Abstract 440: Downregulation of eNOS and Impaired Angiogenesis in Mice with Endothelial-specific Haploinsufficiency of Rac1
[Introduction] Rac1 GTPase is implicated in various cardiovascular disorders including cardiac hypertrophy. However, the precise role of Rac1 in endothelial cells (ECs) is undefined. We hypothesized that Rac1 may be an important mediator of endothelial nitric oxide synthase (eNOS) and endothelial function.
[Methods] EC-specific deletion of Rac1 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 (EC-Rac1+/−) showed 50% reduction in Rac1 expression in ECs as compared to control (Rac1 (flox/+)). Mouse hind limb ischemia was induced by left femoral artery excision.
[Results] The eNOS mRNA expression in EC-Rac1+/− mice brain, as assessed by Northern blot and quantitative RT-PCR analyses, was 50% lower than that of control. The EC-Rac1+/− mice aorta showed reduced eNOS activity (0.66 ± 0.03 vs. 0.88 ± 0.05 fmol L-citrulline/μg protein/min, P < 0.0013), cGMP level (1.59 ± 0.28 vs. 2.70 ± 0.41 fmol/μg protein, P < 0.05) and acetylcholine-induced relaxation (30 nM; 54.4 ± 18.8% vs. 70.4 ± 14.2% of residual tone, P < 0.017). These findings correlated with decrease in NO release in ECs isolated from the EC-Rac1+/− heart. L-NAME treatment (0.2 mg/L in drinking water, 3 days) increased the BP in control mice (pre, 123.8 ± 6.0; post, 145.2 ± 12.5 mmHg, P < 0.04) whereas it had little effect on EC-Rac1+/− mice (pre, 123.2 ± 5.2; post, 125.2 ± 3.4 mmHg). The blood flow recovery following hind limb ischemia was markedly retarded in EC-Rac1+/− mice compared to control (n = 7; 0.53 ± 0.12 vs. 0.91 ± 0.04, ischemic/normal hind limb perfusion ratio; day 14, P < 0.05). L-NAME delayed the blood flow recovery of control mice (0.66 ± 0.12) whereas it had no effect on EC-Rac1+/− mice (0.60 ± 0.18). EC-Rac1+/− aorta explants cultured in Matrigel showed marked delay in microvessel sprouting compared to control, which was partially restored by treatment with 100 μM S-Nitrosoglutathione (GSNO), an NO donor.
[Conclusion] Endothelial Rac1 is required for the expression of eNOS and plays a pivotal role for the maintenance of endothelial function. Our findings further underscore the significance of Rac1-eNOS pathway as an important mediator of angiogenesis.