Abstract 121: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Abstract

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E-Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p<0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p<0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L-NAME induced smaller increases in nocturnal peak SBP and DBP in E-Cul3Δ9 mice (15±1 vs 27±3 mmHg, p<0.01), suggesting their NOS activity is low. Of note, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p<0.05) and cerebral resistance basilar artery (41% vs 77%, p<0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.