Rho Kinase and Endoplasmic Reticulum Stress Mediate Peripheral Vascular Dysfunction in a Model of Small Vessel Disease of the Brain

CADASIL, a monogenic condition due to Notch3 mutations is a small vessel disease of the brain resulting in premature vascular dementia and stroke. CADASIL patients have cerebral vascular narrowing, progressive SMCs loss and granular osmiophilic material (GOM) deposition, however it is unclear whether peripheral arteries also exhibit dysfunction. We studied mice harbouring the CADASIL-causing Notch3 mutation (TgNotch3 R169C ), which recapitulates human CADASIL, to evaluate whether peripheral small arteries exhibit vascular dysfunction. Mesenteric arteries (MA) from male TgNotch3 R169C and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. Vascular structure and function were assessed by myography. No changes were observed in blood pressure and cardiac function in CADASIL mice, measured by tail cuff and echocardiography respectively. Expression of Notch3 and target genes was augmented in MA from CADASIL mice ( Notch3 : 2.0±0.5 vs. 6.0±1.3; HeyL: 1.1±0.4 vs. 2.9±0.6, p<0.01). CADASIL mice exhibited impaired endothelium-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent (pD 2 7.8±0.1 vs 6.8±0.3) relaxation and increased reactivity in response to AngII (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 vs TgNotch3 WT ); effects attenuated by fasudil (Rho-kinase inhibitor) and 4-PBA (ER stress inhibitor) (p<0.05 vs. TgNotch3 R169C ). MA from CADASIL mice also had hypotrophic remodelling. U46619-induced calcium influx was increased in VSMCs (AUC: 1.3 fold increase) from TgNotch3 R169C 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) was increased in MA from TgNotch3 R169C vs. TgNotch3 WT mice (p<0.05). eNOS phosphorylation (Thr 495 ) and BiP expression (ER stress marker) were increased in vessels from CADASIL mice (eNOS: 1.8 fold increase; Bip: 1.7 fold increase; p<0.05 vs. TgNotch3 WT ). In conclusion, our data demonstrated that the vasculopathy associated with Notch3 mutation is also present in peripheral small vessels where the interplay between Notch3, Rho-kinase and ER stress may be important. We identify potential new therapeutic targets in CADASIL, for which there are no disease-specific treatments.