Variants of Rab GTPase–Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke

Abstract

Background and Purpose The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury following large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ~80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine-mapping of C57BL/6 (B6, high extent) and BALB/cBy (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene. Methods We used additional CNG mapping and knockout mice to narrow the number of candidate genes. Subsequent inspection identified a non-synonymous SNP between B6 and BC within Rabep2 (rs33080487). We then created B6 mice with the BC SNP at this locus plus three other lines for predicted alteration or knockout of Rabep2 using gene editing. Results The single amino acid change caused by rs33080487 accounted for the difference in collateral extent and infarct volume between B6 and BC mice attributable to Dce1. Mechanistically, variants of Rabep2 altered collaterogenesis during embryogenesis but had no effect on angiogenesis examined in vivo and in vitro. Rabep2 deficiency altered endosome trafficking known to be involved in VEGF-A→VEGFR2 signaling required for collaterogenesis. Conclusions Naturally occurring variants of Rabep2 are major determinants of variation in collateral extent and stroke severity in mice.

Sex Differences in the Cerebral Collateral Circulation

“…When bilaterally present, PComA and primary cerebral artery diameter was determined as the mean of both sides for each animal. t Tests for sex were one-tailed for infarct volume since in a smaller cohort we previously reported smaller infarctions in females [26] in agreement with other rodent studies; two-tailed for number and diameter of pial collaterals, PComAs, and primary cerebral arteries; and one-tailed for experimental versus control for the effect of comorbidities [30]. ANOVA was used to examine the interaction of sex and genotype for collateral number and diameter in hypertensive animals.…”

“…Eighteen- to 26-month-old B6 mice were from the National Institute of Aging. Congenic (CNG) B6 mice (CNGB6/B6) have varying amounts of B6 genotype spanning the Dce1 locus on chromosome 7 and are BALB/cByJ (BC) (Jackson, #001026) elsewhere [25, 26]. PCR markers were used to identify blocks of B6 genotype.…”

“…PCR markers were used to identify blocks of B6 genotype. Heterozygotes were mated to produce homozygous CNGB6/B6, control CNGBC/BC (essentially wild-type BC due to extensive backcrossing), and heterozygous CNGB6/BC lines [25, 26]. Previously published data from CNG lines 3, 4, 5, 6, and 7 were pooled and segregated by sex.…”

“…Pial collaterals are arteriole anastomoses that cross-connect a small number of the distal branches of adjacent arterial trees. Their extent varies widely in mice due to differences in genetic background, in particular for variants of the recently identified gene within the Dce1 locus, Rabep2 , resulting in large differences in collateral-dependent blood flow and thus infarct volume after pMCAO [24–26]. As well, “environmental” factors, e.g., aging, endothelial dysfunction, hypertension, metabolic syndrome, diabetes, and obesity, cause loss of collateral number and/or a decrease in diameter of those that remain (collateral rarefaction), in association with an increase in infarct volume [27–30].…”

“…We therefore combined newly obtained data with a “meta-like” analysis of our previously published studies [25, 26, 33] wherein data for female and male mice had been obtained but with n-sizes too small to permit statistical comparison. Our aim was to determine if the extent of pial collateral arterioles differ depending on sex in young adult, aged, obese, hypertensive, and genetically different mice.…”