Dr. Raghunathan is a Bioengineer in the Department of Surgical and Radiological Sciences at the University of California, Davis. He is cross trained in biomaterials, biomechanics, and cell and molecular biology with research interests and expertise in drug delivery, force microscopy, toxicology, tissue engineering and medical device development for orthopedic, wound healing, and ocular applications.
Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways.
Invest Ophthalmol Vis Sci. 2020 Aug 03;61(10):41
Authors: Yemanyi F, Vranka J, Raghunathan VK
Purpose: The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways.
Methods: Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/β-catenin and YAP/TAZ pathways were determined.
Results: At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold (P < 0.001) stiffer, total β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear β-catenin.
Conclusions: Increased cytoplasmic TAZ may inhibit β-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.
PMID: 32832971 [PubMed - as supplied by publisher]
Isolation and chasracterization of primary human trabecular meshwork cells from segmental flow regions: New tools for understanding segmental flow.
Exp Eye Res. 2020 May 03;:108046
Authors: Ja S, K D, Ts A, Vk R, Ja V
Segmental flow in the human trabecular meshwork is a well-documented phenomenon but in depth mechanistic investigations of high flow (HF) and low flow (LF) regions are restricted due to the small amount of tissue available from a single donor. To address this issue we have generated and characterized multiple paired HF and LF cell strains. Here paired HF and LF cell strains were generated from single donors. Cells were characterized for growth and proliferation, as well as gene and protein expression of potential segmental region markers. Cells isolated from HF and LF regions have similar growth and proliferation rates. Gene expression data reveals vascular cell adhesion protein 1 (VCAM1), thrombospondin 2 (THBS2), and tissue inhibitor of metalloproteinase 1 (TIMP1) are potential markers of LF cells in vitro. Protein expression of VCAM1, THBS2 and TIMP1 are complex and may reflect the dynamic nature of the TM. Initial protein expression levels of these genes is either similar between HF and LF cells (VCAM1, THBS2), or higher in HF compared to LF in some strains (TIMP1). However, after long term culture LF cells express higher levels of VCAM1, TIMP1 and THBS2 protein compared to HF cells. HF and LF cell strains are a powerful new tool that enable understanding segmental flow allowing for multiple experiments on the same genetic background.
PMID: 32376472 [PubMed - as supplied by publisher]
Stromal Collagen Arrangement Correlates with Stiffness of the Canine Cornea.
Bioengineering (Basel). 2019 Dec 25;7(1):
Authors: Leonard BC, Cosert K, Winkler M, Marangakis A, Thomasy SM, Murphy CJ, Jester JV, Raghunathan VK
The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.
PMID: 31881719 [PubMed]
Biomechanical changes to Descemet's membrane precede endothelial cell loss in an early-onset murine model of Fuchs endothelial corneal dystrophy.
Exp Eye Res. 2018 Nov 21;:
Authors: Leonard BC, Jalilian I, Raghunathan VK, Wang W, Jun AS, Murphy CJ, Thomasy SM
Early-onset Fuchs endothelial corneal dystrophy (FECD) has been associated with nonsynonymous mutations in collagen VIII α2 (COL8A2), a key extracellular matrix (ECM) protein in Descemet's membrane (DM). Two knock-in strains of mice have been generated to each express a mutant COL8A2 protein (Col8a2L450W/L450W and Col8a2Q455K/Q455K) that recapitulate the clinical phenotype of early-onset FECD including endothelial cell loss, cellular polymegathism and pleomorphism, and guttae. Due to abnormalities in ECM protein composition and structure in FECD, the stiffness of DM in Col8a2 knock-in mice and wildtype (WT) controls was measured using atomic force microscopy at 5 and 10 months of age, coinciding with the onset of FECD phenotypic abnormalities. At 5 months, only sporadic guttae were identified via in vivo confocal microscopy (IVCM) in Col8a2Q455K/Q455K mice, otherwise both strains of Col8a2 transgenic mice were indistinguishable from WT controls in terms of endothelial cell density and size. By 10 months of age, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice developed reduced corneal endothelial density, increased endothelial cell area and guttae, with the Col8a2Q455K/Q455K strain exhibiting a more severe phenotype. However, at 5 months of age, prior to the development endothelial cell abnormalities, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice knock-in mice had reduced tissue stiffness of DM that was statistically significant in the Col8a2Q455K/Q455K mice when compared with wildtype controls. These data indicate that alterations in the tissue compliance of DM precede phenotypic changes in endothelial cell count and morphology and may play a role in onset and progression of FECD.
PMID: 30471280 [PubMed - as supplied by publisher]
Species variation and spatial differences in mucin expression from corneal epithelial cells.
Exp Eye Res. 2016 Nov;152:43-48
Authors: Leonard BC, Yañez-Soto B, Raghunathan VK, Abbott NL, Murphy CJ
Mucins are large glycoproteins expressed by epithelial cells of both the conjunctiva and cornea, and principle components of the glycocalyx. They are thought to play an important role in determining the interactions between the cornea/conjunctiva and the overlying tear film. The purpose of this study was to characterize the membrane-associated corneal mucin expression pattern from multiple species commonly used in ophthalmic research and drug development to better define the biochemical attributes of the ocular surface. Humans, rhesus macaques and dogs were found to have a very similar pattern of mucin expression, with mucin 16 (MUC16) being the most prevalent mucin transcript. In contrast, the rabbit had a unique mucin expression pattern with all mucin transcripts expressed at relatively similar levels. To determine if there were spatial differences in expression, peripheral and central corneal epithelium were individually isolated and evaluated for mucin expression. In all species examined, MUC1, MUC4 and MUC16 had higher peripheral corneal expression when compared with central, which reached statistical significance in MUC1 (rhesus and dog). The data demonstrated variation in corneal epithelial membrane-associated mucin expression between species, with the rabbit having a distinct expression pattern. These differences may be reflective of the environment, pathogen exposure or tear film dynamics of the respective species. The species differences, as well as regional mucin expression patterns, characterized in this study further define the biochemical composition of the ocular surface and may play an important role in tear film stability.
PMID: 27614208 [PubMed - in process]
Sensing Conductive Hydrogels for Rapid Detection of Cytokines in Blood.
Adv Healthc Mater. 2016 Mar;5(6):659-64
Authors: Shin DS, Matharu Z, You J, Siltanen C, Vu T, Raghunathan VK, Stybayeva G, Hill AE, Revzin A
Conducting polymer hydrogel is fabricated atop gold or ITO electrodes and is functionalized with monoclonal antibodies. Binding of interferon-γ molecules causes redox properties of conductive hydrogel to change in a concentration-dependent fashion without the need for washing or sample handling steps. This conductive hydrogel remains functional in a fouling media such as whole blood.
PMID: 26799538 [PubMed - in process]
Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix.
Invest Ophthalmol Vis Sci. 2015 Jul 1;56(8):4447-59
Authors: Raghunathan VK, Morgan JT, Park SA, Weber D, Phinney BS, Murphy CJ, Russell P
PURPOSE: Treatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood.
METHODS: Primary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined.
RESULTS: Treatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of α-smooth muscle actin (αSMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM.
DISCUSSION: This integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated αSMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.
PMID: 26193921 [PubMed - in process]
Assessment of platelet-derived growth factor using A splinted full thickness dermal wound model in bearded dragons (Pogona vitticeps).
J Zoo Wildl Med. 2014 Dec;45(4):866-74
Authors: Keller KA, Paul-Murphy J, Weber EP, Kass PH, Guzman SM, Park SA, Raghunathan VK, Gustavsen KA, Murphy CJ
Wounds in reptiles are a common reason for presentation to a veterinarian. At this time there is limited information on effective topical medications to aid in wound closure. The objectives of this study were to translate the splinted, full-thickness dermal wound model, validated in mice, to the bearded dragon (Pogona vitticeps) and to determine the effect of topical becaplermin (BP), a platelet-derived growth factor (0.01%), on the rate of wound closure. Ten bearded dragons were anesthetized and two full-thickness cutaneous wounds were made on the dorsum of each lizard. Encircling splints were applied surrounding each wound and subsequently covered by a semi-occlusive dressing. Five lizards had one wound treated with BP and the adjacent wound treated with a vehicle control. Five additional lizards had one wound treated with saline and the second wound treated with a vehicle control. Wounds were imaged daily, and the wound area was measured using digital image analysis. The change in percentage wound closure over 17 days and the time to 50% wound closure was compared among the four treatment groups. There was no significant difference in wound closure rates between BP-treated and saline-treated wounds or in the time to 50% wound closure between any treatments. Vehicle-treated wounds adjacent to saline-treated wounds closed significantly slower than did BP (P < 0.010), saline (P < 0.001), and vehicle-treated wounds adjacent to BP-treated wounds (P < 0.013). Our preliminary study indicates that the splinted wound model, with modifications, may be used to determine wound closure rates in bearded dragons. When compared with saline, BP did not have a significant effect on wound closure rates, while the vehicle alone delayed wound closure. Histologic analysis of experimentally created wounds throughout the wound healing process is needed to further evaluate the effects of these treatments on reptile dermal wound healing.
PMID: 25632675 [PubMed - indexed for MEDLINE]
Influence of particle size and reactive oxygen species on cobalt chrome nanoparticle-mediated genotoxicity.
Biomaterials. 2013 May;34(14):3559-70
Authors: Raghunathan VK, Devey M, Hawkins S, Hails L, Davis SA, Mann S, Chang IT, Ingham E, Malhas A, Vaux DJ, Lane JD, Case CP
Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.
PMID: 23433773 [PubMed - indexed for MEDLINE]
Changes in protein expression associated with chronic in vitro exposure of hexavalent chromium to osteoblasts and monocytes: a proteomic approach.
J Biomed Mater Res A. 2010 Feb;92(2):615-25
Authors: Raghunathan VK, Grant MH, Ellis EM
Cr (VI) is a well-recognized environmental toxin and carcinogen. It is known to be released from orthopedic metal implants in-situ by biocorrosion and is speculated to play a role in periprosthetic osteolysis. It is hence essential to understand its long-term biological effects. We have assessed the in vitro responses of osteoblasts and monocytes to chronic exposure (3 weeks) to Cr (VI), at concentrations that have been measured in patients with metal implants, using two-dimensional gel electrophoresis. Cr (VI) exposure resulted in a differential time-dependent regulation of glycolytic, stress, and cytoskeletal proteins. The proteins that have been found to be altered in expression play an essential role in normal cellular functioning such as energy metabolism, cell signaling, and proliferation. The results highlight the complex molecular changes that occur in both cell types with long-term exposure to Cr and may be useful in establishing a series of clinically useful biomarkers to monitor long-term use of metallic implants.
PMID: 19235221 [PubMed - indexed for MEDLINE]