Abstract 462: Worsening of Endothelial Function in Mice with Endothelial-specific Haploinsufficiency of Rac1
[Introduction] Rac1 GTPase is implicated in various cardiovascular disorders including cardiac and smooth muscle hypertrophy. However, the precise role of Rac1 in endothelial cells (ECs) is not known. We hypothesized that Rac1 may be an important mediator of endothelial nitric oxide (eNOS) and endothelial function.
[Methods] EC-specific Rac1 deletion in mice was achieved by crossing mice with conditional floxed allele of Rac1 and Tie2 promoter-Cre transgenic mice. Homozygous deletion of Rac1 in ECs was embryonically lethal. Heterozygous mice (Tie2-Cre Rac1(flox/+); EC-Rac1-KO) were phenotypically normal and have similar baseline characteristics (BP, BW, organ weights) as control mice (Rac1(flox/+)). However, Western blot showed 50% reduction in Rac1 protein in EC-Rac1-KO ECs compared to control mice. Vasomotor tone of thoracic aorta ring was assessed using isometric myograph. Statistical significance was determined by ANOVA.
[Results] Mice heart endothelial cells (MHECs) from Rac1(+/−) mice showed upregulation of eNOS mRNA (1.6-fold) and increased phosphorylation of Akt (Ser273) and eNOS (Ser1177) as compared to Rac1(+/+). Surprisingly, nitrite release from Rac1(+/−) MHECs was substantially lower than Rac1(+/+) after 30-min stimulation with acethylcholine (10 μM; 0.36 ± 0.23 vs. 1.81 ± 0.20 nmol/mg/hr, P<0.0001) or calcium ionophore A23187 (10 μM; 0.42 ± 0.23 vs. 1.51 ± 0.42 nmol/mg/hr, P<0.0003). This correlated with reduced contraction to L-NAME (0.3 mM; 0.04 ± 0.06 vs. 0.11 ± 0.12 mN/mm, P=0.035) and relaxation to acethylcholine (30 nM; 70.4 ± 14.2% vs. 54.4 ± 18.8% of residual tone, P=0.017) in aortas of EC-Rac1-KO compared to those of control mice. Furthermore, EC-Rac1-KO aorta exhibited exaggerated contraction to phenylephrine (1 μM; 3.42 ± 1.75 vs. 1.60 ± 1.09 mN/mm, P=0.018).
[Conclusion] Inhibition of endothelial Rac1 increased the expression and phosphorylation of eNOS, while at the same time, blunted both basal and agonist-induced NO release. These findings indicate that Rac1 regulates eNOS activation via post-translational mechanism(s) and suggest that Rac1 is an important mediator of endothelial function.