Browsing by Author "Lopez, Navita"
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Item miR-200b-3p and miR-211-5p downregulate the expression of extracellular matrix and associated proteins in human optic nerve head astrocytes(2018-03-14) Tovar-Vidales, Tara; Clark, Abbot F.; Lopez, NavitaPURPOSE: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is a leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the profibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eyes. Currently there is a lack of knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine: (a) differences in the expression of profibrotic and anti-fibrotic miRNAs in normal ONH astrocytes treated with or without TGFβ2 and (b) whether candidate miRNAs (miR-200b-3p and miR-211-5p) regulate the expression of ECM and ECM associated proteins in the ONH. METHODS: Primary normal human ONH astrocytes (ONA) were grown to 100% confluency. ONA were treated with 5ng/ml TGFβ2 or with control for 24hrs. miRNA qPCR arrays were performed to compare the expression levels of profibrotic and anti-fibrotic miRNAs in normal ONA treated with or without TGFβ2. ONA were transfected with miR-200b-3p and miR-211-5p mimics at 10nM, 5nM, and 1nM concentrations to confirm target predictions based on the TargetScan database. An all stars negative control siRNA was included which will not recognise any mammalian gene. RESULTS: The miRNA qPCR arrays analysed from normal ONA exposed to TGFβ2 showed that TGFβ2 downregulated the expression of hsa-miR-200b-3p and hsa-miR-211-5p. Transfection of miR-200b-3p mim downregulated fibronectin (FN), gremlin, and tissue transglutaminase II in ONA. Transfection of miR-211-5p downregulated FN and gremlin in ONA . CONCLUSIONS: Our results suggest that TGFβ2, which is elevated in the glaucomatous ONH, modulates the expression of miRNAs in ONA. These miRNAs target FN, TGM2, and gremlin to modify the ECM in the ONH. Downregulation of anti-fibrotic miRNAs may contribute to fibrosis of the ONH.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 Role of TGFβ2 and miRNAs in optic nerve head fibrosis(2020) Tovar-Vidales, Tara; Clark, Abbot; Lopez, NavitaPurpose: Glaucoma is characterised by degeneration of retinal ganglion cells (RGCs) and loss of visual function. This is associated with extracellular matrix (ECM) remodelling of the optic nerve head (ONH). The ONH provides mechanical and biochemical support to unmyelinated RGC axons as they exit the eye to form the optic nerve; thus, changes in the ONH affect the physiology of axons and may damage the visual circuitry. In glaucoma, mechanosensitive cells in the ONH respond to elevated pressure by secreting growth factors, ECM and inflammatory cytokines that damage RGCs and remodel the ONH. A key upregulated pathway in glaucoma is TGFβ2, which leads to increased expression of profibrotic genes. We hypothesised that impaired regulation of TGFβ2 and miRNAs is involved in glaucomatous remodelling of the ONH ECM. The purpose of this study was to identify cell-specific changes in glaucoma pathophysiology. Method: ONH astrocytes and lamina cribrosa (LC) were treated with TGFβ2 (5ng/ml) or vehicle and analyzed by miRNA qPCR arrays and NGS. Cells were transfected with candidate miRNA mimics, inhibitors, or non-targeting siRNA (10nM) to determine ECM expression. Results: TGFβ2 treatment decreased the expression of multiple antifibrotic miRNAs, which was associated with increased expression of several profibrotic genes including collagen I and IV and fibronectin. Overexpression of antifibrotic miRNAs including miR-29c-3p and miR-200b-3p decreased TGFβ2 induced collagen I and IV and fibronectin expression. Conclusion: TGFβ2 modulated the expression of several miRNAs to stimulate a profibrotic response; this may lead to pathogenic ONH remodelling.Item Transforming Growth Factor β2 Regulates the Expression of microRNAs (miRNAs) in Human Optic Nerve Head Cells(2017-03-14) Tovar-Vidales, Tara; Clark, Abbot; Lopez, NavitaPurpose: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is the leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the pro-fibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eye. However, there currently is little knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine if there are differences in expression of pro-fibrotic and anti-fibrotic miRNAs in normal ONH cells treated with or without TGFβ2. Methods: Primary human ONH cell strains derived from normal donor eyes were grown to 100% confluency. ONH cells were treated with 5ng/ml TGFβ2 or with control medium for 24hrs. RNA was isolated and cDNA synthesis performed for miRNA qPCR arrays to compare expression levels of pro-fibrotic and anti-fibrotic miRNAs in normal human ONH cells treated with or without TGFβ2. Results: Normal ONH cells exposed to TGFβ2 showed that several anti-fibrotic miRNAs were downregulated (hsa-miR-107, hsa-miR-132-3p, hsa-miR-141-3p hsa-miR-18a-5p, hsa-miR-194-5p, hsa-miR-204-5p) compared to control cells. In contrast, only one pro-fibrotic miRNA was upregulated (hsa-miR-34a-5p) in ONH cells treated with TGFβ2 compared to control. The most prominent targets of these miRNAs include connective tissue growth factor (CTGF), gremlin 2 and lysyl oxidase-like 3 (LOX-L3). Conclusions: Our results suggest that miRNAs expressed by ONH cells may be regulated by TGFβ2. These miRNAs may target CTGF, crosslinking enzymes and BMP antagonists to modify the ECM in the ONH.