Notch3/Hes5 Induces Vascular Dysfunction in Hypoxia-Induced Pulmonary Hypertension Through ER Stress and Redox-Sensitive Pathways

BACKGROUND: Notch3 (neurogenic locus notch homolog protein 3) 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 TgNotch3R169C 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, Ca2+ 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 TgNotch3R169C mice versus controls. TgNotch3R169C mice exhibited enhanced Notch3 activation and expression of Notch3 targets Hes Family BHLH Transcription Factor 5 (Hes5), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca2+ transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC (soluble guanylyl cyclase)/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.

T he Notch (neurogenic locus notch homolog protein) family of 4 transmembrane receptors play a key role in mediating cell-cell communication, regulating diverse cell functions including differentiation, maturation, proliferation, and apoptosis. 1 Notch3 (neurogenic locus notch homolog protein 3) is expressed in vascular smooth muscle cells, regulating proliferation and contraction essential for VSMC function. 2 As such, aberrant Notch3 signaling is implicated in diseases characterized by vascular remodeling, including cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) and pulmonary hypertension (PH). 3,4 Notch3 mutations underlie CADASIL, a cerebral arteriopathy characterized by cerebrovascular dysfunction, stroke and premature vascular dementia. 5 Notch3 mutations cause CADASIL, likely through gain-of-function (GOF) effects over a loss-of-function mechanism. 6 The TgNotch3 R169C mouse, a transgenic model of GOF Notch3 mutation has been used to study the pathophysiology of Notch3 in CADASIL and other conditions. 7,8 Here, we studied this model in PH.
PH is defined as mean pulmonary arterial pressure of ≥20 mm Hg, whereas pulmonary arterial hypertension (PAH) describes the progressive and irreversible remodeling disease. PAH is characterized by increased resistance and pathological remodeling in small pulmonary arteries, mediated by vasoconstriction and a proproliferative VSMC phenotype shift. Notch3 is implicated in both processes; however, previous work focuses on Notch3-mediated proliferation in PH. 4 In human PAH, Notch3 expression correlates positively with vascular resistance. 9 Notch3 mutations described in PAH are also hypothesized to contribute to pulmonary artery smooth muscle cell (PASMC) proliferation and vasoconstriction. 10,11 Additionally, hypoxia upregulates Notch3 signaling, further implicating involvement in PAH. 12 Hypoxia is commonly used to induce experimental PH and increased Notch3 expression and signaling appear necessary for hypoxic PH development in various models, with Notch3 inhibition proving successful as an intervention. 4,9 Transgenic mice overexpressing the GOF R169C Notch3 mutation (TgNotch3 R169C ) therefore provide a useful model for examining Notch3-mediated downstream signaling in the lung.
Mutant Notch3 can aggregate in and damage the endoplasmic reticulum (ER), 13 and contribute to peripheral CADASIL vascular dysfunction. 8,14 ER stress is also implicated in experimental PH 15 and human Notch3 mutations described in PAH patients also indicate a role for ER chaperones. 16 ROCK (Rho-associated kinase) is well documented in the pathogenesis of PH, through its effects on vasoconstriction and remodeling 17 and implicated in the TgNotch3 R169C CADASIL phenotype. 8 Moreover, redox-sensitive RhoA/ROCK acts downstream of Notch3 in myogenic tone regulation. 18 There is significant interplay between reactive oxygen species (ROS)/ reactive nitrogen species and ER stress, 19 and both processes are implicated in PH models. Additionally, chronic hypoxia contributes to vascular dysfunction alongside these pathways. 20,21 Despite this known role for Notch3 in pulmonary vascular dysfunction, there is a paucity of information NOVELTY AND RELEVANCE What Is New? R169C Notch3 (neurogenic locus notch homolog protein 3) mutation induces gain-of-function Notch3-Hes Family BHLH Transcription Factor 5 (Hes5) signaling in the lungs and pulmonary vascular dysfunction. Notch3-induced dysfunction associates with endoplasmic reticulum/oxidative stress and ROCK (Rhoassociated kinase), predisposing to hypoxic pulmonary hypertension (PH). Elevated endoplasmic reticulum stress/reactive oxygen species associates with increased Notch3 signaling in human pulmonary arterial hypertension pulmonary artery smooth muscle cells.

What Is Relevant?
Notch3 contributes to PH by promoting cell proliferation and vascular remodeling. Our data also implicate Notch3 in dysfunctional hypercontractility and vasorelaxation in PH. These findings connect Notch3 to downstream pathways important in PH and vascular dysfunction, including ROCK, endoplasmic reticulum stress, and reactive oxygen species.
Clinical/Pathophysiological Implications? PH/pulmonary arterial hypertension is debilitating with limited treatments and poor prognosis. Dampening of Notch3 signaling or downstream pathways highlighted in this study may constitute useful future therapeutic targets for PH.

Data Availability
See Supplemental Material for detailed methods. Data supporting the findings of this study are available from the corresponding author upon reasonable request.

Notch3 Mutant Mouse Model and Animal Studies
The transgenic TgNotch3 WT and TgNotch3 R169C mouse lines have been characterized previously and TgNotch3 R169C mice exhibit CADASIL features by 6 months. 7,22 Females were excluded for potential transgene mosaicism. 23 Mice were challenged with 14 days hypobaric hypoxia (550 mbar/≈10% O 2 ) to induce moderate PH, previously described. 24 PH was assessed by in vivo hemodynamic measurements, right ventricular hypertrophy, and histopathologic and immunohistochemistry analysis of lung sections, as described 24 and in the Supplemental Material.

PASMC Isolation and Culture
Mouse PASMCs were extracted from TgNotch3 pulmonary arteries and cultured with an adapted method described before. 26 Human PASMCs were isolated from distal pulmonary arteries (≤1 mm) from patients with PAH and controls (non-PAH individuals) as previously described 27 (Professor Nicholas Morrell, Cambridge, United Kingdom).

Quantitative Real-Time Polymerase Chain Reaction
Gene expression analysis was by quantitative real-time polymerase chain reaction with SYBR green reagents, calculated by 2 -ΔΔCt fold change. Primers in Table S1.

Immunoblotting
Immunoblotting was performed for proteins involved in Notch3-Hes Family BHLH Transcription Factor 5 (Hes5), ROCK, NO/ cGMP pathway, ER stress, and redox signaling. Antibodies are detailed in Table S2.

ROS Measurements
ROS was assessed in lung tissue and PASMCs. Lucigeninenhanced chemiluminescence was used for ROS generation as described previously. 19

Statistical Analysis
Data are represented as mean±SEM. Analysis by Student t test or 1-way ANOVA with Bonferroni post hoc test as appropriate, or by nonlinear regression for myography. *P<0.05 was considered significant.

R169C Mutation Is Associated With Increased Pulmonary Notch3 Signaling
We confirmed overexpression of rat Notch3 transgene in TgNotch3 WT and TgNotch3 R169C relative to nontransgenic FVB littermates ( Figure S1A). To assess GOF, we examined elements of the canonical Notch3 pathway, including the full-length Notch3 receptor protein and a transmembrane intracellular fragment produced by receptor cleavage. We confirm increased Notch3 signaling in TgNotch3 R169C lung, in line with work in other peripheral vessels from this model 8 ( Figure S1B). Similarly, downstream Notch3 target Hes5 was shown to be significantly increased in lung from TgNotch3 R169C mice compared to controls ( Figure S1C and S1D). Notch3 signaling also induces its own transcription, and expression of murine Notch3 was found to be increased in TgNotch3 R169C mice ( Figure S1E and S1F). Our results demonstrate that, despite transgene overexpression in both strains, only in TgNotch3 R169C lung was this associated with elevated Notch3 targets, indicating R169C mutation has a GOF effect on Notch3 signaling.

Figure 1. Hypoxia recapitulates increased gain-of-function (GOF) Notch3 (neurogenic locus notch homolog protein 3)-Hes
Family BHLH Transcription Factor 5 (Hes5) axis signaling and impaired vascular function in TgNotch3 WT mice and induces a more pronounced hypoxic pulmonary hypertension (PH) phenotype in TgNotch3 R169C mice. TgNotch3 mice were exposed to 14 days of chronic hypobaric hypoxia (10%). Notch3 signaling was assessed in the lung. A, Notch3 mRNA expression by real-time quantitative polymerase chain reaction normalized to GAPDH (n=5-7; 1-way ANOVA with Bonferroni (Continued ) Gene expression for endogenous murine Notch3, Hes5, and HeyL, downstream targets upregulated by Notch3 signaling, was also higher ( Figure 1B; Figure S2B and S2C). Right ventricular systolic pressure was significantly increased in TgNotch3 R169C animals after 2 weeks of hypoxia, while right ventricular systolic pressure in TgNotch3 WT animals was not significantly increased ( Figure 1C). Additionally, hypertrophic remodeling (right ventricular/[left ventricular+S]) was significantly increased in the hypoxic TgNotch3 R169C but not hypoxic TgNotch3 WT ( Figure S2D). Distal pulmonary artery (<80 μm) remodeling marginally increased following hypoxia, without difference between strains ( Figure S2E). Medial layer thickness, however, was already increased in TgNotch3 R169C pulmonary arteries compared to TgNotch3 WT in normoxia. Hypoxia increased medial layer thickness in both strains, with further elevation in TgNotch3 R169C ( Figure 1D). Hypoxia increased TgNotch3 R169C right ventricular contractility but not relaxation ( Figure S2F Figure 1F) in R169C mutant versus wild-type mice at normoxic baseline. In TgNotch3 WT vessels, hypoxia then increased contractile responses, whereas TgNotch3 R169C vessel responses remained similar to elevated normoxic responses ( Figure 1E; Figure S3A). Both strains exhibited diminished relaxation following hypoxia, with further blunting of vasorelaxation in TgNotch3 R169C arteries (Figure 1F; Figure S3B).

Impaired Vasorelaxation of Pulmonary Arteries in Notch3-Mutant Mice Involves ROS
We then investigated Notch3 GOF mutation effects on vascular relaxation in TgNotch3 mice by pulmonary artery wire myography. In Notch3-mutant mice arteries, acetylcholine-induced endothelial-dependent relaxation was significantly impaired compared with wildtype ( Figure 5A), ameliorating with fasudil or 4-PBA (Figure S10A and S10B). Notch3 mutation did not affect expression of eNOS (endothelial nitric oxide synthase; Figure S10C), or phosphorylation of eNOS at its activator (Ser 1177 ) or inhibitory site (Thr 495 ; Figure S10D and S10E). Nitrosylated tyrosine residues, indicating peroxynitrite (ONOO − ) modification, were significantly more abundant in Notch3-mutant lung ( Figure 5B), alongside lower NO (NOx assay; Figure 5C). Fasudil and 4-PBA had similar effects to restore further impaired acetylcholine-mediated relaxation in arteries from hypoxic TgNotch3 mice, in the context of increased ROS and reduced NO (but without alteration to H 2 O 2 ) in TgNotch3 WT mice (Figure S11A through S11D).

PASMCs From Patients With PAH Reflect Aberrant Signaling in TgNotch3 R169C Mice and Hypoxia
To assess the clinical relevance of our studies we examined pathways elucidated in the TgNotch3 R169C model in the context of human PAH, using PASMCs isolated from individuals with and without PAH (patient characteristics in Table S3). PAH PASMCs show increased expression of total Notch3 and TMIC fragment (Figure 6A and 6B), indicating increased expression and activation in PAH. Previous studies implicated Hes5 as a key Notch3 target in PAH, 9 and expression was higher in PAH-derived versus non-PAH cells ( Figure 6C). Consistent with R169C mouse findings, key ER stress protein BiP was upregulated in PAH cells ( Figure 6D). Elevated NADPH-dependent ROS observed in TgNotch3 R169C lungs was also recapitulated in PAH cells, and was abrogated by γ-secretase inhibitor (GSI) treatment to inhibit Notch ( Figure 6E). However, unlike in our model, H 2 O 2 was increased in PAH PASMCs and unaffected by GSI ( Figure 6F). Nox4 protein expression was concurrently elevated ( Figure S13).

DISCUSSION
Major findings from our study demonstrate that GOF Notch3 mutation exacerbates development of PH with associated hypercontractility and impaired relaxation in pulmonary arteries. These processes involve oxidative and ER stress, Ca 2+ and ROCK signaling, and impaired NO/cGMP signaling. Moreover, molecular changes observed in hypoxia-induced PH in TgNotch3 R169C mice were recapitulated in PASMCs from PAH patients. Our investigations define novel Notch3-redox-regulated pathways underlying pulmonary vascular dysfunction and highlight ER stress and oxidative stress interplay as potential major drivers of these processes in PH.
To explore the potential role of Notch3 in PH pathophysiology in the intact system, we studied TgNotch3 mice exposed to hypoxia. Hypoxia increased pulmonary Notch3-Hes5 signaling in TgNotch3 WT with exaggerated effects in TgNotch3 R169C mice. This was associated with worsening PH, as evidenced by increased right ventricular systolic pressure and hypertrophic remodeling. Together with these changes, pulmonary vessels in hypoxic mice exhibited marked hypereactivity and reduced vasorelaxation, responses that were amplified in TgNotch3 mice   versus wild type. Unlike previous studies demonstrating severe PH in chronic-hypoxic mice, TgNotch3 R169C mice exhibited a mild-moderate phenotype. This may relate to the FVB background strain, previously defined as a lowresponder to hypoxia. 29 It is also possible that a 14-day hypoxia duration was too short to induce robust PH. Nevertheless, the more pronounced phenotype in Notch3mutant mice was consistent with our hypothesis that hypoxia exaggerates aberrant Notch3 signaling, amplifying the GOF vascular response evidenced by baseline TgNotch3 R169C hemodynamic and remodeling changes. This may involve upregulation of α-SMA (α-smooth muscle actin), a Notch3 signaling procontractile target. 30,31 Hypoxia is a potent inducer of oxidative stress 32 as observed in our study. Hypoxic alterations to vascular reactivity improved with fasudil and 4-PBA, suggesting a role for ROCK and ER stress in PH vascular dysfunction. This was further supported through hypoxic upregulation of ROCK and ER stress, with amplified effects in TgNotch3 R169C mice. Reduced NO/cGMP/ PKG (protein kinase G) signaling also promotes PH via ROCK. 33 In TgNotch WT , hypoxia increased ER stress markers (BiP and pIRE1) to levels observed in normoxic TgNotch3 R169C mice. Further supporting a role for ER stress in hypoxic TgNotch3 R169C mice, 4-PBA rescued vascular reactivity changes. Hypoxia and Notch3 upregulation in TgNotch3 WT mice appear to recapitulate aberrant baseline TgNotch3 R169C vascular reactivity, and further affect TgNotch3 R169C phenotype, via ER stress and ROCK. These findings define functional interplay between Notch3, ROCK, and oxidative and ER stress in a hypoxic PH context. Molecular processes underlying pulmonary vascular hypereactivity during Notch3 activation focused on procontractile signaling pathways including Ca 2+ -dependent and Ca 2+ -independent mechanisms. Contraction and Ca 2+ transients in TgNotch3 R169C arteries and PASMCs were enhanced in response to 2 ligands, suggesting that altered vascular reactivity is a generalized phenomenon. In line with this, expression of membrane-associated Ca 2+ channels (Cav1.1, Cav3.1) and subcellular Ca 2+ stores (RyR, IP3R) were increased. Notch3 was previously implicated in regulating store-operated Ca 2+ entry through transient receptor potential cation 6 channels in PASMCs, a process enhanced by hypoxia. 34 Dysregulation of ER Ca 2+ homeostasis can also promote ER stress, linked to Notch3 signaling. ER stress stimulates the unfolded protein response, which may contribute to deposition of granular osmiophilic material, a hallmark feature of small vessels in CADASIL recapitulated in TgNotch3 R169C . 7,8 Our molecular data demonstrate increases in BiP and IRE1 phosphorylation. Notch3 overexpression in cell lines upregulates BiP, 16 and during ER stress, Notch3ICD has been shown to promote IRE1α activation via BiP interaction. 35 Functionally, we confirmed that reducing ER stress with chaperone 4-PBA restores TgNotch3 R169C pulmonary artery dysfunction and Ca 2+ transients. Our findings are consistent with Ca 2+ -dependent and ER stress-induced VSMC contraction. 36 A lack of strain difference in KCl-induced contraction supports our proposal that GOF Notch3 promotes hypercontractility primarily via intracellular Ca 2+ alterations, rather than voltage-gated effects at the membrane.
Ca 2+ -independent regulation of vascular contraction was explored by probing ROCK pathways. Altered TgNotch3 R169C pulmonary artery reactivity was associated with upregulation of Rho-GEFs PDZ and LARG, which enhance ROCK-mediated vascular contraction. 37 To confirm a role for ROCK in Notch3-associated vascular hyperreactivity, we showed that ROCK inhibition attenuated vascular contraction in hypoxic Notch3 mice. Of relevance, Notch signals through ROCK, 38 and RhoA-GTPase is implicated as an ER stress/unfolded protein response modulator. 39 Together these data link Notch3, ROCK and ER stress.
Redox signaling was also altered in TgNotch3 R169C animals. ROS production and markers of oxidative stress were increased in TgNotch3 R169C lung, whereas H 2 O 2 levels were decreased. Expression of Nox4, which produces H 2 O 2 over O 2 -,40 was reduced and may contribute to lower H 2 O 2 generation in TgNotch3 R169C lung. Nox4-derived H 2 O 2 is an endothelial-derived relaxation factor. 41 Thus reduced H 2 O 2 may support reduced vasorelaxation in TgNotch3 R169C mice. N-acetylcysteine correction of Ca 2+ transients in TgNotch3 R169C PASMCs further supports ROS influence in vascular contraction in TgNotch3 R169C lungs. 42 Both endothelium-dependent and -independent relaxation were compromised in TgNotch3 R169C pulmonary arteries, recapitulating previous results in TgNotch3 R169C cerebral arteries 43 and peripheral vessels in patients with CADASIL. 8 We examined eNOS signaling as the regulator of endothelial-dependent relaxation but failed to demonstrate altered activity. However, increased ROS and NO can react to produce injurious radical peroxynitrite (ONOO − ). Decreased acetylcholine-mediated relaxation in TgNotch3 R169C arteries accompanied indications of elevated ROS/ONOO − and reduced NO. We propose increased R169C ONOOis detrimental to pulmonary vasorelaxation through both oxidative stress and reduced NO bioavailability. Together these, findings suggest a prooxidative environment promotes TgNotch3 R169C endothelial dysfunction.
Reduced relaxation to SNP, a direct NO donor, indicated VSMC dysfunction in TgNotch3 R169C mice. To further explore this we investigated redox-sensitive sGC/ cGMP/PKG signaling. sGC oxidation decreases NOmediated cGMP production. 44 In TgNotch3 R169C animals sGCβ1 sulfenylation was increased, consistent with previous findings. 8 This was associated with oxidative stress, lower cGMP levels, and diminished PKG activity, processes involved in impaired vasorelaxation. Unlike sGC, Figure 5. Impaired endothelial-dependent and -independent vasorelaxation in TgNotch3 R169C arteries involves altered NO/cGMP signaling. Vascular reactivity was assessed in TgNotch3 pulmonary arteries by wire myography, responses expressed as percentage relaxation of preconstriction. A, Endothelium-dependent acetylcholine (ACh) vasorelaxation (n=7-9; nonlinear regression). B, Levels of 3-nitrotyrosine modified proteins in TgNotch3 lung, normalized to total protein (n=5; unpaired t test). C, NO levels by total nitrite/nitrate in TgNotch3 lung. D, Endothelium-independent sodium nitroprusside vasorelaxation in TgNotch3 pulmonary arteries (n=9-10; nonlinear regression). E, Reversible sGCβ1 (soluble guanylyl cyclase β1) oxidation by affinity capture of sulfenylated proteins in whole lung from TgNotch3 WT and TgNotch3 R169C mice (pool of 3 animals per sample). F, cGMP ELISA in TgNotch3 pulmonary artery smooth muscle cells±antioxidant N-acetylcysteine (NAC; 10 μM). Results are mean±SEM. *P<0.05 vs TgNotch3 WT , **P<0.01 vs TgNotch3WT. H 2 O 2 oxidation activates PKG independently of cGMP 45 and contributes to PKG-mediated vasorelaxation. 41 Lower H 2 O 2 in TgNotch3 R169C may reduce PKG activity. Together, our findings indicate multiple mechanisms are involved in Notch3-induced redox alterations that may influence NO/sGC/PKG signaling.
To explore the significance of our findings in human pathophysiology, we studied PASMCs from clinically phenotyped PAH patients. Elevated Notch3, TMIC fragment, and Hes5 expression in PAH versus non-PAH PASMCs demonstrated an increased Notch3 signaling, recapitulating findings in our R169C mice and other PH models. 9,46 Upregulation of BiP and ROS generation in PAH PASMCs were also similar to observations in hypoxiatreated TgNotch3 R169C mice. Normalization of oxidative stress by secretase inhibitor GSI suggests a role for Notch3 in ROS generation in PAH PASMCs, corroborating our findings in experimental models.
In contrast to findings in the mice, H 2 O 2 and Nox4 were upregulated in PAH cells as previously described. 47,48 Differences in Nox4/H 2 O 2 between mouse and human PH models may indicate the role of endothelial cells in our whole lung approach, where H 2 O 2 is normally abundant and protective, whereas our human studies examined isolated VSMCs, in which Nox4-derived H 2 O 2 is injurious. 49 A potential switch from low-level H 2 O 2 as a signaling molecule to a damaging ROS at higher levels is also suggested. The exact role of Notch3-regulated Nox4 and H 2 O 2 in endothelial cells versus VSMCs in PH awaits further clarification.

PERSPECTIVES
We provide evidence that GOF Notch3 mutation and increased Notch3 signaling promote pulmonary vascular dysfunction and remodeling, through redox-sensitive pathways involving ROCK and Ca 2+ signaling. These processes are driven by ER and oxidative stress, which also negatively influence the NO/sGC/cGMP relaxation pathway. Hypoxia amplifies these aberrations, predisposing to development of PH. We define a novel Notch3sensitive molecular mechanism involving redox-regulated procontractile and anti-vasodilatory signaling pathways in PH. This notion places Notch3 upstream of other PHassociated molecular mechanisms. These data provide insights on putative novel candidates in PH therapeutics, an area with unmet needs. Our data are particularly interesting in the context of recent studies validating antibody inhibition of Notch3 as a potential PH treatment, 46 which could reduce not only established Notch3 proliferative effects but also vascular hyperreactivity and endothelial dysfunction that we have highlighted.