Browsing by Subject "TGFbeta2"
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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 Effects of TGFβ2 and BMP4 Downstream Targets ID1 and ID3 in Trabecular Meshwork: Implications In Lowering IOP(2017-12-01) Mody, Avani A.; Clark, Abbot F.; Millar, J. Cameron; Krishnamoorthy, Raghu R.Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma, which is a major cause of irreversible vision loss. The major risk factor associated with POAG is increased intra ocular pressure (IOP). Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) increases the deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor I (PAI-1) and ECM cross-linking enzymes. These disruptions in ECM physiology in the TM elevate IOP and decrease aqueous humor outflow facility. Bone morphogenetic proteins (BMPs) regulate TGFβ2 induced profibrotic ECM production. The underlying mechanism for BMP4 inhibition of TGFβ2 induced fibrosis remains undetermined. BMP4 induces inhibitor of DNA binding proteins (ID1, ID3), which are negative transcription regulators that suppress fibrosis by regulating ECM component expression. Our study sought to determine whether ID1and ID3 proteins are downstream targets of BMP4 in TM and thereby attenuate TGFβ2 induction of ECM proteins in TM cells, elevated IOP and decreased outflow facility. Methods: Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. GTM3 cells were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGFβ2 induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression and promoter activities. IOP and AH outflow facility changes were studied in female BALB/cJ mice. Ad5-CMV-ID1 and Ad5-CMV-ID3 viral vectors along with Ad5-CMV-TGFβ2C226S/C288S were injected intravitreally. Ad5-CMV-TGFβ2C226S/C288S was injected along with Ad5- null vector as positive control, while Ad5-null injected mice were included as a negative control. IOP was measured using a Tonolab impact tonometer and AH outflow facility was measured using a constant flow infusion method. In addition we also performed a luciferase reporter activity array assay to study the effect of ID1, ID3 and TGFβ2 on various transcription factor activity in TM. Results: Basal expression of ID1-3 was detected in primary human TM cells. BMP4 significantly induced early expression of ID1 and ID3 mRNA (p [less than] < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGFβ2 induced expression of fibronectin and PAI-1 in TM cells (p< 0.01). Transduction of mouse eyes with ID1 or ID3 significantly blocked TGFβ2 induced ocular hypertension (P [less than] 0.0001) and AH outflow facility changes in living mice. Further, ID1 (P [less than] 0.01) and ID3 increase NFκB, Notch and cAMP/PKA activity, even in presence TGFβ2. Conclusions: BMP4 induced ID1 and ID3 expression suppresses TGFβ2 profibrotic activity in human TM cells. In addition ID1 and ID3 suppresses elevated IOP and decreases AH outflow facility induced by TGFβ2. Further, increased activity of NFκB, Notch and cAMP/PKA suggest that ID1 and ID3 may regulate TGFβ2 effects in TM via activation of the MMP pathway, inhibition of PAI-1 and Rho kinase activity, resisting cytoskeleton changes and lead to increased TM cellularity. This strongly supports ID1 and/or ID3 as robust candidates as a basis for developing disease-modifying IOP lowering therapies in POAG.