Abstract 247: Gender-specific Angiogenesis Suppression By Btg2 May Be Mediated Through Suppressed Renin Expression

Abstract

Angiogenesis is the formation of new microvessels from existing vascular beds. AngII, a downstream product of renin, has been shown to be essential mediator of skeletal muscle angiogenesis in Dahl Salt Sensitive (SS) and Sprague Dawley rats, C57BL/6 mice, and human endothelial cells in vitro. Using partial chromosome introgression from the Brown Norway (BN) rat into the SS rat we have created two congenic lines with small (<300 Kbp) BN substitutions that differ by 23 Kbp. The congenic regions define an angiogenesis locus that contains one gene, Btg2 . Sanger sequencing revealed no sequence variants in exons of Btg2 between the BN and SS strains. Angiogenesis was measured using an in vivo electrical stimulation model of one hindlimb, with the contralateral leg acting as the control, in males and females of both new congenic strains. Males from Btg2 BN have angiogenesis (TA=20.0±4.5% increase in vessel density in stimulated leg relative to unstimulated leg, EDL=15.8±5.8%,) while Btg2 SS males did not have angiogenesis (TA=5.1±2.2%, EDL=4.4±2.7%). Females of both strains had angiogenesis: Btg2 BN (TA=15.8±4.3%, EDL=3.4%), Btg2 SS (TA=14.5±2.0%, EDL=14.6±2.5%). Stimulation significantly increased Btg2 expression in both males and females. Btg2 SS males had a significantly greater increase in Btg2 mRNA in the stimulated muscle relative to unstimulated than Btg2 BN males (5.0±0.9 versus 2.1± 0.3), but there was no difference in stimulated females (Btg2 BN 2.5±0.2, Btg2 SS 3.2±1.5). To test the hypothesis that Btg2 impacted renin expression, we cloned the renin proximal promoter into a vector to drive luciferase, and co-transfected HEK-293 cells with this and vector(s) expressing Btg2 or Hoxb9 and Btg2 . Normalized luciferase activity was 4.52±0.30 (arbitrary units) with an empty vector control and suppressed with the Btg2 vector to 1.45±0.07. Co-expression of Btg2 and Hoxb9 lead to a further reduction in luciferase activity to 0.40±0.04. These data suggest the elevated Btg2 expression observed in Btg2 SS strain may be acting to suppress renin expression through renin proximal promoter transcription factor Hoxb9 , leading to an inhibited angiogenic response.