Notch signalling is critically involved in vascular morphogenesis and function. Four Notch isoforms (Notch1–4) regulating diverse cellular processes have been identified. Of these, Notch3 is expressed almost exclusively in vascular smooth muscle cells (VSMCs), where it is critically involved in vascular development and differentiation. Under pathological conditions, Notch3 regulates VSMC switching between the contractile and synthetic phenotypes. Abnormal Notch3 signalling plays an important role in vascular remodelling, a hallmark of several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Because of the importance of Notch3 in VSMC (de)differentiation, Notch3 has been implicated in the pathophysiology of pulmonary vascular remodelling in PAH. Here we review the current literature on the role of Notch in VSMC function with a focus on Notch3 signalling in pulmonary artery VSMCs, and discuss potential implications in pulmonary artery remodelling in PAH.

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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.

Objective: Notch3 signalling has been associated with changes in the pulmonary vasculature leading to pulmonary arterial hypertension (PAH). Mice with the CADASIL-causing R169C Notch3 mutation (TgNotch3R169C) exhibit gain-of-function Notch3 signalling and develop peripheral vascular dysfunction and injury. Here we investigated whether the R169C mutation contributes to PAH development. Design and method: Briefly, 24-week male and female mice overexpressing either a WT or R169C mutant Notch3 gene were exposed to 2 weeks normoxia (N) or chronic hypobaric hypoxia (10% O2) (H). PAH was assessed by in vivo haemodynamic measurements, Fulton index analysis of right ventricle hypertrophy (RVH), and histological remodelling analysis by elastin staining of pulmonary arteries under 80 microns in diameter. Pulmonary artery wire myography was also performed. Results: RV systolic pressure increased in hypoxic animals vs normoxic (WT H 27.5 ± 1.0 vs WT N 22.8 ± 2.2mmHg); and was exacerbated only in male R169C hypoxic animals (R169C H 32.2 ± 1.1 mmHg vs R169C N 23.6 ± 1.4) (p < 0.05). Male R169C hypoxic animals also exhibited significant RV hypertrophy (H 0.26 ± 0.033 vs N 0.16 ± 0.012), p < 0.05. RVH was not significantly different between female groups. R169C pulmonary arteries from males were significantly hypercontractile to 5HT (Emax: R169C 142 ± 10% vs WT 92 ± 6%); an effect attenuated by ROCK inhibitor Fasudil (Emax: R169C ± Fas. 117 ± 20%). Female R169C arteries were not hypercontractile to 5HT (R169C 113.3 ± 11% vs WT 120.3 ± 8%) but Fasudil reduced contraction (Emax: R169C + Fas. 96.8 ± 10%). Notch3 effector gene Hes5, implicated in PAH, was significantly increased in male R169C lung (8.7 fold change vs WT) suggesting increased Notch3 activation. RhoA/ROCK activating Rho-guanine nucleotide exchange factors LARG and PDZ mRNA were upregulated in R169C lung (R169C 33.5 fold vs WT, R169C 18.7 fold vs WT respectively). These changes were not observed in lungs from female mice. Conclusions: Notch3 R169C mutation may contribute to PAH susceptibility and pulmonary vascular hypercontractility in males. ROCK signaling plays an important role in these processes.

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. Therefore, our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3 R169C ) and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. GOM deposits in MA from CADASIL mice were identified by electron microscopy. mRNA expression of Notch3 (WT: 2.0±0.5 vs. 6.0±1.3) and its downstream target HeyL (WT: 1.1±0.4 vs. 2.9±0.6) was augmented in CADASIL mice (p<0.01), suggesting increased Notch3 activation. CADASIL mice exhibited endothelial-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent dysfunction (pD 2 7.8±0.1 vs. 6.8±0.3); effects associated with increased eNOS inhibition (p-Thr 495 ) (1.8-fold increase) and decreased cGMP levels (1.2±0.2 vs. 0.59±0.2) (p<0.05). Plasma lipid peroxidation (0.8±0.1 vs. 2.0±0.3; p<0.05) and vascular reactive oxygen species (ROS) production (7.2±1.9 vs. 75.4±35.0; p<0.05) were increased in TgNotch3 R169C mice; processes associated with upregulation of soluble guanylate cyclase (sGC) oxidation and decreased sGC activity. H 2 O 2 levels were decreased in TgNotch3 R169C mice (1.9±0.2 vs. 1.1±1.9; p<0.05), which was associated with reduced activation of protein kinase G (PKG). Observations in TgNotch3 R169C mice were recapitulated in human CADASIL, where ROS levels (0.8±0.1 vs. 4.1±2.7; p<0.05) and sGC oxidation were also increased. Our findings demonstrate that the vasculopathy associated with a CADASIL Notch3 gain-of-function mutation in peripheral small vessels involves reduction in eNOS activation and redox-sensitive processes leading to impaired sGC/cGMP signalling pathway. We identify a potential new therapeutic target in CADASIL, for which there are no disease-specific treatments.

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Background: Notch3 is implicated in vascular diseases, including pulmonary hypertension (PH)/pulmonary arterial hypertension. However, molecular mechanisms remain elusive. We hypothesized increased Notch3 activation induces oxidative and endoplasmic reticulum (ER) stress and downstream redox signaling, associated with procontractile pulmonary artery state, pulmonary vascular dysfunction, and PH development. Methods: Studies were performed in TgNotch3 R169C mice (harboring gain-of-function (GOF) Notch3 mutation) exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in pulmonary artery smooth muscle cells from pulmonary arterial hypertension patients and in mouse lung. Notch3-regulated genes/proteins, ER stress, ROCK (Rho-associated kinase) expression/activity, Ca 2+ transients and generation of reactive oxygen species, and nitric oxide were measured. Pulmonary vascular reactivity was assessed in the presence of fasudil (ROCK inhibitor) and 4-phenylbutyric acid (ER stress inhibitor). Results: Hypoxia induced a more severe PH phenotype in TgNotch3 R169C mice versus controls. TgNotch3 R169C mice exhibited enhanced Notch3 activation and Hes5 expression (Notch3 target), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca 2+ transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC/cGMP signaling. These effects were ameliorated by N-acetylcysteine. pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension recapitulated Notch3/Hes5 signaling, ER stress and redox changes observed in PH mice. Conclusions: Notch3 GOF amplifies vascular dysfunction in hypoxic PH. This involves oxidative and ER stress, and ROCK. We highlight a novel role for Notch3/Hes5-redox signaling and important interplay between ER and oxidative stress in PH.

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.