Smooth Muscle Cullin‐3 Deficiency Causes Vascular Dysfunction, Arterial Stiffness and Severe Hypertension
Authors: Agbor Larry N. , Nair Anand R. , Wu Jing , Davis Deborah R. , Keen Henry L. , Lu Ko‐Ting , Quelle Frederick W. , Singer Jeffrey D. , McCormick James A. , Sigmund Curt D. 
Published: June 2021

Cullin‐3 (CUL3) mutations resulting in exon 9 skipping (CUL3D9) cause human hypertension. We recently showed that selective expression of CUL3D9 protein in smooth muscle caused mild hypertension. Herein, we hypothesized that complete genetic deletion of CUL3 in smooth muscle would cause severe hypertension. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen‐inducible CRE‐recombinase driven by a smooth muscle myosin heavy chain promoter. Mice were administered tamoxifen i.p. (75 mg/kg) for 5 consecutive days to generate smooth muscle CUL3 knockout mice (S‐CUL3KO). CUL3 protein was undetectable whereas Cullin‐1 and Cullin‐5 proteins were preserved in aorta. CUL3 protein was also preserved in heart and kidney. We assessed vascular function in the cerebral basilar artery and aorta using pressurized and wire myograph, respectively. Pulse wave velocity (PWV) as index of aortic stiffness was measured by Doppler ultrasound, and blood pressure (BP) was measured by radiotelemetry. Aorta from S‐CUL3KO mice exhibited extremely impaired vasorelaxation to acetylcholine (ACh) compared to controls (at 100 μM: 1±3% vs 77±5%, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 15±4% vs 96±1%, p<0.001). Cerebral basilar artery from S‐CUL3KO mice also exhibited equivalent impairment to ACh‐ and SNP‐mediated vasorelaxation. Although RhoA/Rho‐kinase signaling was significantly elevated in aorta from S‐CUL3KO mice, pre‐incubation with a specific Rho‐kinase inhibitor, Y27632 did not restore vasorelaxation suggesting importance of an alternative pathway in response to CUL3 deletion. Conversely, S‐CUL3KO aorta exhibited only modestly impaired relaxation to a cGMP analogue (8‐pCPT‐cGMP, at 100 μM: 70±3% vs 98±2% controls) and to the heme‐independent soluble guanylate cyclase activator (BAY 58–2667, at 10 μM: 94±2% vs 101±1% controls) with a preservation of maximal relaxation. Aorta from S‐CUL3KO mice and CUL3‐deficient primary aortic smooth muscle cells (AoSMC) exhibited a significant two‐fold reduction in the expression of guanylate cyclase β1 and α1 subunits. As consequence, CUL3‐deficient primary AoSMC exhibited impaired cGMP production following stimulation with SNP (baseline cGMP pmol/mg protein: 0.9±0.1 vs 1.3±0.4 controls, SNP‐induced cGMP pmol/mg protein: 3.4±0.5 vs 8.7±1.6 controls). Interestingly, systolic BP (SBP) progressively increased in S‐CUL3KO mice upon tamoxifen‐induced CUL3 deletion (2 weeks SBP mmHg: 145±1 vs 115±2, p<0.001; 4 weeks SBP: 169±1 vs 115±3, p<0.001, n=6/genotype). Pulse wave velocity was also significantly increased in S‐CUL3KO mice (3.7±0.1 m/s vs 2.2±0.1, P<0.001), suggesting increased arterial stiffness. We conclude that smooth muscle CUL3 is a major BP determinant, and identifying novel CUL3 substrates in smooth muscle would be beneficial in the search for new anti‐hypertensive targets.Support or Funding InformationThis project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.