IMPAIRED NO SIGNALLING, ER STRESS AND RHO KINASE MEDIATE PERIPHERAL VASCULAR DYSFUNCTION IN A MODEL OF SMALL VESSEL DISEASE OF THE BRAIN (CADASIL)

Abstract

Objective: CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Thus our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Design and method: Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3R169C) and wildtype (TgNotch3WT) mice (6 months old) were investigated. Blood pressure (BP) was assessed by tail-cuff and vascular function by myography. Results: BP and cardiac function were unchanged in CADASIL mice. By electron microscopy GOM deposits were identified in MA from CADASIL mice. Notch3 (WT: 2.0 ± 0.5 vs. 6.0 ± 1.3) and HeyL (WT: 1.1 ± 0.4 vs. 2.9 ± 0.6) mRNA were augmented in CADASIL mice (p < 0.01). CADASIL mice exhibited endothelial (Emax 109.9 ± 7.4 vs. 81.3 ± 5.4) and VSMC-dysfunction (pD2 7.8 ± 0.1 vs 6.8 ± 0.3); effects associated with increased eNOS inhibition (p-Thr495) (1.8-fold increase) and decreased cGMP levels (1.2 ± 0.2 vs 0.59 ± 0.2) (p < 0.05). Contraction to Ang II (Emax 33.7 ± 6.8 vs. 72.8 ± 4.4), phenylephrine (Emax: 70.6 ± 7.2 vs. 92.1 ± 4.2) and U46619 (Emax: 123.4 ± 4.4 vs. 215.1 ± 24.4) (p < 0.05) was increased in CADASIL; effects attenuated by fasudil (Rho-kinase inhibitor) and 4-PBA (ER stress inhibitor) (p < 0.05). MA structure and mechanics from CADASIL mice remain unchanged at this stage. U46619-induced calcium influx was increased in TgNotch3R169C VSMCs (AUC: 1.3-fold increase) as well as SERCA (1.5 ± 0.7 vs. 8.1 ± 1.4) and TRPM2 (0.7 ± 0.3 vs. 4.5 ± 1.6) mRNA levels (p < 0.05). BiP protein expression (ER stress marker) (1.7-fold increase) and gene expression of Rho GEFs (LARG: 1.1 ± 0.1 vs. 2.1 ± 0.3; PDZ: 0.9 ± 0.1 vs. 2.9 ± 0.4) were increased in MA from TgNotch3R169C vs. TgNotch3WT mice (p < 0.05). These effects may be associated with the increase in oxidative stress observed in VSMCs from peripheral arteries and brain vessels from CADASIL patients (p < 0.05). Conclusions: Our findings demonstrate that the cerebral vasculopathy associated with Notch3 mutations is also present in peripheral small vessels and identify ER stress and Rho kinase as potential new therapeutic targets in CADASIL, for which there are no disease-specific treatments.