice. However, the effects of splinting on dermal wound healing have not been fully characterized, and there are limited data on the direct comparison of wound parameters in the splinted model between diabetic and normal mice. We compared full-thickness excisional dermal wound healing in db/db and heterozygous mice by investigating the effects of splinting, semi-occlusive dressing, and poly(ethylene glycol) treatment. Two 8-mm full-thickness wounds were made with or without splinting in db/db and heterozygous mice. Body weights, splint maintenance, wound contraction, wound closure, and histopathological parameters including reepithelialization, wound bed collagen deposition, and inflammation were compared between groups. Our results show that silicone splint application effectively reduced wound contraction in heterozygous and db/db mice. Splinted wounds, as opposed to nonsplinted wounds, exhibited no significant differences in wound closure between heterozygous and db/db mice. Finally, polyethylene glycol and the noncontact dressing had no significant effect on wound healing in heterozygous or db/db mice. We believe these findings will help investigators in selection of the appropriate wound model and data interpretation with fully defined parameters.
PMID: 24844336 [PubMed - indexed for MEDLINE]
Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.
J Ocul Pharmacol Ther. 2014 Mar-Apr;30(2-3):254-66
Authors: Morgan JT, Wood JA, Walker NJ, Raghunathan VK, Borjesson DL, Murphy CJ, Russell P
PURPOSE: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs).
METHODS: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue.
RESULTS: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue.
CONCLUSIONS: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.
PMID: 24456002 [PubMed - indexed for MEDLINE]
The influence of substrate topography on the migration of corneal epithelial wound borders.
Biomaterials. 2013 Dec;34(37):9244-51
Authors: Yanez-Soto B, Liliensiek SJ, Gasiorowski JZ, Murphy CJ, Nealey PF
Currently available artificial corneas can develop post-implant complications including epithelial downgrowth, infection, and stromal melting. The likelihood of developing these disastrous complications could be minimized through improved formation and maintenance of a healthy epithelium covering the implant. We hypothesize that this epithelial formation may be enhanced through the incorporation of native corneal basement membrane biomimetic chemical and physical cues onto the surface of the keratoprosthesis. We fabricated hydrogel substrates molded with topographic features containing specific bio-ligands and developed an in vitro wound healing assay. In our experiments, the rate of corneal epithelial wound healing was significantly increased by 50% in hydrogel surfaces containing topographic features, compared to flat surfaces with the same chemical attributes. We determined that this increased healing is not due to enhanced proliferation or increased spreading of the epithelial cells, but to an increased active migration of the epithelial cells. These results show the potential benefit of restructuring and improving the surface of artificial corneas to enhance epithelial coverage and more rapidly restore the formation of a functional epithelium.
PMID: 24016856 [PubMed - indexed for MEDLINE]
Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland, the gland of the third eyelid and the knee joint.
Cytotherapy. 2013 Dec;15(12):1498-510
Authors: Park SA, Reilly CM, Wood JA, Chung DJ, Carrade DD, Deremer SL, Seraphin RL, Clark KC, Zwingenberger AL, Borjesson DL, Hayashi K, Russell P, Murphy CJ
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs.
METHODS: Allogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation.
RESULTS: Clinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations.
CONCLUSIONS: This is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.
PMID: 23992828 [PubMed - indexed for MEDLINE]
Characterizing the effects of heparin gel stiffness on function of primary hepatocytes.
Tissue Eng Part A. 2013 Dec;19(23-24):2655-63
Authors: You J, Park SA, Shin DS, Patel D, Raghunathan VK, Kim M, Murphy CJ, Tae G, Revzin A
In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.
PMID: 23815179 [PubMed - indexed for MEDLINE]
What do mechanotransduction, Hippo, Wnt, and TGFβ have in common? YAP and TAZ as key orchestrating molecules in ocular health and disease.
Exp Eye Res. 2013 Oct;115:1-12
Authors: Morgan JT, Murphy CJ, Russell P
Cells in vivo are exposed to a complex signaling environment. Biochemical signaling modalities, such as secreted proteins, specific extracellular matrix domains and ion fluxes certainly compose an important set of regulatory signals to cells. However, these signals are not exerted in isolation, but rather in concert with biophysical cues of the surrounding tissue, such as stiffness and topography. In this review, we attempt to highlight the biophysical attributes of ocular tissues and their influence on cellular behavior. Additionally, we introduce the proteins YAP and TAZ as targets of biophysical and biochemical signaling and important agonists and antagonists of numerous signaling pathways, including TGFβ and Wnt. We frame the discussion around this extensive signaling crosstalk, which allows YAP and TAZ to act as orchestrating molecules, capable of integrating biophysical and biochemical cues into a broad cellular response. Finally, while we draw on research from various fields to provide a full picture of YAP and TAZ, we attempt to highlight the intersections with vision science and the exciting work that has already been performed.
PMID: 23792172 [PubMed - indexed for MEDLINE]
Substratum stiffness and latrunculin B modulate the gene expression of the mechanotransducers YAP and TAZ in human trabecular meshwork cells.
Exp Eye Res. 2013 Aug;113:66-73
Authors: Thomasy SM, Morgan JT, Wood JA, Murphy CJ, Russell P
The compliance of the human trabecular meshwork (HTM) has been shown to dramatically stiffen in glaucomatous patients. The purpose of this study was to determine the impact of substratum stiffness and latrunculin-B (Lat-B) on the expression and activity of the mechanotransducers, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ), in primary HTM cells as the cells start to recover from Lat-B treatment. Primary human trabecular meshwork (HTM) cells were cultured on hydrogels possessing stiffness values mimicking those found in normal (5 kPa) and glaucomatous meshworks (75 kPa), or tissue culture polystyrene (TCP; >1 GPa). Cells were treated with 2.0 μM Lat-B in DMSO or DMSO alone. RT-PCR was used to determine the impact of substratum stiffness and/or Lat-B treatment on the expression of YAP, TAZ, 14-3-3σ, plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF). Immunoblotting was used to determine the expression of YAP and TAZ as well as the phosphorylation status of YAP. Immunofluorescence was used to determine YAP protein localization. YAP and TAZ mRNA expression were upregulated on the 75 kPa hydrogels in comparison to the 5 kPa hydrogels and TCP. Treatment with Lat-B resulted in a rapid and dramatic downregulation of YAP and TAZ on the 75 kPa hydrogels. On hydrogels, Lat-B treatment increased the phosphorylation of YAP at S127, while decreasing it on TCP. Similarly, Lat-B treatment resulted in markedly decreased nuclear localization of YAP on the hydrogels but elevated nuclear localization on TCP. Lat-B treatment of HTM cells on the 75 kPa hydrogels also increased 14-3-3σ mRNA, a protein important in YAP/TAZ degradation. In addition, Lat-B treatment decreased CTGF and PAI-1 mRNA on the 75 kPa hydrogels. In conclusion, substratum stiffness alters YAP/TAZ expression and YAP localization in primary HTM cells which then may modulate the expression of extracellular matrix proteins important in glaucoma. During the recovery period after Lat-B treatment, gene expression changes are more dramatic on substrates with stiffness similar to glaucomatous meshwork. Use of these hydrogels may more accurately reflect the alterations occurring in HTM cells in glaucoma after treatment with this drug.
PMID: 23727052 [PubMed - indexed for MEDLINE]
Early responses of vascular endothelial cells to topographic cues.
Am J Physiol Cell Physiol. 2013 Aug 1;305(3):C290-8
Authors: Dreier B, Gasiorowski JZ, Morgan JT, Nealey PF, Russell P, Murphy CJ
Vascular endothelial cells in vivo are exposed to multiple biophysical cues provided by the basement membrane, a specialized extracellular matrix through which vascular endothelial cells are attached to the underlying stroma. The importance of biophysical cues has been widely reported, but the signaling pathways that mediate cellular recognition and response to these cues remain poorly understood. Anisotropic topographically patterned substrates with nano- through microscale feature dimensions were fabricated to investigate cellular responses to topographic cues. The present study focuses on early events following exposure of human umbilical vein endothelial cells (HUVECs) to these patterned substrates. In serum-free medium and on substrates without protein coating, HUVECs oriented parallel to the long axis of underlying ridges in as little as 30 min. Immunocytochemistry showed clear differences in the localization of the focal adhesion proteins Src, p130Cas, and focal adhesion kinase (FAK) in HUVECs cultured on topographically patterned surfaces and on planar surfaces, suggesting involvement of these proteins in mediating the response to topographic features. Knockdown experiments demonstrated that FAK was not necessary for HUVEC alignment in response to topographic cues, although FAK knockdown did modulate HUVEC migration. These data identify key events early in the cellular response to biophysical stimuli.
PMID: 23703527 [PubMed - indexed for MEDLINE]
Influence of extracellular matrix proteins and substratum topography on corneal epithelial cell alignment and migration.
Tissue Eng Part A. 2013 Aug;19(15-16):1713-22
Authors: Raghunathan V, McKee C, Cheung W, Naik R, Nealey PF, Russell P, Murphy CJ
The basement membrane (BM) of the corneal epithelium presents biophysical cues in the form of topography and compliance that can impact the phenotype and behaviors of cells and their nuclei through modulation of cytoskeletal dynamics. In addition, it is also well known that the intrinsic biochemical attributes of BMs can modulate cell behaviors. In this study, the influence of the combination of exogenous coating of extracellular matrix proteins (ECM) (fibronectin-collagen [FNC]) with substratum topography was investigated on cytoskeletal architecture as well as alignment and migration of immortalized corneal epithelial cells. In the absence of FNC coating, a significantly greater percentage of cells aligned parallel with the long axis of the underlying anisotropically ordered topographic features; however, their ability to migrate was impaired. Additionally, changes in the surface area, elongation, and orientation of cytoskeletal elements were differentially influenced by the presence or absence of FNC. These results suggest that the effects of topographic cues on cells are modulated by the presence of surface-associated ECM proteins. These findings have relevance to experiments using cell cultureware with biomimetic biophysical attributes as well as the integration of biophysical cues in tissue-engineering strategies and the development of improved prosthetics.
PMID: 23488816 [PubMed - indexed for MEDLINE]
Tryptophan inhibits biofilm formation by Pseudomonas aeruginosa.
Antimicrob Agents Chemother. 2013 Apr;57(4):1921-5
Authors: Brandenburg KS, Rodriguez KJ, McAnulty JF, Murphy CJ, Abbott NL, Schurr MJ, Czuprynski CJ
Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the d and l isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of d and l isoforms producing the greatest inhibitory effect. Addition of d-/l-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa.
PMID: 23318791 [PubMed - indexed for MEDLINE]
Biochemically and topographically engineered poly(ethylene glycol) diacrylate hydrogels with biomimetic characteristics as substrates for human corneal epithelial cells.
J Biomed Mater Res A. 2013 Apr;101(4):1184-94
Authors: Yañez-Soto B, Liliensiek SJ, Murphy CJ, Nealey PF
Incorporation of biophysical and biochemical cues into the design of biomaterials is an important strategy for tissue engineering, the design of biomedical implants and cell culture. Hydrogels synthesized from poly(ethylene glycol) diacrylate (PEGDA) were investigated as a platform to simultaneously present human corneal epithelial cells (HCECs) in vitro with topography and adhesion peptides to mimic the native physical and chemical attributes of the basement membrane underlying the epithelium in vivo. Hydrogels synthesized from aqueous solutions of 20% PEGDA (M(w) = 3400 g/mol) prevented nonspecific cell adhesion and were functionalized with the integrin-binding peptide Arg-Gly-Asp (RGD) in concentrations from 5 to 20 mM. The hydrogels swelled minimally after curing and were molded with ridge and groove features with lateral dimensions from 200 to 2000 nm and 300-nm depth. HCECs were cultured on topographic surfaces functionalized with RGD and compared with control unfunctionalized topographic substrates. HCEC alignment, either parallel or perpendicular to ridges, was influenced by the culture media on substrates promoting nonspecific attachment. In contrast, the alignment of HCECs cultured on RGD hydrogels showed substantially less dependence on the culture media. In the latter case, the moldable RGD-functionalized hydrogels allowed for decoupling the cues from surface chemistry, soluble factors, and topography that simultaneously impact HCEC behavior.
PMID: 23255502 [PubMed - indexed for MEDLINE]
Nuclear and cellular alignment of primary corneal epithelial cells on topography.
J Biomed Mater Res A. 2013 Apr;101(4):1069-79
Authors: Raghunathan VK, McKee CT, Tocce EJ, Nealey PF, Russell P, Murphy CJ
The basement membrane of the corneal epithelium presents biophysical cues in the form of topography and compliance that can modulate cytoskeletal dynamics, which, in turn, can result in altering cellular and nuclear morphology and alignment. In this study, the effect of topographic patterns of alternating ridges and grooves on nuclear and cellular shape and alignment was determined. Primary corneal epithelial cells were cultured on either planar or topographically patterned (400-4000 nm pitch) substrates. Alignment of individual cell body was correlated with respective nucleus for the analysis of orientation and elongation. A biphasic response in alignment was observed. Cell bodies preferentially aligned perpendicular to the 800 nm pitch; and with increasing pitch, cells increasingly aligned parallel to the substratum. Nuclear orientation largely followed this trend with the exception of those on 400 nm. On this biomimetic size scale, some nuclei oriented perpendicular to the topography while their cytoskeleton elements aligned parallel. Both nuclei and cell bodies were elongated on topography compared to those on flat surfaces. Our data demonstrate that nuclear orientation and shape are differentially altered by topographic features that are not mandated by alignment of the cell body. This novel finding suggests that nuanced differences in alignment of the nucleus versus the cell body exist and that these differences could have consequences on gene and protein regulation that ultimately regulate cell behaviors. A full understanding of these mechanisms could disclose novel pathways that would better inform evolving strategies in cell, stem cell, and tissue engineering as well as the design and fabrication of improved prosthetic devices.
PMID: 22965583 [PubMed - indexed for MEDLINE]
The influence of biomimetic topographical features and the extracellular matrix peptide RGD on human corneal epithelial contact guidance.
Acta Biomater. 2013 Feb;9(2):5040-51
Authors: Tocce EJ, Liliensiek SJ, Broderick AH, Jiang Y, Murphy KC, Murphy CJ, Lynn DM, Nealey PF
A major focus in the field of tissue engineering is the regulation of essential cell behaviors through biophysical and biochemical cues from the local extracellular environment. The impact of nanotopographical cues on human corneal epithelial cell (HCEC) contact guidance, proliferation, migration and adhesion have previously been demonstrated. In the current report we have expanded our study of HCEC responses to include both biophysical and controlled biochemical extracellular cues. By exploiting methods for the layer-by-layer coating of substrates with reactive poly(ethylene imine)/poly(2-vinyl-4,4-dimethylazlactone)-based multilayer thin films we have incorporated a single adhesion peptide motif, Arg-Gly-Asp (RGD), on topographically patterned substrates. This strategy eliminates protein adsorption onto the surface, thus decoupling the effects of the HCEC response to topographical cues from adsorbed proteins and soluble media proteins. The direction of cell alignment was dependent on the scale of the topographical cues and, to less of an extent, the culture medium. In EpiLife® medium cell alignment to unmodified-NOA81 topographical features, which allowed protein adsorption, differed significantly from cell alignment on RGD-modified features. These results demonstrate that the surface chemical composition significantly affects how HCECs respond to topographical cues. In summary, we have demonstrated modulation of the HCEC response to environmental cues through critical substrate and soluble parameters.
PMID: 23069317 [PubMed - indexed for MEDLINE]
Role of substratum stiffness in modulating genes associated with extracellular matrix and mechanotransducers YAP and TAZ.
Invest Ophthalmol Vis Sci. 2013 Jan;54(1):378-86
Authors: Raghunathan VK, Morgan JT, Dreier B, Reilly CM, Thomasy SM, Wood JA, Ly I, Tuyen BC, Hughbanks M, Murphy CJ, Russell P
PURPOSE: Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma.
METHODS: HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing.
RESULTS: YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP-1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA.
CONCLUSIONS: The expression of YAP/TAZ and ECM-related-genes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma.
PMID: 23258147 [PubMed - indexed for MEDLINE]
The use of native chemical functional groups presented by wound beds for the covalent attachment of polymeric microcarriers of bioactive factors.
Biomaterials. 2013 Jan;34(2):340-52
Authors: Jain R, Agarwal A, Kierski PR, Schurr MJ, Murphy CJ, McAnulty JF, Abbott NL
The development of versatile methods that provide spatial and temporal control over the presentation of physical and biochemical cues on wound beds can lead to new therapeutic approaches that expedite wound healing by favorably influencing cellular behaviors. Toward that goal, we report that native chemical functional groups presented by wound beds can be utilized for direct covalent attachment of polymeric microbeads. Specifically, we demonstrated the covalent attachment of maleimide-functionalized and catechol-functionalized microbeads, made of either polystyrene (non-degradable) or poly(lactic-co-glycolic acid) ((PLGA), degradable), to sulfhydryl and amine groups present on porcine dermis used here as an ex vivo model wound bed. A pronounced increase (10-70 fold) in the density and persistence of the covalently reactive microbeads was observed relative to microbeads that adsorb via non-covalent interactions. Complementary characterization of the surface chemistry of the ex vivo wound beds using Raman microspectroscopy provides support for our conclusion that the increased adherence of the maleimide-functionalized beads results from their covalent bond formation with sulfhydryl groups on the wound bed. The attachment of maleimide-functionalized microbeads to wounds created in live wild-type and diabetic mice led to observations of differential immobilization of microbeads on them and were consistent with anticipated differences in the presentation of sulfhydryl groups on the two different wound types. Finally, the incorporation of maleimide-functionalized microbeads in wounds created in wild-type mice did not impair the rate of wound closure relative to an untreated wound. Overall, the results presented in this paper enable a general and facile approach to the engineering of wound beds in which microbeads are covalently immobilized to wound beds. Such immobilized microbeads could be used in future studies to release bioactive factors (e.g., antimicrobial agents or growth factors) and/or introduce topographical cues that promote cell behaviors underlying healing and wound closure.
PMID: 23088838 [PubMed - indexed for MEDLINE]
Substratum compliance modulates corneal fibroblast to myofibroblast transformation.
Invest Ophthalmol Vis Sci. 2013;54(8):5901-7
Authors: Dreier B, Thomasy SM, Mendonsa R, Raghunathan VK, Russell P, Murphy CJ
PURPOSE: The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor β1 (TGF-β1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-β1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas.
METHODS: Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (> 1 gigapascal [GPa]). Cells were cultured in media containing TGF-β1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot.
RESULTS: Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-β1.
CONCLUSIONS: These data indicate that substrate compliance modulates TGF-β1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.
PMID: 23860754 [PubMed - indexed for MEDLINE]
Integration of silver nanoparticle-impregnated polyelectrolyte multilayers into murine-splinted cutaneous wound beds.
J Burn Care Res. 2013 Nov-Dec;34(6):e359-67
Authors: Guthrie KM, Agarwal A, Teixeira LB, Dubielzig RR, Abbott NL, Murphy CJ, Singh H, McAnulty JF, Schurr MJ
Silver is a commonly used topical antimicrobial. However, technologies to immobilize silver at the wound surface are lacking, while currently available silver-containing wound dressings release excess silver that can be cytotoxic and impair wound healing. We have shown that precise concentrations of silver at lower levels can be immobilized into a wound bed using a polyelectrolyte multilayer attachment technology. These silver nanoparticle-impregnated polyelectrolyte multilayers are noncytotoxic yet bactericidal in vitro, but their effect on wound healing in vivo was previously unknown. The purpose of this study was to determine the effect on wound healing of integrating silver nanoparticle/polyelectrolyte multilayers into the wound bed. A full-thickness, splinted, excisional murine wound healing model was employed in both phenotypically normal mice and spontaneously diabetic mice (healing impaired model). Gross image measurements showed an initial small lag in healing in the silver-treated wounds in diabetic mice, but no difference in time to complete wound closure in either normal or diabetic mice. Histological analysis showed modest differences between silver-treated and control groups on day 9, but no difference between groups at the time of wound closure. We conclude that silver nanoparticle/polyelectrolyte multilayers can be safely integrated into the wound beds of both normal and diabetic mice without delaying wound closure, and with transient histological effects. The results of this study suggest the feasibility of this technology for use as a platform to affect nanoscale wound engineering approaches to microbial prophylaxis or to augment wound healing.
PMID: 23511285 [PubMed - indexed for MEDLINE]
Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues.
Acta Biomater. 2012 Dec;8(12):4285-94
Authors: Dreier B, Raghunathan VK, Russell P, Murphy CJ
A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.
PMID: 22813850 [PubMed - indexed for MEDLINE]
Compliance profile of the human cornea as measured by atomic force microscopy.
Micron. 2012 Dec;43(12):1293-8
Authors: Last JA, Thomasy SM, Croasdale CR, Russell P, Murphy CJ
The ability to accurately determine the elastic modulus of each layer of the human cornea is a crucial step in the design of better corneal prosthetics. In addition, knowledge of the elastic modulus will allow design of substrates with relevant mechanical properties for in vitro investigations of cellular behavior. Previously, we have reported elastic modulus values for the anterior basement membrane and Descemet's membrane of the human cornea, the surfaces in contact with the epithelial and endothelial cells, respectively. We have completed the compliance profile of the stromal elements of the human cornea by obtaining elastic modulus values for Bowman's layer and the anterior stroma. Atomic force microscopy (AFM) was used to determine the elastic modulus, which is a measure of the tissue stiffness and is inversely proportional to the compliance. The elastic response of the tissue allows analysis with the Hertz equation, a model that provides a relationship between the indentation force and depth and is a function of the tip radius and the modulus of the substrate. The elastic modulus values for each layer of the cornea are: 7.5±4.2 kPa (anterior basement membrane), 109.8±13.2 kPa (Bowman's layer), 33.1±6.1 kPa (anterior stroma), and 50±17.8 kPa (Descemet's membrane). These results indicate that the biophysical properties, including elastic modulus, of each layer of the human cornea are unique and may play a role in the maintenance of homeostasis as well as in the response to therapeutic agents and disease states. The data will also inform the design and fabrication of improved corneal prosthetics.
PMID: 22421334 [PubMed - indexed for MEDLINE]
The modulation of canine mesenchymal stem cells by nano-topographic cues.
Exp Cell Res. 2012 Nov 15;318(19):2438-45
Authors: Wood JA, Ly I, Borjesson DL, Nealey PF, Russell P, Murphy CJ
Mesenchymal stem cells (MSCs) represent a promising cellular therapeutic for the treatment of a variety of disorders. On transplantation, MSCs interact with diverse extracellular matrices (ECMs) that vary dramatically in topographic feature type, size and surface order. In order to investigate the impact of these topographic cues, surfaces were fabricated with either isotropically ordered holes or anisotropically ordered ridges and grooves. To simulate the biologically relevant nano through micron size scale, a series of topographically patterned substrates possessing features of differing pitch (pitch=feature width+groove width) were created. Results document that the surface order and size of substratum topographic features dramatically modulate fundamental MSC behaviors. Topographically patterned (ridge+groove) surfaces were found to significantly impact MSC alignment, elongation, and aspect ratio. Novel findings also demonstrate that submicron surfaces patterned with holes resulted in increased MSC alignment to adjacent cells as well as increased migration rates. Overall, this study demonstrates that the presentation of substratum topographic cues dramatically influence MSC behaviors in a size and shape dependent manner. The response of MSCs to substratum topographic cues was similar to other cell types that have been studied previously with regards to cell shape on ridge and groove surfaces but differed with respect to proliferation and migration. This is the first study to compare the impact of anisotropically ordered ridge and groove topographic cues to isotropically order holed topographic cues on fundamental MSC behaviors across a range of biologically relevant size scales.
PMID: 22771362 [PubMed - indexed for MEDLINE]
Polymeric multilayers that localize the release of chlorhexidine from biologic wound dressings.
Biomaterials. 2012 Oct;33(28):6783-92
Authors: Agarwal A, Nelson TB, Kierski PR, Schurr MJ, Murphy CJ, Czuprynski CJ, McAnulty JF, Abbott NL
Biologic wound dressings contain animal-derived components and are susceptible to high infection rates. To address this issue, we report an approach that permits incorporation of non-toxic levels of the small molecule antiseptic 'chlorhexidine' into biologic dressings. The approach relies on the fabrication of polyelectrolyte multilayer (PEMs) films containing poly(allylaminehydrochloride) (PAH), poly(acrylicacid) (PAA), and chlorhexidine acetate (CX) on elastomeric poly(dimethylsiloxane) (PDMS) sheets. The PEMs (20-100 nm thick) are subsequently stamped onto the wound-contact surface of a synthetic biologic dressing, Biobrane, which contains collagen peptides. Chlorhexidine loading in the PEMs was tailored by tuning the number of (CX/PAA) bilayers deposited, providing burst release of up to 0.98 ± 0.06 μg/cm(2) of CX over 24 h, followed by zero-order release of 0.35 ± 0.04 μg/cm(2)/day for another week. Although the CX concentrations released were below the reported in vitro cytotoxicity limit (5 μg/mL over 24 h) for human dermal fibroblasts, they killed 4 log(10) counts of pathogenic bacteria Staphylococcus aureus in solution. The CX/PEMs could be stamped onto Biobrane with high efficiency to provide CX release kinetics and in vitro antibacterial activity similar to that on PDMS stamps. In a full-thickness 'splinted' dermal wound-model in normal wild-type mice, the CX-functionalized Biobrane showed no decrease in either its adherence to the wound-bed or wound closure rate over 14 days. The murine wounds topically inoculated with ∼10(5) CFU/cm(2) of S. aureus and treated with CX-functionalized Biobrane demonstrated a 3 log(10) decrease in the wound's bacterial burden within 3 days, compared to persistent bacterial colonization found in wounds treated with unmodified Biobrane (n = 10 mice, p < 0.005). Overall, this study presents a promising approach to prevent bacterial colonization in wounds under biologic dressings.
PMID: 22784602 [PubMed - indexed for MEDLINE]
Peptides that anneal to natural collagen in vitro and ex vivo.
Org Biomol Chem. 2012 Aug 14;10(30):5892-7
Authors: Chattopadhyay S, Murphy CJ, McAnulty JF, Raines RT
Collagen comprises ¼ of the protein in humans and ¾ of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds.
PMID: 22522497 [PubMed - indexed for MEDLINE]
Antibacterial efficacy of silver-impregnated polyelectrolyte multilayers immobilized on a biological dressing in a murine wound infection model.
Ann Surg. 2012 Aug;256(2):371-7
Authors: Guthrie KM, Agarwal A, Tackes DS, Johnson KW, Abbott NL, Murphy CJ, Czuprynski CJ, Kierski PR, Schurr MJ, McAnulty JF
OBJECTIVE: To investigate the antibacterial effect of augmenting a biological dressing with polymer films containing silver nanoparticles.
BACKGROUND: Biological dressings, such as Biobrane, are commonly used for treating partial-thickness wounds and burn injuries. Biological dressings have several advantages over traditional wound dressings. However, as many as 19% of wounds treated with Biobrane become infected, and, once infected, the Biobrane must be removed and a traditional dressing approach should be employed. Silver is a commonly used antimicrobial in wound care products, but current technology uses cytotoxic concentrations of silver in these dressings. We have developed a novel and facile technology that allows immobilization of bioactive molecules on the surfaces of soft materials, demonstrated here by augmentation of Biobrane with nanoparticulate silver. Surfaces modified with nanometer-thick polyelectrolyte multilayers (PEMs) impregnated with silver nanoparticles have been shown previously to result in in vitro antibacterial activity against Staphylococcus epidermidis at loadings of silver that are noncytotoxic.
METHODS: We demonstrated that silver-impregnated PEMs can be nondestructively immobilized onto the surface of Biobrane (Biobrane-Ag) and determined the in vitro antibacterial activity of Biobrane-Ag with Staphylococcus aureus. In this study, we used an in vivo wound infection model in mice induced by topical inoculation of S aureus onto full-thickness 6-mm diameter wounds. After 72 hours, bacterial quantification was performed.
RESULTS: Wounds treated with Biobrane-Ag had significantly (P < 0.001) fewer colony-forming units than wounds treated with unmodified Biobrane (more than 4 log10 difference).
CONCLUSIONS: The results of our study indicate that immobilizing silver-impregnated PEMs on the wound-contact surface of Biobrane significantly reduces bacterial bioburden in full-thickness murine skin wounds. Further research will investigate whether this construct can be considered for human use.
PMID: 22609841 [PubMed - indexed for MEDLINE]
Integration of basal topographic cues and apical shear stress in vascular endothelial cells.
Biomaterials. 2012 Jun;33(16):4126-35
Authors: Morgan JT, Wood JA, Shah NM, Hughbanks ML, Russell P, Barakat AI, Murphy CJ
In vivo, vascular endothelial cells (VECs) are anchored to the underlying stroma through a specialization of the extracellular matrix, the basement membrane (BM) which provides a variety of substratum associated biophysical cues that have been shown to regulate fundamental VEC behaviors. VEC function and homeostasis are also influenced by hemodynamic cues applied to their apical surface. How the combination of these biophysical cues impacts fundamental VEC behavior remains poorly studied. In the present study, we investigated the impact of providing biophysical cues simultaneously to the basal and apical surfaces of human aortic endothelial cells (HAECs). Anisotropically ordered patterned surfaces of alternating ridges and grooves and isotropic holed surfaces of varying pitch (pitch = ridge or hole width + intervening groove or planar regions) were fabricated and seeded with HAECs. The cells were then subjected to a steady shear stress of 20 dyne/cm(2) applied either parallel or perpendicular to the direction of the ridge/groove topography. HAECs subjected to flow parallel to the ridge/groove topography exhibited protagonistic effects of the two stimuli on cellular orientation and elongation. In contrast, flow perpendicular to the substrate topography resulted in largely antagonistic effects. Interestingly, the behavior depended on the shape and size of the topographic features. HAECs exhibited a response that was less influenced by the substratum and primarily driven by flow on isotropically ordered holed surfaces of identical pitch to the anistropically ordered surfaces of alternating ridges and grooves. Simultaneous presentation of biophysical cues to the basal and apical aspects of cells also influenced nuclear orientation and elongation; however, the extent of nuclear realignment was more modest in comparison to cellular realignment regardless of the surface order of topographic features. Flow-induced HAEC migration was also influenced by the ridge/groove surface topographic features with significantly altered migration direction and increased migration tortuosity when flow was oriented perpendicular to the topography; this effect was also pitch-dependent. The present findings provide valuable insight into the interaction of biologically relevant apical and basal biophysical cues in regulating cellular behavior and promise to inform improved prosthetic design.
PMID: 22417618 [PubMed - indexed for MEDLINE]
Periocular and intra-articular injection of canine adipose-derived mesenchymal stem cells: an in vivo imaging and migration study.
J Ocul Pharmacol Ther. 2012 Jun;28(3):307-17
Authors: Wood JA, Chung DJ, Park SA, Zwingenberger AL, Reilly CM, Ly I, Walker NJ, Vernau W, Hayashi K, Wisner ER, Cannon MS, Kass PH, Cherry SR, Borjesson DL, Russell P, Murphy CJ
PURPOSE: Immune-mediated diseases affect millions of people worldwide with an economic impact measured in the billions of dollars. Mesenchymal stem cells (MSCs) are being investigated in the treatment of certain immune mediated diseases, but their application in the treatment of the majority of these disorders remains largely unexplored. Keratoconjunctivitis sicca can occur as a result of progressive immune-mediated destruction of lacrimal tissue in dogs and humans, and immune-mediated joint disease is common to both species. In dogs, allogeneic MSC engraftment and migration have yet to be investigated in vivo in the context of repeated injections.
METHODS: With these aims in mind, the engraftment of allogeneic canine MSCs after an injection into the periocular and intra-articular regions was followed in vivo using magnetic resonance and fluorescent imaging.
RESULTS: The cells were shown to be resident near the site of the injection for a minimum of 2 weeks. Analysis of 61 tissues demonstrated preferential migration and subsequent engraftment of MSCs in the thymus as well as the gastrointestinal tract. These results also detail a novel in vivo imaging technique and demonstrate the differential spatial distribution of MSCs after migration away from the sites of local delivery.
CONCLUSION: The active engraftment of the MSCs in combination with their previously documented immunomodulatory capabilities suggests the potential for therapeutic benefit in using MSCs for the treatment of periocular and joint diseases with immune involvement.
PMID: 22175793 [PubMed - indexed for MEDLINE]
Altered stability of mRNAs associated with glaucoma progression in human trabecular meshwork cells following oxidative stress.
Invest Ophthalmol Vis Sci. 2012 Apr;53(4):1734-41
Authors: Mochizuki H, Murphy CJ, Brandt JD, Kiuchi Y, Russell P
PURPOSE: The goals of this study were to determine if oxidative stress on human trabecular meshwork (HTM) cells influences the stability of key mRNAs containing AU rich elements (AREs) known to be associated with glaucoma progression, and if the presence of topographic cue alters the stability of these mRNAs.
METHODS: HTM cells were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 1 hour in the presence of 5 μg/mL actinomycin D and compared with untreated cells. The selected mRNAs (IL-6, IL-8, myocilin, SPARC [secreted protein, acidic and rich in cysteine], matrix metalloproteinase [MMP]-3, and MMP-9) from the cells were analyzed by using relative quantitative PCR. Immunohistochemistry for Hu antigen R (HuR) was performed in addition to Western blots of HuR. HTM cells were also grown on topographically patterned surfaces, and IL-6 mRNA was analyzed by quantitative PCR.
RESULTS: H(2)O(2) increased IL-6 mRNA stability 0.145 (0.095-0.27) to 0.345 (0.2-0.48) (normalized ratio, median [interquartile range]) (n = 5), while IL-8 mRNA was increased from 0.565 (0.408-0.6) to 0.775 (0.486-0.873) (n = 5). These differences were statistically significant (P = 0.0313, for both IL-6 and IL-8; Wilcoxon signed-rank test). The mRNAs of myocilin, SPARC, and MMP-3, which do not have AREs, were more stable after actinomycin D treatment and were not altered with oxidation. Western blot and immunohistochemistry demonstrated that H(2)O(2) treatment induces the translocation of HuR from the nucleus to the cytoplasm. Nanopatterned surfaces did not alter IL-6 mRNA stability.
CONCLUSIONS: Oxidative stress stabilizes IL-6 and IL-8 mRNAs significantly. The decay of certain mRNAs associated with glaucoma may be altered in the trabecular meshwork of glaucoma patients.
PMID: 22395891 [PubMed - indexed for MEDLINE]
The influence of a biologically relevant substratum topography on human aortic and umbilical vein endothelial cells.
Biophys J. 2012 Mar 7;102(5):1224-33
Authors: McKee CT, Wood JA, Ly I, Russell P, Murphy CJ
A topographically patterned substrate with stochastic surface order that closely mimics the topographic features of native basement membranes has been fabricated to investigate the influence of topographic biophysical cueing on human aortic and umbilical vein endothelial cells. The stochastic substrate was fabricated by first generating a highly porous polyelectrolyte multilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) followed by replicate production of this biomimetic topography via soft lithography. These substrates, which are easy to prepare and replicate, possess a number of prominent features associated with in vivo vascular basement membrane (interwoven ridges and grooves, bumps, and pores), which have typically been studied as singular features that frequently possess anisotropic surface order (e.g., alternating ridges and grooves). When compared to a flat surface of identical chemistry, these biomimetic topographies influenced a number of important cellular behaviors associated with the homeostasis and degradation of vascular tissues. These include modulating cell migration rate and directional persistence, proliferation rate, and gene expression associated with regulation and remodeling of vascular tissues as well as inflammation.
PMID: 22404945 [PubMed - indexed for MEDLINE]
Substratum stiffness and latrunculin B regulate matrix gene and protein expression in human trabecular meshwork cells.
Invest Ophthalmol Vis Sci. 2012 Feb;53(2):952-8
Authors: Thomasy SM, Wood JA, Kass PH, Murphy CJ, Russell P
PURPOSE: To determine the impact of substratum stiffness and latrunculin-B (Lat-B), on the expression of several matrix proteins that are associated with glaucoma.
METHODS: Human trabecular meshwork (HTM) cells were cultured on hydrogels possessing stiffness values mimicking those found in normal (5 kPa) and glaucomatous meshworks (75 kPa), or tissue culture polystyrene (TCP; >1 GPa). Cells were treated with 2.0 μM Lat-B in dimethyl sulfoxide (DMSO) or DMSO alone. RT-PCR was used to determine the impact of substratum stiffness and/or Lat-B treatment on the expression of secreted protein, acidic, cysteine rich (SPARC), myocilin, angiopoietin-like factor (ANGPTL)-7, and transglutaminase (TGM)-2. Immunofluorescence was used to assess changes in protein expression.
RESULTS: SPARC and myocilin mRNA expression were dramatically increased on the 75 kPa hydrogels and decreased on the 5 kPa hydrogels in comparison to TCP. In contrast, ANGPTL-7 mRNA and TGM-2 mRNA was decreased on the 75 kPa and 5 kPa hydrogels, respectively, in comparison with TCP. Treatment with Lat-B dramatically downregulated both SPARC and myocilin on 75 kPa hydrogels. In contrast, cells grown on TCP produced greater or similar amounts of SPARC and myocilin mRNA after Lat-B treatment. SPARC and myocilin protein expression paralleled changes in mRNA expression.
CONCLUSIONS: Substratum stiffness impacts HTM matrix gene and protein expression and modulates the impact of Lat-B treatment on the expression of these matrix proteins. Integrating the use of biologically relevant substratum stiffness in the conduction of in vitro experiments gives important insights into HTM cell response to drugs that may more accurately predict responses observed in vivo.
PMID: 22247475 [PubMed - indexed for MEDLINE]
Substratum topography modulates corneal fibroblast to myofibroblast transformation.
Invest Ophthalmol Vis Sci. 2012 Feb;53(2):811-6
Authors: Myrna KE, Mendonsa R, Russell P, Pot SA, Liliensiek SJ, Jester JV, Nealey PF, Brown D, Murphy CJ
PURPOSE: The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFβ-induced myofibroblast transformation of corneal cells.
METHODS: Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFβ at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed.
RESULTS: Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P < 0.002) compared with planar surfaces when exposed to TGFβ; the greatest reduction was seen on the 1400 nm surface. Smad7 mRNA expression was significantly greater on all patterned surfaces exposed to TGFβ (P < 0.002), whereas cells grown on planar surfaces showed equal or reduced levels of Smad7. Western blot analysis and αSMA immunocytochemical staining demonstrated reduced transition to the myofibroblast phenotype on the 1400 nm surface when compared with cells on a planar surface.
CONCLUSIONS: These data demonstrate that nanoscale topographic features modulate TGFβ-induced myofibroblast differentiation and αSMA expression, possibly through upregulation of Smad7. It is therefore proposed that in the wound environment, native nanotopographic cues assist in stabilizing the keratocyte/fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.
PMID: 22232431 [PubMed - indexed for MEDLINE]
Modulation of osteogenic differentiation in hMSCs cells by submicron topographically-patterned ridges and grooves.
Biomaterials. 2012 Jan;33(1):128-36
Authors: Watari S, Hayashi K, Wood JA, Russell P, Nealey PF, Murphy CJ, Genetos DC
Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. Four-hundred nanometer pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of expression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants.
PMID: 21982295 [PubMed - indexed for MEDLINE]
Functionalization of reactive polymer multilayers with RGD and an antifouling motif: RGD density provides control over human corneal epithelial cell-substrate interactions.
J Biomed Mater Res A. 2012 Jan;100(1):84-93
Authors: Tocce EJ, Broderick AH, Murphy KC, Liliensiek SJ, Murphy CJ, Lynn DM, Nealey PF
Our study demonstrates that substrates fabricated using a "reactive" layer-by-layer approach promote well-defined cell-substrate interactions of human corneal epithelial cells. Specifically, crosslinked and amine-reactive polymer multilayers were produced by alternating "reactive" deposition of an azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone)] (PVDMA) and a primary amine-containing polymer [branched poly(ethylene imine)] (PEI). Advantages of our system include a 5- to 30-fold decrease in deposition time compared to traditional polyelectrolyte films and direct modification of the films with peptides. Our films react with mixtures of an adhesion-promoting peptide containing Arg-Gly-Asp (RGD) and the small molecule D-glucamine, a chemical motif which is nonfouling. Resulting surfaces prevent protein adsorption and promote cell attachment through specific peptide interactions. The specificity of cell attachment via immobilized RGD sequences was verified using both a scrambled RDG peptide control as well as soluble-RGD competitive assays. Films were functionalized with monotonically increasing surface densities of RGD which resulted in both increased cell attachment and the promotion of a tri-phasic proliferative response of a human corneal epithelial cell line (hTCEpi). The ability to treat PEI/PVDMA films with peptides for controlled cell-substrate interactions enables the use of these films in a wide range of biological applications.
PMID: 21972074 [PubMed - indexed for MEDLINE]
Hydrogels with well-defined peptide-hydrogel spacing and concentration: impact on epithelial cell behavior().
Soft Matter. 2012;8(2):390-398
Authors: Wilson MJ, Liliensiek SJ, Murphy CJ, Murphy WL, Nealey PF
The spacing of peptides away from a hydrogel matrix dramatically impacts their availability and subsequent interactions with cells. Peptides were synthesized with monodisperse poly(ethylene glycol) spacers of different lengths that separate the peptide from the monomeric functionality which reacts during hydrogel polymerization. Specifically, bioactive RGD ligands were conjugated to PEG(5), PEG(11) or PEG(27) spacers via solid phase techniques and then functionalized with an acryloyl end group. These acryloyl-PEGx-RGD conjugates were then copolymerized with PEGDA to form an inert hydrogel network decorated with RGD ligands for cell interactions. As the PEG spacer length increases, the RGD concentration required to support cell attachment and spreading decreases. The competitive detachment of hTCEpi cells in the presence of soluble linear RGD also shows non-linear dependence on the PEG spacer length, as more cells remained attached and spread on gels functionalized with longer PEG-RGD conjugates in comparison to the shorter PEG-RGD conjugates. The strategy and synthetic techniques developed here allow for reproducible control over peptide-hydrogel spacing and peptide concentration, and may be extended for incorporation of multiple peptides and to other hydrogel platforms.
PMID: 23264803 [PubMed - as supplied by publisher]
Topographic modulation of the orientation and shape of cell nuclei and their influence on the measured elastic modulus of epithelial cells.
Biophys J. 2011 Nov 2;101(9):2139-46
Authors: McKee CT, Raghunathan VK, Nealey PF, Russell P, Murphy CJ
The influence of nucleus shape and orientation on the elastic modulus of epithelial cells was investigated with atomic force microscopy. The shape and orientation were controlled by presenting the epithelial cells with anisotropic parallel ridges and grooves of varying pitch at the cell substratum. As the cells oriented to the underlying topography, the volume of the nucleus increased as the pitch of the topography increased from 400 nm to 2000 nm. The increase in nucleus volume was reflected by an increase in the measured elastic modulus of the topographically aligned cells. Significant alterations in the shape of the nucleus, by intimate contact with the topographic ridge and grooves of the underlying cell, were also observed via confocal microscopy, indicating that the nucleus may also act as a direct mechanosensor of substratum topography.
PMID: 22067151 [PubMed - indexed for MEDLINE]
The role of substratum compliance of hydrogels on vascular endothelial cell behavior.
Biomaterials. 2011 Aug;32(22):5056-64
Authors: Wood JA, Shah NM, McKee CT, Hughbanks ML, Liliensiek SJ, Russell P, Murphy CJ
Cardiovascular disease (CVD) remains a leading cause of death both within the United States (US) as well as globally. In 2006 alone, over one-third of all deaths in the US were attributable to CVD. The high prevalence, mortality, morbidity, and socioeconomic impact of CVD has motivated a significant research effort; however, there remain significant knowledge gaps regarding disease onset and progression as well as pressing needs for improved therapeutic approaches. One critical area of research that has received limited attention is the role of biophysical cues on the modulation of endothelial cell behaviors; specifically, the impact of local compliance, or the stiffness, of the surrounding vascular endothelial extracellular matrix. In this study, the impact of substratum compliance on the modulation of cell behaviors of several human primary endothelial cell types, representing different anatomic sites and differentiation states in vivo, were investigated. Substrates used within our studies span the range of compliance that has been reported for the vascular endothelial basement membrane. Differences in substratum compliance had a profound impact on cell attachment, spreading, elongation, proliferation, and migration. In addition, each cell population responded differentially to changes in substratum compliance, documenting endothelial heterogeneity in the response to biophysical cues. These results demonstrate the importance of incorporating substratum compliance in the design of in vitro experiments as well as future prosthetic design. Alterations in vascular substratum compliance directly influence endothelial cell behavior and may participate in the onset and/or progression of CVDs.
PMID: 21501863 [PubMed - indexed for MEDLINE]
Endotoxin-induced structural transformations in liquid crystalline droplets.
Science. 2011 Jun 10;332(6035):1297-300
Authors: Lin IH, Miller DS, Bertics PJ, Murphy CJ, de Pablo JJ, Abbott NL
The ordering of liquid crystals (LCs) is known to be influenced by surfaces and contaminants. Here, we report that picogram per milliliter concentrations of endotoxin in water trigger ordering transitions in micrometer-size LC droplets. The ordering transitions, which occur at surface concentrations of endotoxin that are less than 10(-5) Langmuir, are not due to adsorbate-induced changes in the interfacial energy of the LC. The sensitivity of the LC to endotoxin was measured to change by six orders of magnitude with the geometry of the LC (droplet versus slab), supporting the hypothesis that interactions of endotoxin with topological defects in the LC mediate the response of the droplets. The LC ordering transitions depend strongly on glycophospholipid structure and provide new designs for responsive soft matter.
PMID: 21596951 [PubMed - indexed for MEDLINE]
Indentation versus tensile measurements of Young's modulus for soft biological tissues.
Tissue Eng Part B Rev. 2011 Jun;17(3):155-64
Authors: McKee CT, Last JA, Russell P, Murphy CJ
In this review, we compare the reported values of Young's modulus (YM) obtained from indentation and tensile deformations of soft biological tissues. When the method of deformation is ignored, YM values for any given tissue typically span several orders of magnitude. If the method of deformation is considered, then a consistent and less ambiguous result emerges. On average, YM values for soft tissues are consistently lower when obtained by indentation deformations. We discuss the implications and potential impact of this finding.
PMID: 21303220 [PubMed - indexed for MEDLINE]
Polymeric Multilayers that contain Silver Nanoparticles can be Stamped onto Biological Tissues to Provide Antibacterial Activity.
Adv Funct Mater. 2011 May 24;21(10):1863-1873
Authors: Agarwal A, Guthrie KM, Czuprynski CJ, Schurr MJ, McAnulty JF, Murphy CJ, Abbott NL
We report the design of polyelectrolyte multilayers (PEMs) that can be prefabricated on an elastomeric stamp and mechanically transferred onto biomedically-relevant soft materials, including medical-grade silicone elastomers (E'~450-1500 kPa; E'-elastic modulus) and the dermis of cadaver-skin (E'~200-600 kPa). Whereas initial attempts to stamp PEMs formed from poly(allylamine hydrochloride) and poly(acrylic acid) resulted in minimal transfer onto soft materials, we report that integration of micrometer-sized beads into the PEMs (thicknesses of 6-160 nm) led to their quantitative transfer within 30 seconds of contact at a pressure of ~196 kPa. To demonstrate the utility of this approach, PEMs were impregnated with a range of loadings of silver-nanoparticles and stamped onto the dermis of human cadaver-skin (a wound-simulant) that was subsequently incubated with bacterial cultures. Skin-dermis stamped with PEMs that released 0.25±0.01 μg cm(-2) of silver ions caused a 6 log10 reduction in colony forming units of Staphylococcus epidermidis and Pseudomonas aeruginosa within 12 h. Significantly, this level of silver release is below that which is cytotoxic to NIH 3T3 mouse fibroblast cells. Overall, this study describes a general and facile approach for the functionalization of biomaterial surfaces without subjecting them to potentially deleterious processing conditions.
PMID: 25558188 [PubMed - as supplied by publisher]
The pharmacologic assessment of a novel lymphocyte function-associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in dogs.
Invest Ophthalmol Vis Sci. 2011 May;52(6):3174-80
Authors: Murphy CJ, Bentley E, Miller PE, McIntyre K, Leatherberry G, Dubielzig R, Giuliano E, Moore CP, Phillips TE, Smith PB, Prescott E, Miller JM, Thomas P, Scagliotti R, Esson D, Gadek T, O'Neill CA
PURPOSE: Keratoconjunctivitis sicca (KCS) is characterized by inflammation and decreased production of tears containing increased levels of cytokines. The release occurs in the setting of conjunctival and lacrimal gland inflammation, potentially mediated by the interaction between lymphocyte function-associated antigen (LFA)-1, a cell surface protein found on lymphocytes, and its cognate ligand intercellular adhesion molecule (ICAM)-1. SAR 1118 is a novel LFA-1 antagonist and may be an effective therapeutic agent for the treatment of KCS. The following studies were performed to assess the in vitro activity of SAR 1118 and to evaluate the clinical efficacy of topical SAR 1118 for the treatment of idiopathic canine KCS.
METHOD: Pharmacodynamics were assessed by measuring the ability of SAR 1118 to inhibit Jurkat T-cell binding with recombinant human ICAM-1 and to inhibit cytokine release from human peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin B. For the assessment of clinical efficacy, 10 dogs diagnosed with idiopathic KCS were treated with SAR 1118 1% topical ophthalmic solution three times daily for 12 weeks. Schirmer's tear test (STT) was used to measure tear production.
RESULTS: SAR 1118 demonstrated concentration-dependent inhibition of Jurkat T-cell attachment, inhibition of lymphocyte activation, and release of inflammatory cytokines, particularly the Th1, Th2, and Th17 T-cell cytokines IFN-γ, IL-2, and IL-17F, respectively. Mean STT values increased from 3.4 mm during week 1 to 5.8 mm at week 12 (P < 0.025). No SAR 1118-related adverse events were observed.
CONCLUSIONS: SAR 1118 appears to be an effective anti-inflammatory treatment for KCS. Additional studies are warranted to establish the efficacy of SAR 1118 for the treatment of KCS in humans.
PMID: 21330663 [PubMed - indexed for MEDLINE]
Elastic modulus determination of normal and glaucomatous human trabecular meshwork.
Invest Ophthalmol Vis Sci. 2011 Apr;52(5):2147-52
Authors: Last JA, Pan T, Ding Y, Reilly CM, Keller K, Acott TS, Fautsch MP, Murphy CJ, Russell P
PURPOSE: Elevated intraocular pressure (IOP) is a risk factor for glaucoma. The principal outflow pathway for aqueous humor in the human eye is through the trabecular meshwork (HTM) and Schlemm's canal (SC). The junction between the HTM and SC is thought to have a significant role in the regulation of IOP. A possible mechanism for the increased resistance to flow in glaucomatous eyes is an increase in stiffness (increased elastic modulus) of the HTM. In this study, the stiffness of the HTM in normal and glaucomatous tissue was compared, and a mathematical model was developed to predict the impact of changes in stiffness of the juxtacanalicular layer of HTM on flow dynamics through this region.
METHODS: Atomic force microscopy (AFM) was used to measure the elastic modulus of normal and glaucomatous HTM. According to these results, a model was developed that simulated the juxtacanalicular layer of the HTM as a flexible membrane with embedded pores.
RESULTS: The mean elastic modulus increased substantially in the glaucomatous HTM (mean = 80.8 kPa) compared with that in the normal HTM (mean = 4.0 kPa). Regional variation was identified across the glaucomatous HTM, possibly corresponding to the disease state. Mathematical modeling suggested an increased flow resistance with increasing HTM modulus.
CONCLUSIONS: The data indicate that the stiffness of glaucomatous HTM is significantly increased compared with that of normal HTM. Modeling exercises support substantial impairment in outflow facility with increased HTM stiffness. Alterations in the biophysical attributes of the HTM may participate directly in the onset and progression of glaucoma.
PMID: 21220561 [PubMed - indexed for MEDLINE]
The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents.
Biomaterials. 2011 Mar;32(9):2417-23
Authors: McKee CT, Wood JA, Shah NM, Fischer ME, Reilly CM, Murphy CJ, Russell P
Glaucoma is a devastating neurodegenerative disease, which can lead to vision loss and is associated with irreversible damage to retinal ganglion cells. Although the mechanism of disease onset remains unknown, we have recently demonstrated that the stiffness of the ocular trabecular meshwork (HTM) increases dramatically in human donor eyes with a history of glaucoma. Here we report that polyacrylamide hydrogels, which mimic the compliant conditions of normal and glaucomatous HTM, profoundly modulate cytoskeletal dynamics and the elastic modulus of the overlying HTM cells. Substratum compliance also modulates HTM cell response to Latrunculin-B, a cytoskeletal disrupting agent currently in human clinical trials for the treatment of glaucoma. Additionally, we observed a compliance-dependent rebound effect of Latrunculin-B with an unexpected increase in HTM cell elastic modulus being observed upon withdrawal of the drug. The results predict that cytoskeletal disrupting drugs may be more potent in advanced stages of glaucoma.
PMID: 21220171 [PubMed - indexed for MEDLINE]
Ocular components in three breeds of dogs with high prevalence of myopia.
Optom Vis Sci. 2011 Feb;88(2):269-74
Authors: Williams LA, Kubai MA, Murphy CJ, Mutti DO
PURPOSE: Experimental models of human myopia have been developed using animals of various species. However, most of these are an induced rather than a spontaneous, naturally occurring myopia. This study was conducted to evaluate whether the spontaneous myopia found in three canine breeds was axial in nature and therefore similar to humans.
METHODS: Refractive error was measured by cycloplegic retinoscopy and ocular components by A-scan ultrasound (ocular axial dimensions) and videophakometry (corneal and lens radii and powers) in 83 dogs of three breeds [English Springer Spaniels (n = 33), Toy Poodles (n = 36), and Collies (n = 14)]. Dogs with refractive errors equal to or more myopic than -0.5 diopters spherical equivalent were considered myopic.
RESULTS: Myopia was most common in Toy Poodles (63.9%), followed by English Springer Spaniels (36.4%) and Collies (35.7%). Axial lengths and vitreous chamber depths were not different between myopic and non-myopic dogs (p = 0.84 and 0.63, respectively). The anterior crystalline lens radius was steeper and the lens power was greater in myopic compared with non-myopic dogs (p = 0.048 for each).
CONCLUSIONS: Spontaneous myopia was very common in all three breeds in this sample of dogs, with Toy Poodles being most affected. However, the cause of the myopia appeared to be refractive, that is from a steeper, more powerful crystalline lens, rather than from excess axial elongation. These breeds do not appear to be promising models for human axial myopia.
PMID: 21169876 [PubMed - indexed for MEDLINE]
Substratum compliance regulates human trabecular meshwork cell behaviors and response to latrunculin B.
Invest Ophthalmol Vis Sci. 2011;52(13):9298-303
Authors: Wood JA, McKee CT, Thomasy SM, Fischer ME, Shah NM, Murphy CJ, Russell P
PURPOSE: To determine the impact of substratum compliance and latrunculin-B (Lat-B), both alone and together, on fundamental human trabecular meshwork (HTM) cell behavior. Lat-B is a reversible actin cytoskeleton disruptor that decreases resistance to aqueous humor outflow and decreases intraocular pressure.
METHODS: HTM cells were cultured on polyacrylamide hydrogels possessing values for compliance that mimic those reported for normal and glaucomatous HTM, or tissue culture plastic (TCP). Cells were treated with 0.2 μM or 2.0 μM Lat-B in dimethyl sulfoxide (DMSO) or DMSO alone. The impact of substratum compliance and/or Lat-B treatment on cell attachment, proliferation, surface area, aspect ratio, and migration were investigated.
RESULTS: HTM cells had profoundly decreased attachment and proliferation rates when cultured on hydrogels possessing compliance values that mimic those found for healthy HTM. The effect of Lat-B treatment on HTM cell surface area was less for cells cultured on more compliant hydrogels compared with TCP. HTM cell migration was increased on stiffer hydrogels that mimic the compliance of glaucomatous HTM and on TCP in comparison with more compliant hydrogels. Lat-B treatment decreased cellular migration on all surfaces for at least 7 hours after treatment.
CONCLUSIONS: Substratum compliance profoundly influenced HTM cell behaviors and modulated the response of HTM cells to Lat-B. The inclusion of substratum compliance that reflects healthy or glaucomatous HTM results in cell behaviors and responses to therapeutic agents in vitro that may more accurately reflect in vivo conditions.
PMID: 22064990 [PubMed - indexed for MEDLINE]
The applications of atomic force microscopy to vision science.
Invest Ophthalmol Vis Sci. 2010 Dec;51(12):6083-94
Authors: Last JA, Russell P, Nealey PF, Murphy CJ
The atomic force microscope (AFM) is widely used in materials science and has found many applications in biological sciences but has been limited in use in vision science. The AFM can be used to image the topography of soft biological materials in their native environments. It can also be used to probe the mechanical properties of cells and extracellular matrices, including their intrinsic elastic modulus and receptor-ligand interactions. In this review, the operation of the AFM is described along with a review of how it has been thus far used in vision science. It is hoped that this review will serve to stimulate vision scientists to consider incorporating AFM as part of their research toolkit.
PMID: 21123767 [PubMed - indexed for MEDLINE]
Alterations in gene expression of human vascular endothelial cells associated with nanotopographic cues.
Biomaterials. 2010 Dec;31(34):8882-8
Authors: Gasiorowski JZ, Liliensiek SJ, Russell P, Stephan DA, Nealey PF, Murphy CJ
Human cells in vivo are exposed to a topographically rich, 3-dimenisional environment which provides extracellular cues initiating a cascade of biochemical signals resulting in changes in cell behavior. One primary focus of our group is the development of biomimetic substrates with anisotropic nanoscale topography to elucidate the mechanisms by which physical surface cues are translated into biochemical signals. To investigate changes in gene expression as a result of nanotopographic cues, Human Umbilical Vein Endothelial Cells (HUVECs) were cultured on chemically identical flat and 400 nm pitch nanogrooved surfaces. After 12 h, RNA was harvested for an Affymetrix HG U133 Plus 2.0 gene array. Of over 47,000 possible gene probes, 3171 had at least a two-fold difference in expression between the control flat and 400 nm pitch. The gene ontology groups with the most significant increase in expression are involved in protein modification and maintenance, similar to cells upregulating chaperone and protein synthesis genes in response to physical stresses. The most significant decreases in expression were observed with cell cycle proteins, including cyclins and checkpoint proteins. Extracellular matrix proteins, including integrins, collagens, and laminins, are almost uniformly downregulated on the 400 nm pitch surfaces compared to control. The downregulation of one of these genes, integrin beta 1, was confirmed via quantitative PCR. Together, these gene array data, in addition to our studies of cell behavior on nanoscale surfaces, contribute to our understanding of the signaling pathways modulated by topographical surface cues.
PMID: 20832112 [PubMed - indexed for MEDLINE]
Kinetics of immune cell infiltration in vaccinia virus keratitis.
Invest Ophthalmol Vis Sci. 2010 Sep;51(9):4541-8
Authors: Altmann S, Toomey M, Nesbit B, McIntyre K, Covert J, Dubielzig RR, Leatherberry G, Adkins E, Murphy CJ, Brandt CR
PURPOSE: Vaccinia virus keratitis leading to blindness is a severe complication of smallpox vaccination. The clinical manifestations of vaccinia virus keratitis are similar to those of herpes simplex virus keratitis, a well-studied immunopathologic disease. Vaccinia virus keratitis is likely to involve an immunopathologic component, but little is known about the pathogenesis of the disease. The goal of this study was to determine type and kinetics of immune cell infiltration in the cornea during vaccinia virus keratitis.
METHODS: Rabbit eyes were trephined and inoculated with 1x10(5) pfu of the Dryvax strain of the vaccinia virus. On days 2, 4, 7, 10, 14, and 28 after infection, the animals were scored for clinical disease and eye sections were stained for B cells, CD4+ cells, CD8+ cells, and neutrophils. The eyelid, ciliary body, cornea, iris, iridocorneal angle, and choroid were examined.
RESULTS: Corneal vaccinia virus challenge resulted in the infiltration of B cells, CD4+ cells, CD8+ cells, and neutrophils into the cornea and eyelids. Neutrophils were the predominant cell type on days 2 and 3 after infection, whereas CD4+ cells were the predominant cell type detected in corneas on days 4 through 10. CD8+ cells and B cells peaked on day 10, but at lower levels than CD4+ cells and neutrophils.
CONCLUSIONS: These results suggest that sequential migration of neutrophils, then CD4+ cells, plays an important role in vaccinia virus keratitis.
PMID: 20375330 [PubMed - indexed for MEDLINE]
Biosynthetic corneal substitute implantation in dogs.
Cornea. 2010 Aug;29(8):910-6
Authors: Bentley E, Murphy CJ, Li F, Carlsson DJ, Griffith M
PURPOSE: To assess integration of a biosynthetic corneal implant in dogs.
METHODS: Three normal adult laboratory Beagles underwent ophthalmic examinations, including slit-lamp biomicroscopy, indirect ophthalmoscopy, applanation tonometry, and Cochet-Bonnet aesthesiometry. Biosynthetic corneas fabricated from glutaraldehyde crosslinked collagen and copolymers of collagen and poly(N-isopropylacrylamide-co-acrylic acid-co-acryloxysuccinimide, denoted as TERP) were implanted into dogs by a modified epikeratoplasty technique. Ophthalmic examinations and aesthesiometry were performed daily for 5 days and then weekly thereafter for 16 weeks. Corneal samples underwent histopathological and transmission electron microscopy examination at 16 weeks.
RESULTS: Implants were epithelialized by 7 days. Intraocular pressure was within normal range throughout the study. Aesthesiometry values dropped from an average of 3.67 cm preoperatively to less than 1 mm for all dogs for the first postoperative weeks. By week 16, the average Cochet-Bonnet value was 1.67 cm, demonstrating partial recovery of functional innervation of the implant. No inflammation or rejection of the implant occurred, and minimal haze formation was noted. Light microscopy revealed thickened but normal epithelium over the implant with fibroblast migration into the scaffold. On transmission electron microscopy, the basement membrane was irregular but present and adhesion complexes were noted.
CONCLUSION: Biosynthetic corneal implantation is well tolerated in dogs, and the collagen-polymer hybrid construct holds promise for clinical application in animals and humans.
PMID: 20539221 [PubMed - indexed for MEDLINE]
The ability of corneal epithelial cells to recognize high aspect ratio nanostructures.
Biomaterials. 2010 May;31(14):4064-72
Authors: Tocce EJ, Smirnov VK, Kibalov DS, Liliensiek SJ, Murphy CJ, Nealey PF
The basement membrane of the human corneal epithelium comprises topographic features including fibers, pores, and elevations with feature dimensions on the order of 20-400 nm. Understanding the impact of sub-micron and nanotopography on corneal cell behavior will contribute to our understanding of biomechanical cues and will assist in the design of improved synthetic corneal implants. We utilized well defined ridge and groove wave-like nanostructures (wave ordered structures, WOS) of 60-140 pitches (30-70 nm ridge widths) and 200 nm depths to assess human corneal epithelial cell (HCEC) contact guidance and to establish HCEC contact acuity defined as the lower limit in feature dimensions at which cells respond to biomimetic topographic cues. Results using the WOS substrates demonstrate that HCEC contact acuity is in the range of 60 nm pitch for cells in a serum-free basal medium (EpiLife) and in the range of 90 nm pitch for cells in epithelial medium. To further investigate the influence of HCEC contact acuity in the presence of larger topographic cues, we fabricated 70 nm pitch WOS-overlaid parallel to the top of the ridges of 800-4000 nm pitch. HCEC cultured in epithelial medium demonstrate a significant increase in the percent of cells aligning to 4000 nm pitch topography with WOS-overlay compared to controls (both flat and 70 nm WOS alone) and 4000 nm pitch topography alone. These results highlight the significance of the lower range of basement membrane scale topographic cues on cell response and allow for improved prosthetic design.
PMID: 20153044 [PubMed - indexed for MEDLINE]
Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells.
Biomaterials. 2010 Feb;31(4):680-90
Authors: Agarwal A, Weis TL, Schurr MJ, Faith NG, Czuprynski CJ, McAnulty JF, Murphy CJ, Abbott NL
Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (approximately 100 microg/cm(2)) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with approximately 0.4 microg/cm(2) to approximately 23.6 microg/cm(2) of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as approximately 0.4 microg/cm(2) of silver in the polymeric films caused a reduction of 6log(10)CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing approximately 0.4 microg/cm(2) of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.
PMID: 19864019 [PubMed - indexed for MEDLINE]
The PPCD1 mouse: characterization of a mouse model for posterior polymorphous corneal dystrophy and identification of a candidate gene.
PLoS One. 2010;5(8):e12213
Authors: Shen AL, O'Leary KA, Dubielzig RR, Drinkwater N, Murphy CJ, Kasper CB, Bradfield CA
The PPCD1 mouse, a spontaneous mutant that arose in our mouse colony, is characterized by an enlarged anterior chamber resulting from metaplasia of the corneal endothelium and blockage of the iridocorneal angle by epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human posterior polymorphous corneal dystrophy (PPCD) and the sporadic condition, iridocorneal endothelial syndrome. Affected eyes exhibit epithelialized corneal endothelial cells, with inappropriate cytokeratin expression and proliferation over the iridocorneal angle and posterior cornea. We have termed this the "mouse PPCD1" phenotype and mapped the mouse locus for this phenotype, designated "Ppcd1", to a 6.1 Mbp interval on Chromosome 2, which is syntenic to the human Chromosome 20 PPCD1 interval. Inheritance of the mouse PPCD1 phenotype is autosomal dominant, with complete penetrance on the sensitive DBA/2J background and decreased penetrance on the C57BL/6J background. Comparative genome hybridization has identified a hemizygous 78 Kbp duplication in the mapped interval. The endpoints of the duplication are located in positions that disrupt the genes Csrp2bp and 6330439K17Rik and lead to duplication of the pseudogene LOC100043552. Quantitative reverse transcriptase-PCR indicates that expression levels of Csrp2bp and 6330439K17Rik are decreased in eyes of PPCD1 mice. Based on the observations of decreased gene expression levels, association with ZEB1-related pathways, and the report of corneal opacities in Csrp2bp(tm1a(KOMP)Wtsi) heterozygotes and embryonic lethality in nulls, we postulate that duplication of the 78 Kbp segment leading to haploinsufficiency of Csrp2bp is responsible for the mouse PPCD1 phenotype. Similarly, CSRP2BP haploinsufficiency may lead to human PPCD.
PMID: 20808945 [PubMed - indexed for MEDLINE]