Cover Image

Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh

Trabeculectomy is the gold standard procedure performed in glaucoma when topical medication and laser intervention fail to maintain the ideal intraocular pressure (IOP) of patient’s eye. However, excessive accumulation of extracellular matrix components (ECM) mediated by Tenon’s fibroblast (HTF) lea...

Full description

Saved in:
Bibliographic Details
Main Author: Md Noh, Siti Munirah
Format: Thesis
Language:English
Published: 2017
Subjects:
RE Ophthalmology
Online Access:https://ir.uitm.edu.my/id/eprint/62204/1/62204.pdf
https://ir.uitm.edu.my/id/eprint/62204/
Tags: Add Tag
No Tags, Be the first to tag this record!
id my.uitm.ir.62204
record_format eprints
spelling my.uitm.ir.622042022-06-27T11:36:48Z https://ir.uitm.edu.my/id/eprint/62204/ Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh Md Noh, Siti Munirah RE Ophthalmology Trabeculectomy is the gold standard procedure performed in glaucoma when topical medication and laser intervention fail to maintain the ideal intraocular pressure (IOP) of patient’s eye. However, excessive accumulation of extracellular matrix components (ECM) mediated by Tenon’s fibroblast (HTF) leads to significant cases of surgical failure. Anti-vascular endothelial growth factor (VEGF) has become the focus in current scar modulation strategy. Improved bleb morphology following trabeculectomy augmented with ranibizumab has been reported. However, mechanism of actions of ranibizumab on HTF is not well understood. Therefore, this in vitro study was conducted to elucidate mechanism of actions of ranibizumab on HTF. HTF used in this study were propagated from Tenon’s capsule obtained from patients undergoing trabeculectomy. Firstly, isolated and characterized HTF were treated with different concentrations of ranibizumab in serum and serum-free media for 24 and 48 hours and then HTF viability was measured using MTT assay. Then, HTF were extracted to measure the expression of collagen Type 1 (COL1A1), fibronectin (FN), transforming growth factor-β1 and -β2 (TGF-β1 &TGF-β2) using qRT-PCR and ELISA. The experiment was followed with metabolomics profiling which was performed to identify the most significant metabolite regulated by ranibizumab. Finally, the expression of regulatory genes and proteins involved in the cell cycle regulation and angiogenesis including p21, p53, CDK2, CDK4, PTEN, AKT1 and THBS1 were measured by RT² Profiler PCR Array and Western Blot. Findings from the MTT assay showed that ranibizumab at the concentration of 0.5 mg/ml induce significant reduction in HTFs viability. The optimum degree of reduction was observed in serum-free media incubated for 48 hours. Furthermore, results suggested that ranibizumab mediates the down-regulation of COL1A1 and TGF-β1at gene level, but not at the protein level. No relevant changes were observed in FN and TGF-β2 mRNA level, but the proteins level was up-regulated. In metabolomics study, ranibizumab was shown to induce significant reduction in spermidine level. Therefore in subsequent experiment, ranibizumab effects were compared to DFMO, a potent irreversible inhibitor in spermidine synthesis. Findings show that ranibizumab exerts similar mechanism to DFMO in regulating spermidine expression by HTF, where it reduces PTEN, AKT1 and THBS1 expression. Moreover, ranibizumab administration increase p53 and p21 expression and reduces CDK2 and CDK4 expression. These observations suggest that ranibizumab might exert its anti-scarring property by enhancing the activities of p53 and p21, thus lead to reduction in CDK2 and CDK4. This shows that cell cycle of ranibizumab-treated HTF could be arrested, particularly at G1 phase. 2017-09 Thesis NonPeerReviewed text en https://ir.uitm.edu.my/id/eprint/62204/1/62204.pdf Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh. (2017) PhD thesis, thesis, Universiti Teknologi MARA (Kampus Sg. Buloh).
institution Universiti Teknologi Mara
building Tun Abdul Razak Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Teknologi Mara
content_source UiTM Institutional Repository
url_provider http://ir.uitm.edu.my/
language English
topic RE Ophthalmology
spellingShingle RE Ophthalmology
Md Noh, Siti Munirah
Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh
description Trabeculectomy is the gold standard procedure performed in glaucoma when topical medication and laser intervention fail to maintain the ideal intraocular pressure (IOP) of patient’s eye. However, excessive accumulation of extracellular matrix components (ECM) mediated by Tenon’s fibroblast (HTF) leads to significant cases of surgical failure. Anti-vascular endothelial growth factor (VEGF) has become the focus in current scar modulation strategy. Improved bleb morphology following trabeculectomy augmented with ranibizumab has been reported. However, mechanism of actions of ranibizumab on HTF is not well understood. Therefore, this in vitro study was conducted to elucidate mechanism of actions of ranibizumab on HTF. HTF used in this study were propagated from Tenon’s capsule obtained from patients undergoing trabeculectomy. Firstly, isolated and characterized HTF were treated with different concentrations of ranibizumab in serum and serum-free media for 24 and 48 hours and then HTF viability was measured using MTT assay. Then, HTF were extracted to measure the expression of collagen Type 1 (COL1A1), fibronectin (FN), transforming growth factor-β1 and -β2 (TGF-β1 &TGF-β2) using qRT-PCR and ELISA. The experiment was followed with metabolomics profiling which was performed to identify the most significant metabolite regulated by ranibizumab. Finally, the expression of regulatory genes and proteins involved in the cell cycle regulation and angiogenesis including p21, p53, CDK2, CDK4, PTEN, AKT1 and THBS1 were measured by RT² Profiler PCR Array and Western Blot. Findings from the MTT assay showed that ranibizumab at the concentration of 0.5 mg/ml induce significant reduction in HTFs viability. The optimum degree of reduction was observed in serum-free media incubated for 48 hours. Furthermore, results suggested that ranibizumab mediates the down-regulation of COL1A1 and TGF-β1at gene level, but not at the protein level. No relevant changes were observed in FN and TGF-β2 mRNA level, but the proteins level was up-regulated. In metabolomics study, ranibizumab was shown to induce significant reduction in spermidine level. Therefore in subsequent experiment, ranibizumab effects were compared to DFMO, a potent irreversible inhibitor in spermidine synthesis. Findings show that ranibizumab exerts similar mechanism to DFMO in regulating spermidine expression by HTF, where it reduces PTEN, AKT1 and THBS1 expression. Moreover, ranibizumab administration increase p53 and p21 expression and reduces CDK2 and CDK4 expression. These observations suggest that ranibizumab might exert its anti-scarring property by enhancing the activities of p53 and p21, thus lead to reduction in CDK2 and CDK4. This shows that cell cycle of ranibizumab-treated HTF could be arrested, particularly at G1 phase.
format Thesis
author Md Noh, Siti Munirah
author_facet Md Noh, Siti Munirah
author_sort Md Noh, Siti Munirah
title Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh
title_short Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh
title_full Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh
title_fullStr Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh
title_full_unstemmed Mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / Siti Munirah Md Noh
title_sort mechanisms of ranibizumab as an anti- scarring agent in trabeculectomy / siti munirah md noh
publishDate 2017
url https://ir.uitm.edu.my/id/eprint/62204/1/62204.pdf
https://ir.uitm.edu.my/id/eprint/62204/
_version_ 1736837383801798656
score 13.211869

Similar Items