Abstract
Purpose :
Kcne3, formerly named Mink-related Peptide 2 (MiRP2), is a member of the Kcne3 family of ancillary b-subunits that assemble with a-subunits of voltage-gated potassium (K+) ion channels, which modulates the gating kinetics and enhances the stability of the channel complex (Abbott Gene 2016). We showed that its expression is responsive to exogenous VEGFA (Deckelbaum, Angiogenesis, 2019). The purpose of this study was to evaluate the expression and effects of Kcne3 gene deletion on normal retinal vascular development (RVD), and on vaso-obliteration and neovascularization in the murine model of oxygen-induced ischemic retinopathy (OIR).
Methods :
The coding region of the Kcne3 gene was replaced with the β-galactosidase reporter gene. Mice heterozygous for the Kcne3 gene deletion were bred to generate litters containing Kcne3 null (KO), heterozygous (HET), wild-type (WT) mice. Retinas were collected at postnatal day (P)6 and P7, flatmounted and stained with X-gal staining solution (KPL) and FITC-labeled Griffonia simplicfolia (GS) lectin I (Vector Labs). For the OIR model, litters were placed in a 75% O2 environment at P6 and returned to room air at P11. Retinas were collected at P11 and P16, flatmounted and stained with X-gal or anti-NG2 (Millipore), a mural cell marker and GS Lectin I.
Results :
In early RVD, Kcne3 was mostly expressed in the tip cells at P6 and P7. At P6, Kcne3 KO mice exhibited an incomplete vessel coverage of the retina, suggesting a reduced rate of vessel outgrowth compared to WTs (-24.6%, n=4, p <0.005). In OIR, Kcne3 expression also found in the tip cells in the growing edge of P11 and P16, not in the neovascular tufts. At P11 KO mice showed a decreased vaso-obliteration (-29%, n=4, p <0.01), and at P16 exhibited a decrease in avascular area (-30%, n=3, p <0.001) and in pathological neovascularization (-50%, n=3, p <0.001) compared to WTs. Kcne3 HETs exhibited an intermediate phenotype.
Conclusions :
This study identifies Kcne3 as a good cell marker to study tip cells in the growing and developing retina in mice. This study also illustrates its role in normal and ischemic retinal vessel growth and development. These results are consistent with observations in other disease models, which suggest that Kcne3 plays a role in VEGFA driven angiogenesis.
This is a 2020 ARVO Annual Meeting abstract.