Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21624
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Browsing Eye / Vision by Author "Clark, Abbot F."
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Item Role of miR-29c-3p in regulation of extracellular matrix synthesis(2019-03-05) Clark, Abbot F.; Tovar-Vidales, Tara; Lopez, NavitaPurpose Glaucoma is a group of optic neuropathies characterized by cupping of the optic nerve head (ONH) and degeneration of retinal ganglion cell (RGC) axons that lead to loss of visual function. Primary open angle glaucoma (POAG) is the most common form of glaucoma, with a global prevalence of 65.5 million, approximately 74% of glaucoma cases. The initial site of damage in POAG is within the lamina cribrosa (LC) region of the ONH. There is upregulation of the pro-fibrotic cytokine, TGFβ2, and marked disparity in the distribution and organisation of extracellular matrix (ECM) proteins. TGFβ2 induced downregulation of miR-29 has been shown, in part, to drive ECM protein synthesis in trabecular meshwork cells. Our purpose was to determine the effect of TGFβ2 on miRNA expression, in cells that populate the LC. We hypothesise that increased TGFβ2 signalling downregulates the expression of anti-fibrotic miRNAs, stimulating a fibrotic response and remodelling of the glaucomatous LC. Methods Primary human LC cells were grown to 100% confluency, treated with TGFβ2 (5ng/ml) or control for 24hours and differences in expression of miRNAs were analysed by PCR arrays. LC cells were transfected with miR-29c-3p (10nM) mimic, inhibitor or non-targeting controls and analysed by Q-PCR to confirm overexpression or knockdown of miR-29c-3p. mRNA targets of miR-29c-3p were determined through protein expression analysis by immunocytochemistry. The effects of miR-29c-3p and TGFβ2 on collagen type (COL) I and IV protein expression were evaluated in cells transfected with miR-29c-3p mimic, inhibitor or control and treated with TGFβ2 expression. Results TGFβ2 treatment downregulated the expression of miR-29c-3p in LC cells (n=4, pa-smooth muscle actin,COL (collagen) I and IV. Transfection of miR-29c-3p mimic or inhibitor showed upregulation and downregulation of miR-29c-3p respectively, confirming transfection efficiency. miR-29c-3p was found to be a key regulator of COL I and IV synthesis. Overexpression of miR-29c-3p decreased TGFβ2 induced COL I and IV expression in LC cells. Inhibition of miR-29c-3p exacerbated the effects of TGFβ2 on COL I and IV expression. Conclusion This suggests that elevated TGFβ2 signalling may stimulate a pro-fibrotic response through downregulation of miR-29c-3p. Although miR-29c-3p may be protective by decreasing the effects of TGFβ2 induced ECM protein synthesis, we will need to further elucidate the role of TGFβ2 and miR-29c-3p in maintaining the balance of ECM synthesis.Item TGFβ2-TLR4 Crosstalk Signaling in the Glaucomatous Trabecular Meshwork(2019-03-05) Curry, Stacy; Clark, Abbot F.; McDowell, Colleen; Roberts, AmandaPurpose: Glaucoma is a group of optic neuropathies and the leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the most prevalent type of glaucoma. Elevated intraocular pressure (IOP) is a major risk factor for the development of POAG. Elevated IOP is caused by aqueous humor fluid not draining properly through the drainage structures in the eye and leads to vision loss. Discovering potential new targets to lower IOP is necessary to develop novel and effective drug therapies. Here we explore a novel molecular mechanism involved in the development of glaucomatous trabecular meshwork (TM) damage. The TM regulates aqueous humor outflow and IOP. The effects of transforming growth factor beta (TGFβ)signaling pathways on the TM’s extracellular matrix (EÇM) have been extensively studied. Recently, we identified TGFβ2 and toll-like receptor 4 (TLR4) signaling crosstalk regulates changes in the TM ECM and mutation in Tlr4 rescues TGFβ2-induced ocular hypertension in mice. Here, we investigated the role of an endogenous TLR4 ligand, FN-EDA, and a downstream signaling molecule of TLR4, NFκB, in TGFβ2-induced ocular hypertension in mice. Methods: B6.FN-EDA+/+, B6.TLR4-/-, B6.FN-EDA-/-, B6.FN-EDA+/+/TLR4-/-, B6.FN-EDA-/-/TLR4-/-, and C57BL/6J mice were intravitreally injected with 2.0μL Ad5.TGFβ2 (2.5x107pfu) in one eye and the contralateral uninjected eye was used as a negative control. Likewise, we tested mice lacking the p50 subunit of NFκB (B6.Cg-NFκB1tm1Bal/J) and C57BL/6J mice. IOP was measured once per week using a TonoLab rebound tonometer on isoflurane-anesthetized mice 42 or 49 days post-injection. Significance determined by one-way ANOVA at each time point. Eyes were harvested, fixed in 4% paraformaldehyde, and sectioned for immunohistochemistry to access total fibronectin and FN-EDA isoform expression. Results: Ad5.TGFβ2significantly induced ocular hypertension in C57BL/6J mice and enhanced ocular hypertension in B6.EDA+/+ mice. Mutations in Tlr4,FN-EDA, and NFκB blocked Ad5.TGFβ2induced ocular hypertension with no significant IOP elevation at any time point. Total FN and FN-EDA isoform expression increased in Ad5.TGFβ2 injected C57BL/6J mice. Data suggest the onset of ocular hypertension developed in uninjected B6.EDA+/+ mice at 14 weeks of age and Ad5.TGFβ2 enhanced elevated IOP levels. Conclusions: TLR4, FN-EDA, and NFkB are necessary for TGFβ2 induced ocular hypertension in mice. In the absence of Ad5.TGFb2 constitutively active EDA (FN-EDA+/+) mice develop ocular hypertension and in the presence of Ad5.TGFβ2 ocular hypertension is enhanced. These data demonstrate that the crosstalk between TGFβ2 and TLR4 is involved in the glaucomatous development within the trabecular meshwork. In addition, it provides potential new targets to lower IOP and to further explore mechanisms involved in the development of glaucomatous TM damage.