Browsing by Author "Soundararajan, Avinash"

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    Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway
    (MDPI, 2021-10-24) Soundararajan, Avinash; Ghag, Sachin Anil; Vuda, Sai Supriya; Wang, Ting; Pattabiraman, Padmanabhan Paranji; Ophthalmology, School of Medicine
    The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFβ2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.
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    Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms
    (Association for Research in Vision and Ophthalmology, 2022) McDowell, Colleen M.; Kizhatil, Krishnakumar; Elliott, Michael H.; Overby, Darryl R.; van Batenburg-Sherwood, Joseph; Millar, J. Cameron; Kuehn, Markus H.; Zode, Gulab; Acott, Ted S.; Anderson, Michael G.; Bhattacharya, Sanjoy K.; Bertrand, Jacques A.; Borras, Terete; Bovenkamp, Diane E.; Cheng, Lin; Danias, John; De Ieso, Michael Lucio; Du, Yiqin; Faralli, Jennifer A.; Fuchshofer, Rudolf; Ganapathy, Preethi S.; Gong, Haiyan; Herberg, Samuel; Hernandez, Humberto; Humphries, Peter; John, Simon W.M.; Kaufman, Paul L.; Keller, Kate E.; Kelley, Mary J.; Kelly, Ruth A.; Krizaj, David; Kumar, Ajay; Leonard, Brian C.; Lieberman, Raquel L.; Liton, Paloma; Liu, Yutao; Liu, Katy C.; Lopez, Navita N.; Mao, Weiming; Mavlyutov, Timur; McDonnell, Fiona; McLellan, Gillian J.; Mzyk, Philip; Nartey, Andrews; Pasquale, Louis R.; Patel, Gaurang C.; Pattabiraman, Padmanabhan P.; Peters, Donna M.; Raghunathan, Vijaykrishna; Rao, Ponugoti Vasantha; Rayana, Naga; Raychaudhuri, Urmimala; Reina-Torres, Ester; Ren, Ruiyi; Rhee, Douglas; Chowdhury, Uttio Roy; Samples, John R.; Samples, E. Griffen; Sharif, Najam; Schuman, Joel S.; Sheffield, Val C.; Stevenson, Cooper H.; Soundararajan, Avinash; Subramanian, Preeti; Sugali, Chenna Kesavulu; Sun, Yang; Toris, Carol B.; Torrejon, Karen Y.; Vahabikashi, Amir; Vranka, Janice A.; Wang, Ting; Willoughby, Colin E.; Xin, Chen; Yun, Hongmin; Zhang, Hao F.; Fautsch, Michael P.; Tamm, Ernst R.; Clark, Abbot F.; Ethier, C. Ross; Stamer, W. Daniel; Ophthalmology, School of Medicine
    Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.
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    Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms
    (ARVO, 2022-02) McDowell, Colleen M.; Kizhatil, Krishnakumar; Elliott, Michael H.; Overby, Darryl R.; Van Batenburg-Sherwood, Joseph; Millar, J. Cameron; Kuehn, Markus H.; Zode, Gulab; Acott, Ted S.; Anderson, Michael G.; Bhattacharya, Sanjoy K.; Bertrand, Jacques A.; Borras, Terete; Bovenkamp, Diane E.; Cheng, Lin; Danias, John; De Ieso, Michael Lucio; Du, Yiqin; Faralli, Jennifer A.; Fuchshofer, Rudolf; Ganapathy, Preethi S.; Gong, Haiyan; Herberg, Samuel; Hernandez, Humberto; Humphries, Peter; John, Simon W. M.; Kaufman, Paul L.; Keller, Kate E.; Kelley, Mary J.; Kelly, Ruth A.; Krizaj, David; Kumar, Ajay; Leonard, Brian C.; Lieberman, Raquel L.; Liton, Paloma; Liu, Yutao; Liu, Katy C.; Lopez, Navita N.; Mao, Weiming; Mavlyutov, Timur; McDonnell, Fiona; McLellan, Gillian J.; Mzyk, Philip; Nartey, Andrews; Pasquale, Louis R.; Patel, Gaurang C.; Pattabiraman, Padmanabhan P.; Peters, Donna M.; Raghunathan, Vijaykrishna; Rao, Ponugoti Vasantha; Rayana, Naga; Raychaudhuri, Urmimala; Reina-Torres, Ester; Ren, Ruiyi; Rhee, Douglas; Chowdhury, Uttio Roy; Samples, John R.; Samples, E. Griffen; Sharif, Najam; Schuman, Joel S.; Sheffield, Val C.; Stevenson, Cooper H.; Soundararajan, Avinash; Subramanian, Preeti; Sugali, Chenna Kesavulu; Sun, Yang; Toris, Carol B.; Torrejon, Karen Y.; Vahabikashi, Amir; Vranka, Janice A.; Wang, Ting; Willoughby, Colin E.; Xin, Chen; Yun, Hongmin; Zhang, Hao F.; Fautsch, Michael P.; Tamm, Ernst R.; Clark, Abbot F.; Ethier, C. Ross; Stamer, W. Daniel; Ophthalmology, School of Medicine
    Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.
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    High Glucose-Induced Transcriptomic Changes in Human Trabecular Meshwork Cells
    (Research Square, 2024-12-24) Singh, Shivendra; Patel, Niketa A.; Soundararajan, Avinash; Pattabiraman, Padmanabhan P.; Ophthalmology, School of Medicine
    Glaucoma is a leading cause of irreversible blindness, often associated with elevated intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction. Diabetes mellitus (DM) is recognized as a significant risk factor for glaucoma; however, the molecular mechanisms through which hyperglycemia affects TM function remain unclear. This study investigated the impact of high glucose on gene expression in human TM (HTM) cells to uncover pathways that contribute to TM dysfunction and glaucoma pathogenesis under diabetic conditions. Primary HTM cells were cultured under normoglycemic (5.5 mM) and hyperglycemic (30 mM) conditions for seven days, followed by mRNA sequencing (mRNA-seq) to identify differentially expressed genes, with quantitative PCR (qPCR) used for confirmatory analysis. STRING network analysis was performed to predict interactions among upregulated and downregulated proteins. mRNA-seq analysis revealed 25 significantly differentially expressed genes in high glucose conditions, including upregulated genes associated with oxidative stress, apoptosis, autophagy, immune response, and fibrosis. Notably, TXNIP was significantly upregulated, indicating increased oxidative stress and apoptosis in TM cells, while downregulation of autophagy-related genes, such as HSPA6 and LAMP3, suggests compromised protein quality control. Immune response genes, including CCL7 and CHI3L1, were upregulated, suggesting an inflammatory response to oxidative stress. Increased expression of fibrosis-related genes, such as SNAI1, FGF7, and KRT19, supports the hypothesis of ECM accumulation in diabetic conditions, potentially elevating IOP. Chronic hyperglycemia in diabetic patients could therefore lead to TM dysfunction, impair aqueous humor outflow, and elevate IOP, thereby increasing glaucoma risk. Targeting oxidative stress and fibrosis pathways offers therapeutic strategies to mitigate glaucoma progression in diabetic populations.
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    Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation
    (Wiley, 2023) Wang, Ting; Soundararajan, Avinash; Rabinowitz, Jeffrey; Jaiswal, Anant; Osborne, Timothy; Pattabiraman, Padmanabhan Paranji; Ophthalmology, School of Medicine
    Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.
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    Multiomics analysis reveals the mechanical stress-dependent changes in trabecular meshwork cytoskeletal-extracellular matrix interactions
    (Frontiers, 2022-09) Soundararajan, Avinash; Wang, Ting; Sundararajan, Rekha; Wijeratne, Aruna; Mosley, Amber; Harvey, Faith Christine; Bhattacharya, Sanjoy; Pattabiraman, Padmanabhan Paranji; Ophthalmology, School of Medicine
    Trabecular meshwork (TM) tissue is subjected to constant mechanical stress due to the ocular pulse created by the cardiac cycle. This brings about alterations in the membrane lipids and associated cell–cell adhesion and cell–extracellular matrix (ECM) interactions, triggering intracellular signaling responses to counter mechanical insults. A loss of such response can lead to elevated intraocular pressure (IOP), a major risk factor for primary open-angle glaucoma. This study is aimed to understand the changes in signaling responses by TM subjected to mechanical stretch. We utilized multiomics to perform an unbiased mRNA sequencing to identify changes in transcripts, mass spectrometry- (MS-) based quantitative proteomics for protein changes, and multiple reaction monitoring (MRM) profiling-based MS and high-performance liquid chromatography (HPLC-) based MS to characterize the lipid changes. We performed pathway analysis to obtain an integrated map of TM response to mechanical stretch. The human TM cells subjected to mechanical stretch demonstrated an upregulation of protein quality control, oxidative damage response, pro-autophagic signal, induction of anti-apoptotic, and survival signaling. We propose that mechanical stretch-induced lipid signaling via increased ceramide and sphingomyelin potentially contributes to increased TM stiffness through actin-cytoskeleton reorganization and profibrotic response. Interestingly, increased phospholipids and diacylglycerol due to mechanical stretch potentially enable cell membrane remodeling and changes in signaling pathways to alter cellular contractility. Overall, we propose the mechanistic interplay of macromolecules to bring about a concerted cellular response in TM cells to achieve mechanotransduction and IOP regulation when TM cells undergo mechanical stretch.
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    Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork
    (Wiley, 2022) Soundararajan, Avinash; Wang, Ting; Ghag, Sachin A.; Kang, Min H.; Pattabirama, Padmanabhan P.; Ophthalmology, School of Medicine
    This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and β5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and β5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-β2 (TGFβ2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFβ2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.