Abstract P145: Notch3 Mutation is Involved in Pulmonary Vascular Dysfunction and Development of Pulmonary Arterial Hypertension

Abstract

Notch3 is implicated in CADASIL and pulmonary arterial hypertension (PAH), two diseases characterized by vascular dysfunction and remodelling. Mice harbouring the CADASIL-causing Notch3 mutation (TgNotch3 R169C ) exhibit gain-of-function Notch3 signalling. As increased Notch3 signalling has been demonstrated in several PAH models, we investigated the contribution of R169C mutation to pulmonary vascular dysfunction and PAH development. Briefly, vascular reactivity was studied by wire myography using pulmonary intralobar arteries from 24-week male mice overexpressing either a WT or R169C mutant Notch3 gene. Further mice were exposed to 2 weeks of normoxia or chronic hypobaric hypoxia (10% O 2 ) before assessing PAH development by in vivo haemodynamic measurements. Notch3 effector gene Hes5, implicated in PAH development, was significantly increased in R169C but not WT lung (8.7 fold change vs WT) suggesting increased pulmonary Notch3 activation. Hypercontractility of R169C pulmonary arteries to both 5HT (Emax R169C 146 ± 10% vs WT 92 ± 6%) and ET-1 (Emax R169C 211 ± 15% vs WT 154 ± 32%) was observed; an effect attenuated by ROCK inhibitor Fasudil (5HT Emax R169C + Fas. 117% ± 20%, ET-1 R169C + Fas. 154 ± 11%) (p<0.05). In addition, gene expression for Rho-guanine nucleotide exchange factors LARG and PDZ, which can activate RhoA/ROCK signalling, was upregulated in R169C lung (R169C 33.5 fold vs WT, R169C 18.7 fold vs WT respectively), with no changes to ROCK gene or protein expression. Reduced endothelial-dependent (ACh Emax WT 43 ± 3% vs R169C 36 ± 3%) and -independent relaxation (SNP Emax WT 62 ± 4% vs R169C 57 ± 4%) were also observed in R169C vessels (p<0.05). Chronic hypoxia did not affect systemic haemodynamic parameters. RV systolic pressure increased in hypoxic animals vs normoxic; this was exacerbated in R169C hypoxic animals (WT hypoxic 27.46 ± 0.97 vs WT normoxic 22.76 ± 2.17mmHg, R169C hypoxic 32.18 ± 1.13 mmHg vs R169C normoxic 23.60 ± 1.44) (p<0.05). R169C hypoxic animals also exhibited RV hypertrophy (RV/LV+S) (hypoxic 0.26 ± 0.033 vs normoxic 0.16 ± 0.012). These data demonstrate Notch3 R169C mutation may contribute to pulmonary vascular hypercontractility and PAH susceptibility, and highlight ROCK as a potential mechanism.