Slc38A1 blockade inhibits angiogenesis and laser-induced choroidal neovascularization

Abstract

Purpose : We have recently identified Slc38A1 (Solute carrier 38A1) as one of the candidate genes involved in angiogenic response by genome-wide association. Here, we determined the effect of targeting Slc38A1 in human microvascular endothelial cell (HMVEC) functional assays and on experimental choroidal neovascularization (CNV) induced by laser photocoagulation in mice.

Methods : We initially evaluated the role of Slc38A1 in angiogenesis by in vitro functional assays in HMVECs. Subsequently, we evaluated the effect of targeting Slc38A1 with 6-amino acid blocking peptides (SL1 and SL2) in HMVEC migration to full serum media supplemented with L-Citrulline (100 μM) in 3 dosages of 100 nM, 1 μM and 10 μM. Expression of endothelial nitric oxide synthase (eNOS), phospho-eNOS (peNOS Ser1177) and Slc38A1 was quantified by Western blot. Then, CNV was induced by laser photocoagulation in C57BL/6J mice. The mice received 1 mM of scrambled peptide control or Slc38A1 blocking peptides intravitreally 2 days after laser application. Choroids were immunostained and CNV area (µm²) was quantified at one week after laser induction.

Results : We observed a significant decrease in the ability of HMVECs to migrate to full serum-containing media in Slc38A1 siRNA transfected cells compared to scrambled control siRNA transfected cells. Furthermore, we found a significant decrease (P < 0.001) in HMVEC migration when treated with blocking peptides SL1 or SL2 in a dose dependent manner vs. the scrambled controls. Total eNOS and Slc38A1 expression did not differ significantly between the treated and control groups. However, the protein levels of peNOS were decreased significantly in the treated group compared with the control group. In vivo, the mean CNV area from 3 independent experiments was significantly smaller in the SL1 (14,333 ± 1,834 µm²) group compared with control eyes (25,289 ± 2,968 µm²; P < 0.005).

Conclusions :
Slc38A1 blocking peptide (SL1) effectively altered endothelial cell activity and inhibited laser-induced CNV in mice. This was associated specifically with down-regulation of phosphorylated eNOS suggesting that it may be a potent inhibitor of endothelial cell function and angiogenesis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.