Abstract MP14: Endothelial Cullin3 Mutation Causes Decreased Nitric Oxide (NO) Bioavailability And Vascular Dysfunction Through Protein Phosphatase 2A

Abstract

Mutations in CULLIN3 gene (in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT) driven by a combination of renal tubular and vascular mechanisms. 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 arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, n=5-7, p<0.05) and a trend towards elevated nighttime blood pressure (peak systolic BP, E-Cul3Δ9 136±3 vs control 128±3 mmHg, n=9-11) that were not associated with any alterations in locomotion, food/water intake or sleep/wake behaviors. No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 , n=5-9, p<0.05) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (max ACh relaxation: 69% vs 84%, n=5-7, p<0.05) and cerebral resistance basilar artery (41% vs 77%, n=4-6, p<0.05). However, no dilatory impairment in mesenteric resistance artery and no difference in smooth muscle function were observed, suggesting that the effects of Cul3Δ9 are arterial bed specific. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS and NO production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Therefore, 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 (4nM), but not by a PP1 inhibitor tautomycetin (4nM). Thus, CUL3 mutations in the endothelium may contribute to human HT in part through decreased endothelial NO bioavailability, arterial stiffening and secondary sympathoexcitation.