Abstract
Purpose :
Pathological angiogenesis and subretinal fibrosis are two major causes of neovascular age-related macular degeneration (nvAMD), the main cause of vision loss and blindness in AMD patients. Our purpose was to explore the role of Sigma 1 receptor (Sig1R) in the abnormal vascular growth and subretinal fibrosis after laser-induced CNV, and the modulation of Sig1R in inflammatory/angiogenic factors and fibrosis-related genes.
Methods :
Laser-induced CNV was introduced in ♂&♀ C57BL/6J (WT) mice (6-8 wk) by laser photocoagulation (532nm, 280mW, 70ms) of Bruch’s Membrane using image-guided system (Micro IV). 4 laser spots/eye were applied for retinal vasculature evaluation by Fluorescein Angiography (FA), retinal structure by spectral-optical coherence tomography(OCT) and immunostaining in choroid/RPE flatmounts. 20 laser spots/eye were for gene assay. (+)-pentazocine ((+)-PTZ), a specific Sig1R agonist (0.5mg/kg) was administered i.p. every other day starting 4hr after laser. Age-matched WT mice served as CT, total 3 groups: CT, CNV-non, PTZ+CNV. After 1 wk, the mice were subjected to FA&OCT for retinal vasculature/structure and CNV lesion evaluation. Pathological angiogenesis was determined by IMH with Isolectin B4 (IB4), fibrotic progression with collagen I (Col1), epithelial-to-mesenchymal transition (EMT) by reactivity to α-SMA antibodies in choroid/RPE flatmounts. Injury severity was quantified by the staining area of the maximum projection with respect to each antibody. Gene assay by qRT-PCR was to assess inflammatory cytokines, proangiogenic, and profibrotic factors in choroid/RPE tissue.
Results :
FA&OCT data show that CNV lesion in PTZ+CNV mice reduced significantly compared to CNV-non. IB4 staining in choroid/RPE flatmounts showed that angiogenesis in PTZ+CNV mice decreased by ~65% vs. CNV-non. Col1 and α-SMA staining showed that fibrotic and EMT areas of PTZ+CNV mice decreased by ~70%. Levels of inflammatory genes (IL10, IL1β and TNFα), angiogenic (Mmp2, Mmp9 and Hbegf) and fibrosis-related genes (Tgfβ2, Fgf2, Acta2 and Col1) remarkably increased in CNV-non mice. However, (+)-PTZ treatment significantly decreased their levels, only slightly greater than CT.
Conclusions :
Our data provide the first evidence that Sig1R activation is protective in laser-induced CNV mice by inhibition of abnormal angiogenesis and subretinal fibrosis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.