Conditional deletion of smooth muscle Cullin-3 causes severe progressive hypertension
Larry N. Agbor, Anand R. Nair, Jing Wu, Ko-Ting Lu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, James A. McCormick, Jeffrey D. Singer, Curt D. Sigmund
Larry N. Agbor, Anand R. Nair, Jing Wu, Ko-Ting Lu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, James A. McCormick, Jeffrey D. Singer, Curt D. Sigmund
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Research Article Vascular biology

Conditional deletion of smooth muscle Cullin-3 causes severe progressive hypertension

Abstract

Patients with mutations in Cullin-3 (CUL3) exhibit severe early-onset hypertension, but the contribution of the smooth muscle remains unclear. Conditional genetic ablation of CUL3 in vascular smooth muscle (S-CUL3KO) causes progressive impairment in response to NO, rapid development of severe hypertension, and increased arterial stiffness. Loss of CUL3 in primary aortic smooth muscle cells or aorta resulted in decreased expression of the NO receptor soluble guanylate cyclase (sGC), a marked reduction in cGMP production, and impaired vasodilation to cGMP analogs. Vasodilation responses to a selective large-conductance Ca2+-activated K+ channel activator were normal, suggesting that downstream signals that promote smooth muscle–dependent relaxation remained intact. We conclude that smooth muscle–specific CUL3 ablation impairs both cGMP production and cGMP responses and that loss of CUL3 function selectively in smooth muscle is sufficient to cause severe hypertension by interfering with the NO/sGC/cGMP pathway. Our study provides evidence that vascular smooth muscle CUL3 is a major regulator of BP. CUL3 mutations cause severe vascular dysfunction, arterial stiffness, and hypertension due to defects in vascular smooth muscle.

Authors

Larry N. Agbor, Anand R. Nair, Jing Wu, Ko-Ting Lu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, James A. McCormick, Jeffrey D. Singer, Curt D. Sigmund

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Figure 1

Smooth muscle–specific ablation of CUL3.

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Smooth muscle–specific ablation of CUL3. (A) Mice carrying 2 copies of a...
(A) Mice carrying 2 copies of a conditional CUL3 allele were bred with mice expressing an inducible Cre recombinase driven by a smooth muscle myosin heavy chain promoter (Myh11-CreERT2). The resultant offspring heterozygous for the CUL3 floxed allele (CUL3fl/+) and also expressing Myh11-CreERT2 (Cre+/CUL3fl/+) were backcrossed to CUL3fl/fl mice. Cre+/CUL3fl/fl mice were administered corn oil (vehicle) as controls, and Cre+/CUL3fl/fl mice or Cre+/CUL3fl/+ mice were administered tamoxifen (75 mg/kg) i.p. for 5 consecutive days to generate smooth muscle–specific CUL3 knockout (S-CUL3KO) or heterozygous mice (S-CUL3het), respectively. (B) Western blots showing expression levels of the indicated proteins in aorta of control and S-CUL3KO mice 2 weeks after administration of tamoxifen. Aortas were cleaned of perivascular adipose fat and adventitia. A representative of 5 independent experiments is shown. (C) Western blots showing expression of the indicated proteins in heart and kidney 2 weeks after tamoxifen or corn oil treatment. (D) Cross sections of aorta from control and S-CUL3KO mice 2 weeks after tamoxifen administration. Immunofluorescence for CUL3 was performed using a validated CUL3 antibody (red), and coimmunostained for the endothelial marker CD31 (green). Slides were counterstained with DAPI (blue) and visualized using a Zeiss 710 confocal microscope. Scale bars: 200 μm in panels 1 and 3; 15 μm in panels 2, 4, and 5. Panel 4 is CUL3 + DAPI. Panel 5 is a merge of CUL3, CD31, and DAPI.