Peripheral arteriopathy caused by Notch3 gain-of-function mutation involves ER and oxidative stress and blunting of NO∕sGC∕cGMP pathway.

Notch3 mutations cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoenephalopathy (CADASIL), which predisposes to stroke and dementia. CADASIL is characterized by vascular dysfunction and granular osmiophilic material (GOM) accumulation in cerebral small vessels. Systemic vessels may also be impacted by Notch3 mutations. However vascular characteristics and pathophysiological processes remain elusive. We investigated mechanisms underlying the peripheral vasculopathy mediated by CADASIL-causing Notch3 gain-of-function mutation. We studied: i) small arteries and vascular smooth muscle cells (VSMC) from TgNotch3R169C mice (CADASIL model), ii) VSMCs from peripheral arteries from CADASIL patients, and iii) post-mortem brains from CADASIL individuals. TgNotch3R169C vessels exhibited GOM deposits, increased vasoreactivity and impaired vasorelaxation. Hypercontractile responses were normalized by fasudil (Rho kinase inhibitor) and 4-PBA (endoplasmic-reticulum (ER) stress inhibitor). Ca2+ transients and Ca2+ channel expression were increased in CADASIL VSMCs, with increased expression of Rho GEFs and ER stress proteins. Vasorelaxation mechanisms were impaired in CADASIL, evidenced by decreased eNOS phosphorylation and reduced cGMP levels, with associated increased guanylate cyclase (sGC) oxidation, decreased sGC activity and reduced levels of the vasodilator H2O2. In VSMCs from CADASIL patients, sGC oxidation was increased and cGMP levels decreased, effects normalized by fasudil and 4-PBA. Cerebral vessels in CADASIL patients exhibited significant oxidative damage. In conclusion, peripheral vascular dysfunction in CADASIL is associated with altered Ca2+ homeostasis,  oxidative stress and blunted eNOS/sGC/cGMP signaling, processes involving Rho kinase and ER stress. We identify novel pathways underlying the peripheral arteriopathy induced by Notch3 gain-of-function mutation, phenomena that may also be important in cerebral vessels.