In IPAH, c-kit(+) cells infiltrate pulmonary arterial lesions and may participate to vascular remodeling. Therefore, c-kit may represent a potential target for innovative PAH therapy.

Haematopoietic c-kit+ progenitor cells may contribute to pulmonary vascular remodelling and pulmonary hypertension (PH). Stromal derived factor-1 (SDF-1/CXCL12) and its receptors CXCR4 and CXCR7 have been shown to be critical for homing and mobilisation of haematopoietic c-kit+ progenitor cells in the perivascular niche.We administered AMD3100, a CXCR4 antagonist, and CCX771, a CXCR7 antagonist, to chronic hypoxia exposed mice in order to study the role of c-kit+ progenitor cells in PH. CXCL12, CXCR4 and CXCR7 protein expression, haemodynamic parameters, right ventricular mass, extent of vascular remodelling and perivascular progenitor cell accumulation were studied.Chronic hypoxia-exposed mice showed increased total lung tissue expression of CXCR4, CXCR7 and CXCL12 after development of PH. This was associated with significantly increased right ventricular systolic pressure and evidence of right ventricular hypertrophy, vascular remodelling and perivascular c-kit+/sca-1+ progenitor cell accumulation. CCX771 administration did not abrogate these effects. In contrast, administration of AMD3100, whether alone or combined with CCX771, prevented vascular remodelling, PH and perivascular accumulation of c-kit+/sca-1+ progenitor cells, with a synergistic effect of these agents.This study offers important pathophysiological insights into the role of haematopoietic c-kit+ progenitors in hypoxia-induced vascular remodelling and may have therapeutic implications for PH.

Immune mechanisms and autoimmunity seem to play a significant role in idiopathic pulmonary arterial hypertension (IPAH) pathogenesis and/or progression, but the pathophysiology is still unclear. Recent evidence has demonstrated a detrimental involvement of leptin in promoting various autoimmune diseases by controlling regulatory T-lymphocytes. Despite this knowledge, the role of leptin in IPAH is currently unknown. We hypothesised that leptin, synthesised by dysfunctional pulmonary endothelium, might play a role in the immunopathogenesis of IPAH by regulating circulating regulatory T-lymphocytes function.First, we collected serum and regulatory T-lymphocytes from controls, and IPAH and sclerodermaassociated pulmonary arterial hypertension (SSc-PAH) patients; secondly, we recovered tissue samples and cultured endothelial cells after either surgery or transplantation in controls and IPAH patients, respectively.Our findings indicate that serum leptin was higher in IPAH and SSc-PAH patients than controls. Circulating regulatory T-lymphocyte numbers were comparable in all groups, and the percentage of those expressing leptin receptor was higher in IPAH and SSc-PAH compared with controls, whereas their function was reduced in IPAH and SSc-PAH patients compared with controls, in a leptin-dependent manner. Furthermore, endothelial cells from IPAH patients synthesised more leptin than controls.Our data suggest that endothelial-derived leptin may play a role in the immunopathogenesis of IPAH.

ObjectivesTo assess activation of the inflammatory transcription factor NF-kappa B (NF-κB) in human idiopathic pulmonary arterial hypertension (PAH).BackgroundIdiopathic PAH is a severe progressive disease characterized by pulmonary vascular remodeling and excessive proliferation of vascular cells. Increasing evidence indicates that inflammation is important in disease pathophysiology.MethodsNF-κB-p65 and CD68, CD20 and CD45 were measured by immunohistochemistry and confocal microscopy on lung specimens from patients with idiopathic PAH (n = 12) and controls undergoing lung surgery (n = 14). Clinical data were recorded for all patients including invasive pulmonary hemodynamics for the PAH patients. Immunohistochemical images were analyzed by blinded observers to include standard pulmonary vascular morphometry; absolute macrophage counts/mm2 and p65-positivity (p65+) using composite images and image-analysis software; and cytoplasmic:nuclear p65+ of individual pulmonary arterial endothelial and smooth muscle cells (PASMC) in 10–20 pulmonary arteries or arterioles per subject. The expression of ET-1 and CCL5 (RANTES) in whole lung was determined by RT-qPCR.ResultsMacrophage numbers were increased in idiopathic PAH versus controls (49.0±4.5 vs. 7.95±1.9 macrophages/100 mm2, p<0.0001): these macrophages demonstrated more nuclear p65+ than in macrophages from controls (16.9±2.49 vs. 3.5±1.25%, p<0.001). An increase in p65+ was also seen in perivascular lymphocytes in patients with PAH. Furthermore, NF-κB activation was increased in pulmonary arterial endothelial cells (62.3±2.9 vs. 14.4±3.8, p<0.0001) and PASMC (22.6±2.3 vs. 11.2±2.0, p<0.001) in patients with PAH versus controls, with similar findings in arterioles. Gene expression of both ET-1 mRNA ((0.213±0.069 vs. 1.06±0.23, p<0.01) and CCL5 (RANTES) (0.16±0.045 vs. 0.26±0.039, p<0.05) was increased in whole lung homogenates from patients with PAH.ConclusionsNF-κB is activated in pulmonary macrophages, lymphocytes, endothelial and PASMC in patients with end-stage idiopathic PAH. Future research should determine whether NF-κB activation is a driver or bystander of pulmonary vascular inflammation and if the former, its potential role as a therapeutic target.

Pulmonary arterial hypertension (PAH) is associated with dysregulated bone morphogenetic protein receptor (BMPR)-II signaling and pulmonary vascular inflammation. We evaluated the effects of dexamethasone on monocrotaline (MCT)-induced PAH in rats for potential reversal of PAH at late time-points.Saline-treated control, MCT-exposed, MCT-exposed and dexamethasone-treated rats (5 mg?kg ) were evaluated at day 28 and day 35 following MCT for haemodynamic parameters, right ventricular hypertrophy, morphometry, immunohistochemistry, and IL6 and BMPR2 expression.Dexamethasone improved haemodynamics and pulmonary vascular remodelling, preventing PAH development at early (day 1-14 and 1-28) and reversing PAH at late (day 14-28 and 21-35) time-points following MCT, as well as improving survival in MCT-exposed rats compared with controls. Both MCT-induced pulmonary IL6 overexpression and interleukin (IL)-6-expressing adventitial inflammatory cell infiltration were reduced with dexamethasone. This was associated with pulmonary BMPR2 downregulation following MCT, which was increased with dexamethasone, in whole lung and control pulmonary artery smooth muscle cells. Dexamethasone also reduced proliferation of rat pulmonary artery smooth muscle cells in vitro.Experimental PAH can be prevented and reversed by dexamethasone, and survival is improved. In this model, mechanisms may involve reduction of IL-6-expressing inflammatory cells, restoration of pulmonary BMPR2 expression and reduced proliferation of vascular smooth muscle cells.

PVOD is characterized by alterations of circulating cytotoxic cell subpopulations and by epigenetic dysregulation within the GNLY gene. Our findings may be helpful in the quest to develop needed diagnostic tools, including flow cytometry analyses, to screen for suspected PVOD in patients with pulmonary hypertension.

Background and objectiveNuclear factor kappa B (NF‐kB)‐mediated inflammatory gene expression and vascular endothelial cell proliferation/remodelling are implicated in the pathophysiology of the fatal disease, pulmonary arterial hypertension (PAH). Bromodomain and extra‐terminal (BET) proteins are essential for the expression of a subset of NF‐kB‐induced inflammatory genes. BET mimics including JQ1+ prevent binding of BETs to acetylated histones and down‐regulate the expression of selected genes.MethodsThe effects of JQ1+ on the proliferation of primary human pulmonary microvascular endothelial cells (HPMECs) from healthy subjects were measured by bromodeoxyuridine (BrdU) incorporation. Cell cycle progression was assessed by flow cytometry; mRNA and protein levels of cyclin‐dependent kinases (CDKs), inhibitors and cytokines were determined by reverse transcription‐quantitative PCR (RT‐qPCR), Western blotting or ELISA. Histone acetyltransferase (HAT) and deacetylase (HDAC) activities were determined in nuclear extracts from whole lung of PAH and control patients.Results JQ1+ significantly inhibited IL6 and IL8 (IL6 and CXCL8) mRNA and protein in HPMECs compared with its inactive enantiomer JQ1−. JQ1+ decreased NF‐kB p65 recruitment to native IL6 and IL8 promoters. JQ1+ showed a concentration‐dependent decrease in HPMEC proliferation compared with JQ1−‐treated cells. JQ1+ induced G1 cell cycle arrest by increasing the expression of the CDK inhibitors (CDKN) 1A (p21cip) and CDKN2D (p19INK4D) and decreasing that of CDK2, CDK4 and CDK6. JQ1+ also inhibited serum‐stimulated migration of HPMECs. Finally, HAT activity was significantly increased in the lung of PAH patients.ConclusionInhibition of BETs in primary HPMECs decreases inflammation and remodelling. BET proteins could be a target for future therapies for PAH.

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. by interacting with proapoptotic proteins, or inhibiting caspase-3 activation. 10,12,15 In cardiomyocytes, it was shown that CRYAB silencing promoted cell death after exposure to H 2 O 2 , and that CRYAB has a protective role against apoptosis, by a mechanism involving translocation of CRYAB into mitochondria and by interacting with different proteins, including voltage-dependent anion channels and caspase-3. 16 Here, we show that BMPs induce CRYAB expression by a transcriptional, indirect molecular mechanism, which requires the BMPR2 and ALK1 receptors and the transcription factor Id1. BMPs protect EC from serum starvation and hypoxiainduced apoptosis and the protecting effect is, in part, because of a strong upregulation of the antiapoptotic protein CRYAB. cryab is downregulated in the lung of a mouse model of pulmonary hypertension induced by hypoxia, a condition in which the BMP signaling pathway has decreased activity. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results BMPs Upregulate CRYAB in Human Microvascular ECsMicroarray RNA profiling experiments were performed to identify BMP target genes in human microvascular EC-1 (HMEC-1) stimulated for 7 or 24 hours by 10 ng/mL of BMP4. Among the modulated genes, including ID1, ID2, and ID3, which were markedly upregulated (data not shown), we observed a strong upregulation of CRYAB expression by BMP4 at both 7 and 24 hours of stimulation.We confirmed, by real-time polymerase chain reaction (PCR), the upregulation of CRYAB expression in HMEC-1 treated by 10 ng/mL of BMP4 for 24, 48, and 72 hours (4.3±4.1, 8.6±0.7, and 7.5±1, respectively; Figure 1A). CRYAB mRNA was also increased by other members of the BMP growth factors family, BMP7 (fold increase at 4.8±0.9 at 24 hours, 4.8±1.8 at 48 hours, and 3.4±0.06 at 72 hours) and BMP9 (fold increase at 86.5±6.3 at 24 hours, 25.1±0.8 at 48 hours, and 15.1±1.5 at 72 hours), similar to BMP4 ( Figure 1A). In human lung microvascular ECs (HL...