Disruption of GCN2 Pathway Aggravates Vascular and Parenchymal Remodeling during Pulmonary Fibrosis.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 (general control nonderepressible 2) gene (EIF2AK4 [eukaryotic translation initiation factor 2 alpha kinase 4]) were recently associated with pulmonary veno-occlusive disease. The aim of this study is to explore the involvement of the GCN2/eIF2α (eukaryotic initiation factor 2α) pathway in the development of PH during PF, in both human disease and in a laboratory animal model. Lung tissue from patients with PF with or without PH was collected at the time of lung transplantation, and control tissue was obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4-mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies and organ collection were performed 3 weeks after instillation. Only significant results (P < 0.05) are presented. In PF lung tissue, GCN2 protein expression was decreased compared with control tissue. GCN2 expression was reduced in CD31+ endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats compared with saline. EIF2AK4-mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline concentrations) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure compared with wild-type animals. Our data show that GCN2 is dysregulated in both humans and in an animal model of combined PF and PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied.

T-cell dysregulation and inflammatory process in Gcn2 (Eif2ak4-/-)-deficient rats in basal and stress conditions.

Hereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2-/- rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2-/- rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2-/- rat lungs revealed increases in two T-cell populations, a LAG3+ T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2-/- rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development.

Lineage Tracing Reveals the Dynamic Contribution of Pericytes to the Blood Vessel Remodeling in Pulmonary Hypertension.

OBJECTIVE: Excessive accumulation of resident cells within the pulmonary vascular wall represents the hallmark feature of the remodeling occurring in pulmonary arterial hypertension (PAH). Furthermore, we have previously demonstrated that pulmonary arterioles are excessively covered by pericytes in PAH, but this process is not fully understood. The aim of our study was to investigate the dynamic contribution of pericytes in PAH vascular remodeling. Approach and Results: In this study, we performed in situ, in vivo, and in vitro experiments. We isolated primary cultures of human pericytes from controls and PAH lung specimens then performed functional studies (cell migration, proliferation, and differentiation). In addition, to follow up pericyte number and fate, a genetic fate-mapping approach was used with an NG2CreER;mT/mG transgenic mice in a model of pulmonary arteriole muscularization occurring during chronic hypoxia. We identified phenotypic and functional abnormalities of PAH pericytes in vitro, as they overexpress CXCR (C-X-C motif chemokine receptor)-7 and TGF (transforming growth factor)-ßRII and, thereby, display a higher capacity to migrate, proliferate, and differentiate into smooth muscle-like cells than controls. In an in vivo model of chronic hypoxia, we found an early increase in pericyte number in a CXCL (C-X-C motif chemokine ligand)-12-dependent manner whereas later, from day 7, activation of the canonical TGF-ß signaling pathway induces pericytes to differentiate into smooth muscle-like cells. CONCLUSIONS: Our findings reveal a pivotal role of pulmonary pericytes in PAH and identify CXCR-7 and TGF-ßRII as 2 intrinsic abnormalities in these resident progenitor vascular cells that foster the onset and maintenance of PAH structural changes in blood lung vessels.

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

Widening the landscape of heritable pulmonary hypertension mutations in paediatric and adult cases.

BACKGROUND: Heritable forms of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease/pulmonary capillary haemangiomatosis (PVOD/PCH) diverge by lung histopathological lesions, clinical and para-clinical presentation, their responsible genes, and mode of transmission. Since the identification of the BMPR2 gene in families affected by PAH, mutations in several other genes have been discovered for both forms. The mutation landscape in these new genes is not yet well known. METHODS: We set up a next-generation sequencing-based targeted sequencing gene panel allowing known genes for PAH and PVOD/PCH to be analysed simultaneously. Genetic analysis was prospectively performed on 263 PAH and PVOD/PCH patients (adult and paediatric cases). RESULTS: Pathogenic mutations were identified in 19.5% of sporadic PAH patients (n=180), 54.5% of familial PAH patients and 13.3% of PVOD/PCH patients. BMPR2 was the most frequently mutated gene, followed by TBX4 in both paediatric and adult PAH. BMP9 mutations were identified in 1.2% of adult PAH cases. EIF2AK4 biallelic mutations were restricted to PVOD/PCH. A truncating mutation and a predicted loss-of-function variant were also identified in BMP10 in two severely affected sporadic PAH female patients. CONCLUSION: Our results confirm that mutations are found in genes beyond BMPR2 in heritable PAH, emphasise the role of TBX4 and BMP9, and designate BMP10 as a new PAH gene.

Resident PW1+ Progenitor Cells Participate in Vascular Remodeling During Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1(+) progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. OBJECTIVE: To determine the role of pulmonary PW1(+) progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. METHODS AND RESULTS: We investigated their contribution during chronic hypoxia-induced vascular remodeling in Pw1(nLacZ+/-) mouse expressing ß-galactosidase in PW1(+) cells and in differentiated cells derived from PW1(+) cells. PW1(+) progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1(+) cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1(+) cells and the proportion of ß-gal(+) vascular SMC were increased, indicating a recruitment of PW1(+) cells and their differentiation into vascular SMC during early chronic hypoxia-induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1(+) cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed ß-gal(+) SMC were not derived from circulating bone marrow-derived PW1(+) progenitor cells, confirming a resident origin of the recruited PW1(+) cells. The number of pulmonary PW1(+) cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. CONCLUSIONS: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension-associated vascular remodeling.

Genetic analyses in a cohort of children with pulmonary hypertension.

The prevalence of germline mutations in paediatric pulmonary hypertension (PH) is poorly documented. The objective of this study was to determine the mutation frequency in PH genes in a paediatric cohort and describe the clinical characteristics of mutation carriers.The study involved 66 index cases with PH: 35 children with idiopathic pulmonary arterial hypertension (IPAH); five children with familial PAH (FPAH); three children with pulmonary veno-occlusive disease (PVOD); and 23 children with PAH associated with congenital heart disease (APAH-CHD).No mutations were found in the 23 children with APAH-CHD. In the 40 children with IPAH or FPAH, 12 mutations were found: five on BMPR2; four on ACVRL1; and three on TBX4. In the three PVOD cases, two carried the EIF2AK4 mutation. Mutation carriers had a more severe disease at diagnosis and more aggressive first-line therapy was required. The three patients with PVOD had a very severe disease at diagnosis and required a lung transplantation.The genetic architecture of paediatric PAH is enriched in ACVRL1 and TBX4 mutations compared to adult PAH, but further studies are required to confirm these results. Childhood-onset PAH in children carrying a mutation in one of the genes tested has a more severe presentation at diagnosis but a similar outcome to that observed in non-carriers.

Leptin signalling system as a target for pulmonary arterial hypertension therapy.

Excessive proliferation of pulmonary arterial smooth muscle cells (PA-SMCs) and perivascular inflammation lead to pulmonary arterial hypertension (PAH) progression, but they are not specifically targeted by the current therapies. Since leptin (Ob) and its main receptor ObR-b contribute to systemic vascular cell proliferation and inflammation, we questioned whether targeting Ob/ObR-b axis would be an effective antiproliferative and anti-inflammatory strategy against PAH. In idiopathic PAH (iPAH), using human lung tissues and primary cell cultures (early passages ≤5), we demonstrate that pulmonary endothelial cells (P-ECs) over produce Ob and that PA-SMCs overexpress ObR-b. Furthermore, we obtain evidence that Ob enhances proliferation of human PA-SMCs in vitro and increases right ventricular systolic pressure in Ob-treated mice in the chronic hypoxia-induced pulmonary hypertension (PH) model. Using human cells, we also show that Ob leads to monocyte activation and increases cell adhesion molecule expression levels in P-ECs. We also find that Ob/ObR-b axis contributes to PH susceptibility by using ObR-deficient rats, which display less severe hypoxia-induced PH (pulmonary haemodynamics, arterial muscularisation, PA-SMC proliferation and perivascular inflammation). Importantly, we demonstrate the efficacy of two curative strategies using a soluble Ob neutraliser and dichloroacetate in hypoxia-induced PH. We demonstrate here that Ob/ObR-b axis may represent anti-proliferative and anti-inflammatory targets in PAH.

Bone morphogenetic proteins protect pulmonary microvascular endothelial cells from apoptosis by upregulating α-B-crystallin.

OBJECTIVE: To investigate the role of bone morphogenetic proteins (BMPs) on α-B-crystallin (CRYAB) expression and its physiological consequences on endothelial cells (ECs). APPROACH AND RESULTS: We report that the gene encoding for the small heat shock protein, CRYAB, is a transcriptional target of the BMP signaling pathway. We demonstrate that CRYAB expression is upregulated strongly by BMPs in an EC line and in human lung microvascular ECs and human umbilical vein ECs. We show that BMP signals through the BMPR2-ALK1 pathway to upregulate CRYAB expression through a transcriptional indirect mechanism involving Id1. We observed that the known antiapoptotic effect of the BMPs is, in part, because of the upregulation of CRYAB expression in EC. We also show that cryab is downregulated in vivo, in a mouse model of pulmonary arterial hypertension induced by chronic hypoxia where the BMP pathway is downregulated. CONCLUSIONS: We demonstrate a cross-talk between BMPs and CRYAB and a major effect of this regulatory interaction on resistance to apoptosis.

A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70(-) and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70(+) and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70(-)) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease.

Small platelet microparticle levels are increased in pulmonary arterial hypertension.

BACKGROUND: The various aetiologies and risk factors for pulmonary arterial hypertension (PAH) lead to close phenotypes with small differences. Plasma microparticles have been shown to be increased in vascular pathologies including PAH. The aim of this study was to determine whether the levels of endothelial and platelet-derived microparticles could vary between different forms of PAH: idiopathic PAH (iPAH), heritable PAH associated with BMPR2 (Bone morphogenetic protein receptor, type II) mutation (hPAH) and PAH associated with connective tissue diseases (aPAH). MATERIALS AND METHODS: Microparticles were analysed using flow cytometry in plasma from controls and iPAH, hPAH and aPAH patients. Platelet-derived MP (PMP) were defined as CD31(+)/CD41(+) and endothelial-derived MP (EMP) as CD31(+)/CD41(-). Two populations of PMP were isolated according to their size, defining small PMP (0·3-0·5 µm) and large PMP (0·5-0·9 µm). BMPR2 genotype, clinical and biologic parameters were recorded. RESULTS: EMP and small PMP levels in iPAH, hPAH and aPAH were similar and were significantly increased as compared with controls. No differences in large PMP levels were observed. After adjusting for age, sex, proBNP and CRP, EMP and small PMP levels did not correlate with clinical parameters. CONCLUSIONS: iPAH, hPAH and aPAH were characterized by increased levels of EMP and of small PMP, a new class of PMP which seems to be differentially produced than large PMP.

Inhibition of apelin expression by BMP signaling in endothelial cells.

The transforming growth factor-ß/bone morphogenic protein (BMP) system is a major pathway for angiogenesis and is involved in hereditary vascular diseases. Here we report that the gene encoding the vasoactive and vascular cell growth-regulating peptide apelin is a target of the BMP pathway. We demonstrate that apelin expression is strongly downregulated by BMP in an endothelial cell line as well as in lung endothelial microvascular cells. We show that BMP signals through the BMPR2-SMAD pathway to downregulate apelin expression and that a transcriptional direct and indirect mechanism is required. The BMP-induced downregulation of apelin expression was found to be critical for hypoxia-induced growth of endothelial cells, because the growth inhibitory effect of BMP in this condition is suppressed by enforced expression of apelin. Thus, we describe an important link between a signaling pathway involved in angiogenesis and vascular diseases and a peptide regulating vascular homeostasis.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation.

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with ß-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

Early Protective Role of Inflammation in Cardiac Remodeling and Heart Failure: Focus on TNFα and Resident Macrophages.

Cardiac hypertrophy, initiated by a variety of physiological or pathological stimuli (hemodynamic or hormonal stimulation or infarction), is a critical early adaptive compensatory response of the heart. The structural basis of the progression from compensated hypertrophy to pathological hypertrophy and heart failure is still largely unknown. In most cases, early activation of an inflammatory program reflects a reparative or protective response to other primary injurious processes. Later on, regardless of the underlying etiology, heart failure is always associated with both local and systemic activation of inflammatory signaling cascades. Cardiac macrophages are nodal regulators of inflammation. Resident macrophages mostly attenuate cardiac injury by secreting cytoprotective factors (cytokines, chemokines, and growth factors), scavenging damaged cells or mitochondrial debris, and regulating cardiac conduction, angiogenesis, lymphangiogenesis, and fibrosis. In contrast, excessive recruitment of monocyte-derived inflammatory macrophages largely contributes to the transition to heart failure. The current review examines the ambivalent role of inflammation (mainly TNFα-related) and cardiac macrophages (Mφ) in pathophysiologies from non-infarction origin, focusing on the protective signaling processes. Our objective is to illustrate how harnessing this knowledge could pave the way for innovative therapeutics in patients with heart failure.

Transcriptomic and Lipidomic Mapping of Macrophages in the Hub of Chronic Beta-Adrenergic-Stimulation Unravels Hypertrophy-, Proliferation-, and Lipid Metabolism-Related Genes as Novel Potential Markers of Early Hypertrophy or Heart Failure.

Sympathetic nervous system overdrive with chronic release of catecholamines is the most important neurohormonal mechanism activated to maintain cardiac output in response to heart stress. Beta-adrenergic signaling behaves first as a compensatory pathway improving cardiac contractility and maladaptive remodeling but becomes dysfunctional leading to pathological hypertrophy and heart failure (HF). Cardiac remodeling is a complex inflammatory syndrome where macrophages play a determinant role. This study aimed at characterizing the temporal transcriptomic evolution of cardiac macrophages in mice subjected to beta-adrenergic-stimulation using RNA sequencing. Owing to a comprehensive bibliographic analysis and complementary lipidomic experiments, this study deciphers typical gene profiles in early compensated hypertrophy (ECH) versus late dilated remodeling related to HF. We uncover cardiac hypertrophy- and proliferation-related transcription programs typical of ECH or HF macrophages and identify lipid metabolism-associated and Na+ or K+ channel-related genes as markers of ECH and HF macrophages, respectively. In addition, our results substantiate the key time-dependent role of inflammatory, metabolic, and functional gene regulation in macrophages during beta-adrenergic dependent remodeling. This study provides important and novel knowledge to better understand the prevalent key role of resident macrophages in response to chronically activated beta-adrenergic signaling, an effective diagnostic and therapeutic target in failing hearts.

The Platelet-Derived Growth Factor Pathway in Pulmonary Arterial Hypertension: Still an Interesting Target?

The lack of curative options for pulmonary arterial hypertension drives important research to understand the mechanisms underlying this devastating disease. Among the main identified pathways, the platelet-derived growth factor (PDGF) pathway was established to control vascular remodeling and anti-PDGF receptor (PDGFR) drugs were shown to reverse the disease in experimental models. Four different isoforms of PDGF are produced by various cell types in the lung. PDGFs control vascular cells migration, proliferation and survival through binding to their receptors PDGFRα and ß. They elicit multiple intracellular signaling pathways which have been particularly studied in pulmonary smooth muscle cells. Activation of the PDGF pathway has been demonstrated both in patients and in pulmonary hypertension (PH) experimental models. Tyrosine kinase inhibitors (TKI) are numerous but without real specificity and Imatinib, one of the most specific, resulted in beneficial effects. However, adverse events and treatment discontinuation discouraged to pursue this therapy. Novel therapeutic strategies are currently under experimental evaluation. For TKI, they include intratracheal drug administration, low dosage or nanoparticles delivery. Specific anti-PDGF and anti-PDGFR molecules can also be designed such as new TKI, soluble receptors, aptamers or oligonucleotides.

Platelet-Derived Growth Factor Receptor Type α Activation Drives Pulmonary Vascular Remodeling Via Progenitor Cell Proliferation and Induces Pulmonary Hypertension.

Background Platelet-derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1+) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet-derived growth factor receptor type α (PDGFRα) pathway in progenitor cell-dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1+ cells expressing PDGFRα. PW1nLacZ reporter mice were used to follow the fate of pulmonary PW1+ progenitor cells in a model of chronic hypoxia-induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1+ progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1+ progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1+ progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell-dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add-on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.

Progenitor/Stem Cells in Vascular Remodeling during Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by an important occlusive vascular remodeling with the production of new endothelial cells, smooth muscle cells, myofibroblasts, and fibroblasts. Identifying the cellular processes leading to vascular proliferation and dysfunction is a major goal in order to decipher the mechanisms leading to PAH development. In addition to in situ proliferation of vascular cells, studies from the past 20 years have unveiled the role of circulating and resident vascular in pulmonary vascular remodeling. This review aims at summarizing the current knowledge on the different progenitor and stem cells that have been shown to participate in pulmonary vascular lesions and on the pathways regulating their recruitment during PAH. Finally, this review also addresses the therapeutic potential of circulating endothelial progenitor cells and mesenchymal stem cells.

Anti-integrin αv therapy improves cardiac fibrosis after myocardial infarction by blunting cardiac PW1+ stromal cells.

There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1+ cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1+ cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1+ cells (93% ± 1%), predominantly as the αVß1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFß at the surface of cardiac PW1+ cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1+CD51+ cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.

Cardiac inflammatory CD11b/c cells exert a protective role in hypertrophied cardiomyocyte by promoting TNFR2- and Orai3- dependent signaling.

Early adaptive cardiac hypertrophy (EACH) is initially a compensatory process to optimize pump function. We reported the emergence of Orai3 activity during EACH. This study aimed to characterize how inflammation regulates store-independent activation of Orai3-calcium influx and to evaluate the functional role of this influx. Isoproterenol infusion or abdominal aortic banding triggered EACH. TNFα or conditioned medium from cardiac CD11b/c cells activated either in vivo [isolated from rats displaying EACH], or in vitro [isolated from normal rats and activated with lipopolysaccharide], were added to adult cardiomyocytes before measuring calcium entry, cell hypertrophy and cell injury. Using intramyocardial injection of siRNA, Orai3 was in vivo knockdown during EACH to evaluate its protective activity in heart failure. Inflammatory CD11b/c cells trigger a store-independent calcium influx in hypertrophied cardiomyocytes, that is mimicked by TNFα. Pharmacological or molecular (siRNA) approaches demonstrate that this calcium influx, depends on TNFR2, is Orai3-driven, and elicits cardiomyocyte hypertrophy and resistance to oxidative stress. Neutralization of Orai3 inhibits protective GSK3ß phosphorylation, impairs EACH and accelerates heart failure. Orai3 exerts a pathophysiological protective impact in EACH promoting hypertrophy and resistance to oxidative stress. We highlight inflammation arising from CD11b/c cells as a potential trigger of TNFR2- and Orai3-dependent signaling pathways.