Inhibition of the Hippo Pathway Prevents Arterial Stiffness and Hypertension in Vascular Smooth Muscle Cell Specific Kdm6a Knockout Mice

Abstract

Arterial stiffness (AS) results from the progressive loss of elasticity of large vessels due to aging and is correlated with hypertension and other cardiovascular co‐morbidities. Increased collagen deposition and contractile protein loss are key contributors to diminished flexibility. The histone 3 lysine demethylase 6a (Kdm6a) transcriptionally activates target genes via removal of tri‐methyl residues on lysine 27 of histone 3 (H3K27me3). The Hippo signaling pathway is crucial for regulating cell proliferation, cell survival, and organ size. The relationship between H3K27me3 and the Hippo pathway in vascular smooth muscle cells (VSMCs) and their effects on arterial compliance and blood pressure (BP) regulation remains unclear. We generated VSMC‐specific Kdm6a knockout (KO) mice of C57BL/6 background and confirmed Kdm6a KO and elevation of H3K27me3 levels by Western blot. Pilot studies (unpublished) indicate that Kdm6a KO results in increased systolic BP and AS due to aortic ECM remodeling and downregulation of contractile protein expression. Further, increased activity of mammalian sterile 20‐like kinase 2 (MST2), a core kinase of the Hippo pathway, was found in Kdm6a KO VSMCs. The objectives of this study were to investigate the effects of Hippo pathway inhibition on arterial compliance and sBP in a VSMC‐specific Kdm6a KO model. Based on the increased MST2 activity noted in Kdm6a KO VSMCs, we hypothesized that Hippo inhibition would prevent the onset of Kdm6a KO‐induced AS and hypertension or blunt the severity of symptoms. Male Myh11Cre/Kdm6aLoxP and Myh11Cre controls were randomly assigned to four groups (n=7‐8): Kdm6a KO‐treated, Kdm6a KO‐vehicle, control‐treated, or control‐vehicle. Immediately after gene deletion, Kdm6a KO mice and controls were administered the MST1/2 inhibitor XMU‐MP‐1 (1mg/kg, i.p.), or vehicle (2% DMSO, i.p.), daily for 4 weeks. Pulse wave velocity (PWV), the gold‐standard method for determination of degree of arterial compliance, was measured weekly. At the study endpoint, sBPs were obtained via direct carotid artery cannulation. The aortic media were collected for further analyses. After one week of treatment, PWVs of Kdm6a KO‐treated mice (3.05 + 0.26 mm/ms) were significantly lower compared with Kdm6a KO‐vehicle (4.10 + 0.29 mm/ms; p<0.01) and remained so throughout the study. The sBPs of Kdm6a KO‐treated mice (106.3 + 2.9 mmHg) were significantly less than those of the Kdm6a KO‐vehicle mice (118.0 + 6 mmHg; p<0.01). Analysis of aortic media by Western blot and immunohistochemistry revealed that treatment with XMU‐MP‐1 effectively inhibits MST2 phosphorylation and activates the downstream Hippo effector protein YAP. Additionally, treated Kdm6a KO mice produced significantly less collagen (p<0.05) and more Myh11 (p<0.05) and calponin (p<0.05) than the Kdm6a KO‐vehicle group. This study suggests that Kdm6a expression by VSMCs is essential for maintaining arterial compliance and regulating BP. Inhibiting the Hippo signaling pathway prevented Kdm6a KO‐induced vascular deregulation by lowering collagen production and rescuing contractile protein expression. These results demonstrate the therapeutic potential of the Hippo pathway in preservation of arterial compliance and BP.