Abstract 693: Rgs Homology Domain Of GRK5 Regulates Postischemic Neoangiogenesis By Inhibiting NFkB Trascriptional Activity
We recently showed that RGS homology domain (RH) in the NT region of GRK5 inhibits NFkB transcription activity through binding and stabilization of the IkB-NFkB complex. Through the release of cytokines, NFkB regulates events leading to angiogenesis, in particular cell survival, migration and network formation in vitro, and capillary formation and tissue perfusion in vivo. Cytokine production was assessed in bovine aorta endothelial cells (EC) as LPS induced TNFα mRNA by northern blot. GRK5RH overexpression inhibits TNFα mRNA expression induced after LPS stimulation (GRK5RH: −61 ± 0.7% vs LPS). Apoptosis was assessed in EC by cleaved caspase 3 levels visualized by western blot. GRK5RH increases caspase level (GRK5RH: ± 53 ± 0.4% vs control), suggesting that GRK5RH-induced NFkB inhibition results in loss of apoptosis protection. Serum (FBS) induced cell migration was assessed in EC; GRK5RH inhibits FBS induced ECs migration into the wounded area (GRK5RH: −99.9 ± 0.1% vs FBS). Finally, network formation of EC on matrigel matrix was also inhibited by GRK5RH. Using the rat ischemic hindlimb (HL), we assessed the effects on neoangiogenesis of intrafemoral artery infusion of adenovirus encoding the GRK5NT which contains the RH domain (n = 5) or an empty vector (n = 5). After 14 days, HL blood flow assessed by digital angiographies, showed a longer time to perfusion in ischemic AD-GRK5NT vs AdEmpty rat HLs (ADGRK5NT:40 ± 1.6; ADEmpty:22 ± 2.1; P < 0.05, ANOVA). Capillary bed capacity assessed by died microspheres perfusion through abdominal aorta injection confirmed that ADGRK5NT ischemic HLs presented a reduced neoangiogenesis response compared to AdEmpty (AD-GRK5NT: 0.1 ± 0.02; AD-Empty: 0.5 ± 0.04%; P < 0.05, ANOVA). Our results reveal kinase independent role of GRK5 in the regulation of angiogenesis by means of NFkB transcription activity inhibition and identify the NT/RH domain of GRK5 as a possible therapeutic target.