Abstract
Purpose :
7-Ketocholesterol (7KC) is an oxidized form of cholesterol that accumulates in drusen in age-related macular degeneration (AMD). We previously found that 7KC induces endothelial-mesenchymal transition (EndMT) of choroidal endothelial cells (CECs) partially by activating Rac1GTP. When Rac1GTP binds to the RasGAP-related scaffolding protein, IQGAP1, its activation is sustained, thereby enabling CEC transmigration of the RPE and neurosensory retinal invasion. We tested the hypothesis that 7KC promotes EndMT of CECs through IQGAP1.
Methods :
Primary human CECs were cultured and transfected with IQGAP1 siRNA or control siRNA. 24 hours later, CECs were treated with 7KC (10 μg/ml) or its solvent, HPBCD (control) for another 48 hours. Expression of IQGAP1, endothelial markers (VEGFR2, vWF), mesenchymal markers (α-SMA, FAP) was determined with qRT-PCR and western blot. The relative expression was quantified by comparing its expression to GAPDH (qRT-PCR) or β-actin (western blot). Experiments were repeated 5-6 times (n=6-9) in CECs from 3 different donor eyes. Statistical analyses were with mixed effect linear regression models using Stata-18 software.
Results :
In CECs, transfection with IQGAP1 siRNA reduced its expression by 57% (p<0.001) at 72 hrs as indicated by qRT-PCR. 7KC down-regulated endothelial markers VEGFR2 (p<0.001 at both mRNA and protein levels) and vWF (p= 0.014 at protein level), and up-regulated mesenchymal markers, α-SMA (p=0.032 at protein level, p=0.209 at mRNA level) and FAP (p<0.001 at mRNA and protein levels). Knockdown of IQGAP1 abrogated 7KC-induced down-regulation of VEGFR2 (p<0.001 at mRNA level, p=0.163 at protein level) and vWF (p<0.001 at protein level). Additionally, IQGAP1 knockdown reduced 7KC mediated up-regulation of α-SMA (p=0.028 at mRNA level, p=0.074 at protein level) and FAP (p<0.001 at mRNA level, p=0.004 at protein level).
Conclusions :
Knockdown of IQGAP1 inhibits 7KC-induced EndMT of CECs, suggesting that IQGAP1 mediates 7KC-induced EndMT. These data provide insights into fibrosis in neovascular AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.