Endothelial Jag1-RBPJ signalling promotes inflammatory leucocyte recruitment and atherosclerosis.

AIM: To determine the role of NOTCH during the arterial injury response and the subsequent chronic arterial-wall inflammation underlying atherosclerosis. METHODS AND RESULTS: We have generated a mouse model of endothelial-specific (Cdh5-driven) depletion of the Notch effector recombination signal binding protein for immunoglobulin kappa J region (RBPJ) [(ApoE-/-); homozygous RBPJk conditional mice (RBPJflox/flox); Cadherin 5-CreERT, tamoxifen inducible driver mice (Cdh5-CreERT)]. Endothelial-specific deletion of RBPJ or systemic deletion of Notch1 in athero-susceptible ApoE-/- mice fed a high-cholesterol diet for 6 weeks resulted in reduced atherosclerosis in the aortic arch and sinus. Intravital microscopy revealed decreased leucocyte rolling on the endothelium of ApoE-/-; RBPJflox/flox; Cdh5-CreERT mice, correlating with a lowered content of leucocytes and macrophages in the vascular wall. Transcriptome analysis revealed down-regulation of proinflammatory and endothelial activation pathways in atherosclerotic tissue of RBPJ-mutant mice. During normal Notch activation, Jagged1 signalling up-regulation in endothelial cells promotes nuclear translocation of the Notch1 intracellular domain (N1ICD) and its physical interaction with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). This N1ICD-NF-κB interaction is required for reciprocal transactivation of target genes, including vascular cell adhesion molecule-1. CONCLUSIONS: Notch signalling pathway inactivation decreases leucocyte rolling, thereby preventing endothelial dysfunction and vascular inflammation. Attenuation of Notch signalling might provide a treatment strategy for atherosclerosis.This study was funded by grants SAF2013-45543R, RD12/0042/0005 (RIC) and RD12/0019/0003 (TERCEL) from the Spanish Ministry of Economy and Competitiveness (MINECO) to J.L.dlP, RD12/0042/0028 (RIC) to V.A. and RD12/0042/0053 (RIC) and SAF2012-40127 to J.M.G. M.N. held a Sara Borrell post-doctoral contract (CD09/00452) and D.M. holds a post-doctoral contract associated with grant RD12/0042/0005, both awarded by The Instituto de Salud Carlos III; B.M.P. holds a Juan de la Cierva post-doctoral contract (JCI-2010-06343). The CNIC is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro CNIC Foundation, and is a ‘Severo Ochoa’ Center of Excellence (MINECO award SEV-2015-0505).This is the author accepted manuscript. The final version is available from Oxford University Press via http://dx.doi.org/10.1093/cvr/cvw19

Profiling the Role of Mammalian Target of Rapamycin in the Vascular Smooth Muscle Metabolome in Pulmonary Arterial Hypertension

Abstract. Increased proliferation and resistance to apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs), coupled with metabolic reprogramming, are key components of pulmonary vascular remodeling, a major and currently irreversible pathophysiological feature of pulmonary arterial hypertension (PAH). We recently reported that activation of mammalian target of rapamycin (mTOR) plays a key role in increased energy generation and maintenance of the proliferative, apoptosis-resistant PAVSMC phenotype in human PAH, but the downstream effects of mTOR activation on PAH PAVSMC metabolism are not clear. Using liquid and gas chromatography–based mass spectrometry, we performed pilot metabolomic profiling of human microvascular PAVSMCs from idiopathic-PAH subjects before and after treatment with the selective adenosine triphosphate–competitive mTOR inhibitor PP242 and from nondiseased lungs. We have shown that PAH PAVSMCs have a distinct metabolomic signature of altered metabolites—components of fatty acid synthesis, deficiency of sugars, amino sugars, and nucleotide sugars—intermediates of protein and lipid glycosylation, and downregulation of key biochemicals involved in glutathione and nicotinamide adenine dinucleotide (NAD) metabolism. We also report that mTOR inhibition attenuated or reversed the majority of the PAH-specific abnormalities in lipogenesis, glycosylation, glutathione, and NAD metabolism without affecting altered polyunsaturated fatty acid metabolism. Collectively, our data demonstrate a critical role of mTOR in major PAH PAVSMC metabolic abnormalities and suggest the existence of de novo lipid synthesis in PAVSMCs in human PAH that may represent a new, important component of disease pathogenesis worthy of future investigation

Mammalian Target of Rapamycin Complex 2 (mTORC2) Coordinates Pulmonary Artery Smooth Muscle Cell Metabolism, Proliferation, and Survival in Pulmonary Arterial Hypertension

BackgroundEnhanced proliferation, resistance to apoptosis, and metabolic shift to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (PAH). The role of the distinct mammalian target of rapamycin (mTOR) complexes mTORC1 (mTOR-Raptor) and mTORC2 (mTOR-Rictor) in PAVSMC proliferation and survival in PAH and their therapeutic relevance are unknown.Methods and resultsImmunohistochemical and immunoblot analyses revealed that mTORC1 and mTORC2 pathways are markedly upregulated in small remodeled pulmonary arteries and isolated distal PAVSMCs from subjects with idiopathic PAH that have increased ATP levels, proliferation, and survival that depend on glycolytic metabolism. Small interfering RNA- and pharmacology-based analysis showed that although both mTORC1 and mTORC2 contribute to proliferation, only mTORC2 is required for ATP generation and survival of idiopathic PAH PAVSMCs. mTORC2 downregulated the energy sensor AMP-activated protein kinase, which led to activation of mTORC1-S6 and increased proliferation, as well as a deficiency of the proapoptotic protein Bim and idiopathic PAH PAVSMC survival. NADPH oxidase 4 (Nox4) protein levels were increased in idiopathic PAH PAVSMCs, which was necessary for mTORC2 activation, proliferation, and survival. Nox4 levels and mTORC2 signaling were significantly upregulated in small pulmonary arteries from hypoxia-exposed rats at days 2 to 28 of hypoxia. Treatment with the mTOR kinase inhibitor PP242 at days 15 to 28 suppressed mTORC2 but not Nox4, induced smooth muscle-specific apoptosis in small pulmonary arteries, and reversed hypoxia-induced pulmonary vascular remodeling in rats.ConclusionsThese data provide a novel mechanistic link of Nox4-dependent activation of mTORC2 via the energy sensor AMP-activated protein kinase to increased proliferation and survival of PAVSMCs in PAH, which suggests a new potential pathway for therapeutic interventions

Mammalian Target of Rapamycin Complex 2 (mTORC2) Coordinates Pulmonary Artery Smooth Muscle Cell Metabolism, Proliferation, and Survival in Pulmonary Arterial Hypertension

BackgroundEnhanced proliferation, resistance to apoptosis, and metabolic shift to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (PAH). The role of the distinct mammalian target of rapamycin (mTOR) complexes mTORC1 (mTOR-Raptor) and mTORC2 (mTOR-Rictor) in PAVSMC proliferation and survival in PAH and their therapeutic relevance are unknown.Methods and resultsImmunohistochemical and immunoblot analyses revealed that mTORC1 and mTORC2 pathways are markedly upregulated in small remodeled pulmonary arteries and isolated distal PAVSMCs from subjects with idiopathic PAH that have increased ATP levels, proliferation, and survival that depend on glycolytic metabolism. Small interfering RNA- and pharmacology-based analysis showed that although both mTORC1 and mTORC2 contribute to proliferation, only mTORC2 is required for ATP generation and survival of idiopathic PAH PAVSMCs. mTORC2 downregulated the energy sensor AMP-activated protein kinase, which led to activation of mTORC1-S6 and increased proliferation, as well as a deficiency of the proapoptotic protein Bim and idiopathic PAH PAVSMC survival. NADPH oxidase 4 (Nox4) protein levels were increased in idiopathic PAH PAVSMCs, which was necessary for mTORC2 activation, proliferation, and survival. Nox4 levels and mTORC2 signaling were significantly upregulated in small pulmonary arteries from hypoxia-exposed rats at days 2 to 28 of hypoxia. Treatment with the mTOR kinase inhibitor PP242 at days 15 to 28 suppressed mTORC2 but not Nox4, induced smooth muscle-specific apoptosis in small pulmonary arteries, and reversed hypoxia-induced pulmonary vascular remodeling in rats.ConclusionsThese data provide a novel mechanistic link of Nox4-dependent activation of mTORC2 via the energy sensor AMP-activated protein kinase to increased proliferation and survival of PAVSMCs in PAH, which suggests a new potential pathway for therapeutic interventions

Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway

Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and β-catenin were required for expression of Pdgfrα and Pdgfrβ and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfrα and Pdgfrβ as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfrα/β expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung