Browsing by Subject "Actins / metabolism"
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Item A Novel Mouse Model of TGFbeta2-Induced Ocular Hypertension Using Lentiviral Gene Delivery(MDPI, 2022-06-21) Patil, Shruti V.; Kasetti, Ramesh B.; Millar, J. Cameron; Zode, Gulab S.Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) beta2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFbeta2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFbeta2 in the TM. We developed an LV vector-encoding active hTGFbeta2(C226,228S) under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFbeta2(C226,228S). We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFbeta2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFbeta2(C226,228S) exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFbeta2(C226,228S) that induces TM dysfunction and outflow resistance.Item TGFbeta2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways(ARVO Journals, 2017-02) Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot F.; Mao, WeimingPurpose: Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor beta2 (TGFbeta2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFbeta2 signaling pathways in CLAN formation. Methods: Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFbeta2, with or without the inhibitors of TGFbeta receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFbeta2 plus inhibitors for 10 days or pretreated with TGFbeta2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4',6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results: TGFbeta2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFbeta receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFbeta receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions: TGFbeta2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.