PASMC Proliferation Research Articles

Abstract 9511: Implication of the KATP SUR2B/kir6.1 in the Physiopathology of Pulmonary Arterial Hypertension

Introduction: PAH is a severe and rare cardiopulmonary disease which is defined by an elevation of the mean pulmonary artery pressure >25mmHg. ABCC9 mutations are responsible of Cantu syndrome, a disease characterised by several cardiac disorders and for some case pulmonary hypertension (PH). ABCC9 gene encodes for SUR2 protein, a regulatory subunit of KATP channels. KATP are composed of SUR1 or SUR2 and Kir6.1 or Kir6.2 subunits. We hypothesized that SUR2 / Kir6.1 channel contributes to PAH pathogenesis and could be a therapeutic target against PAH. Methods: Using a combination of in vitro , ex vivo and in vivo approaches, we analysed the localisation and expression of SUR2A, SUR2B and Kir6.1 in pulmonary vasculature from control and PAH patients as well as in experimental PH rat-models. We also analysed the role of SUR2/Kir6.1 in PAH physiopathology (PASMCs proliferation, migration and contraction). Finally, we deciphered the consequences of in vivo pharmacological activation of SUR2/Kir6.1 in experimental model of PH. Results: We demonstrated that SUR2A, SUR2B and Kir6.1 are expressed (mRNA and protein) in lungs from control and PAH patients and in MCT induced-PH model.Myograph experiments demonstrated that pharmacological activation of SUR2 by pinacidil induced pulmonary arterial relaxation in human and rat isolated pulmonary arteries. PA relaxation was still present in PA from MCT-rats but reduced compared to control rats. In vitro experiments on human PASMCs (hPASMCs) from control and PAH patients show a decrease of the cell proliferation and migration after SUR2 activation (pinacidil). Using Patch-clamp technique, we demonstrated that SUR2 activator (pinacidil) in adult rat RV cardiomyocytes reduces RV action potential. Finally, pharmacological activation in vivo of SUR2 by pinacidil (1mg/kg/day) on MCT induced-PH rats show an improvement of the hemodynamic parameters as the mean right ventricular systolic pressure during preventive and curative protocol. Conclusions: We demonstrated that SUR2A, SUR2B and Kir6.1 are expressed in hPASMCs and hPECs from control and iPAH patients. The in vivo SUR2 activation by pinacidil treatment reduced MCT induced-PH phenotype suggesting that SUR2 pharmacological activation should be considered for PAH.

Abstract 14210: Impaired Mitophagy and Reduced Parkin Expression in Human Pulmonary Arterial Smooth Muscle Cells (PASMCs) in Pulmonary Arterial Hypertension (PAH)

Introduction: Mitophagy is the key mitochondrial quality control process of selective removal of damaged mitochondria which is necessary for maintaining cellular homeostasis. While studies have demonstrated that abnormalities in mitochondrial metabolism drive a hyperproliferative phenotype of pulmonary vasculature in PAH, there is limited understanding of mitophagy dysregulation in PAH. Recent studies have shown that hypoxia induced Parkin/Pink1-mediated mitophagy promotes vascular remodeling, however another report suggests that loss of Pink1 and reduced mitophagy in hypoxia promotes PASMC proliferation. The role and mechanisms of altered mitochondrial dynamics/mitophagy dysregulation in driving pulmonary vascular remodeling in PAH remain incompletely understood. Hypothesis: We hypothesized that impaired mitophagy and reduced Parkin expression may drive the apoptosis-resistant, hyperproliferative phenotype in PAH PASMCs. Methods: Mitophagy was assessed in human PASMCs from control donors and PAH patients stably expressing mtKeima protein and in lungs from hypoxia-exposed mtKeima transgenic mice. Parkin expression was assessed by Western blotting and immunocytochemistry. Immunofluorescence staining for Parkin was performed in lungs from mice exposed to chronic hypoxia. Human PASMCs from control donors were transfected with siControl and siParkin and assessed for proliferation and apoptosis by Ki67 and Annexin V/PI flow cytometry and PCNA expression. Results: PASMCs from PAH patients showed reduced KeimaRed vs. KeimaGreen ratio compared to control donors indicating reduced mitophagy i.e. further confirmed by decreased Parkin in PAH PASMCs. Parkin deficient PASMCs demonstrated more Ki67 and less Annexin V/PI positive cells and increased PCNA. Interestingly, lungs from hypoxia-exposed mtKeima transgenic mice showed increased KeimaRed vs. KeimaGreen ratio and increased Parkin in lungs of hypoxia-exposed mice. Conclusions: PAH PASMCs demonstrate impaired mitophagy and reduced Parkin expression; Parkin deficiency promotes proliferation and inhibits apoptosis in PASMCs. Our findings suggest that Parkin may regulate remodeling phenotypes in PASMCs and thus may represent a potential therapeutic target in PAH.

Osthole alleviates pulmonary vascular remodeling by modulating microRNA-22–3p mediated lipid metabolic reprogramming

BackgroundPulmonary vascular remodeling is the key pathological feature of pulmonary arterial hypertension (PAH) characterized by a pattern of lipid-related insulin resistance(IR), hormonal derangements and metabolic reprogramming. Our previous studies have demonstrated osthole as natural coumarin compound derived from traditional Chinese medicine is a promising agent for the treatment of pulmonary vascular remodeling in PAH. PurposeThe present study sought to delineate lipid metabolic modulatory mechanism of osthole against pulmonary vascular remodeling by employing an interdisciplinary strategy. MethodsRat model with PAH induced with MCT and PASMCs proliferation model induced with PDGF-BB were established in this study. Serum and lung tissues were used to lipid-related IR, hormone related indexes, pulmonary vascular remodeling analysis. Then, lipid metabolic gene, key enzymes, metabolites and cell proliferation indexes were examined to investigate metabolic regulatory mechanism in vivo and vitro model of PAH. ResultsOsthole significantly showed improvement of lipid-related IR and hormone dysregulation in rats with PAH evidenced by elevating testosterone, androgen receptor and cyclic guanosine monophosphate (cGMP), inhibiting phosphodiesterase-5(PDE-5), modulating lipid-related IR indexes total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), triglyceride (TG)/HDL-C ratio. Additionally, osthole limited key metabolic gene and enzymes to inhibit accumulation of decadienyl-l-carnitine in lipid metabolism, thus to promote oxidative phosphorylation and ATP production through inhibition of miRNA-22–3p, fatty acid translocase (CD36), fatty acid synthase (FAS), phospholipase A2 (PLA2), carnitine palmitoyltransferase 1A (CPT1A), hexokinase 2 (HK2), activation of metabolic switch isocitrate dehydrogenase 3α (IDH3α), NADH dehydrogenase 1 (ND1). We found for the first time miRNA-22–3p modulated PASMCs proliferation and vascular remodeling by regulating lipid metabolism reprogramming. Those modifications uncovered therapeutic mechanism of osthole against pulmonary vascular remodeling. ConclusionOur findings revealed the function of miRNA-22–3p in PASMCs and demonstrated a novel mechanism that miRNA-22–3p as a regulator can be targeted by osthole to greatly restore dysregulated lipid metabolism thus to alleviate pulmonary vascular remodeling in PAH, which provides novel insight into the potential therapeutic target for PAH, further highlights the development potential of osthole derived new drug against PAH.

Deficiency of Axl aggravates pulmonary arterial hypertension via BMPR2

Pulmonary arterial hypertension (PAH), is a fatal disease characterized by a pseudo-malignant phenotype. We investigated the expression and the role of the receptor tyrosine kinase Axl in experimental (i.e., monocrotaline and Su5416/hypoxia treated rats) and clinical PAH. In vitro Axl inhibition by R428 and Axl knock-down inhibited growth factor-driven proliferation and migration of non-PAH and PAH PASMCs. Conversely, Axl overexpression conferred a growth advantage. Axl declined in PAECs of PAH patients. Axl blockage inhibited BMP9 signaling and increased PAEC apoptosis, while BMP9 induced Axl phosphorylation. Gas6 induced SMAD1/5/8 phosphorylation and ID1/ID2 increase were blunted by BMP signaling obstruction. Axl association with BMPR2 was facilitated by Gas6/BMP9 stimulation and diminished by R428. In vivo R428 aggravated right ventricular hypertrophy and dysfunction, abrogated BMPR2 signaling, elevated pulmonary endothelial cell apoptosis and loss. Together, Axl is a key regulator of endothelial BMPR2 signaling and potential determinant of PAH.

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca2+] i mobilization. In the present study, hypoxia exposure of rat's model was established. Two‐pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh‐TPC1 or Lsh‐TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP‐activated [Ca2+] i shows to be mediated via two‐pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia‐induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh‐TPC1 or Lsh‐TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP‐AM‐induced PASMC proliferation and [Ca2+] i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP‐AM‐ induced [Ca2+] i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.

ATT has positive effects on pulmonary hypertension hallmarks

Pulmonary hypertension (PH) is a multifactorial disease characterized by pulmonary arteries (PA) hyperreactivity and remodelling involving an imbalance of contractile and proliferative agents such as 5-hydroxytryptamine (5-HT). Mitochondria and associated reactive oxygen species (ROS) production were shown to contribute to these phenomena. OP2 Drugs recently demonstrated that Anethole Trithione (ATT), the active ingredient of its principal lead OP2113, specifically inhibits mitochondrial ROS (mtROS) production from complex I in its reverse mode. In this context, this study aims to assess the effects of ATT on PH hallmarks. Male rats were either exposed to normoxia or chronic hypoxia (10% O2) during 3 weeks to induce PH (CH-PH). The effect of ATT on PA reactivity was determined ex vivo by isometric tension measurements of arterial rings. PA contraction was induced by 5-HT, prostaglandin F2α (PGF2α), phenylephrine (Phe) or endothelin-1 (ET-1) whereas relaxation was induced by carbachol (Cch) on preconstricted vessels with PGF2α. The effect of ATT on calcium responses induced by 5-HT was assessed in rat PA smooth muscle cells using confocal imaging. Proliferative responses induced by 5-HT were evaluated by ELISA BrdU on human PASMC in presence or absence of ATT. In rats with CH-PH, ATT reduced contractile hyperreactivity induced by 5-HT and PGF2α. In control rats, ATT treatment also decreased contraction to 5-HT and Phe, but not contraction to ET-1 and PGF2α as well as relaxation to Cch. Interestingly, acute ATT exposure did not modify basal cytosolic calcium signal in control rat PASMC. However, the number of 5-HT responding cells was reduced in presence of ATT whereas neither maximal peak of cytosolic calcium responses nor the global calcium variations were affected. Finally, 5-HT-induced proliferation was reduced by ATT in control human PASMC. In conclusion, ATT improves vascular hyperreactivity of PA from rats with CH-PH whereas in control condition ATT altered 5-HT-induced contraction in rat PA, calcium signalling to 5-HT in rat PASMC and 5-HT-induced human PASMC proliferation. Although mechanisms underlying ATT influence on PH hallmarks need to be further elucidated, these Results suggest that OP2113 may be used to develop new PH therapies.

The Influence of Bosentan on MicroRNA-27a/PPARγ/ET-1 Signaling Pathway in Pulmonary Artery Hypertension.

Pulmonary artery hypertension (PAH) is a common and serious disease which is characterized by pulmonary vascular remodeling. Bosentan (BST) is the first approved oral targeted drug of endothelin-1 (ET-1) receptor antagonists for the treatment of PAH. MicroRNA-27a (miR-27a) and peroxisome proliferator-activated receptor γ (PPARγ) were found to be related to the pathogenesis of PAH. To further explore the signal transduction mechanism of BST in the treatment of PAH, we examined the effects of BST on endothelin receptors, miR-27a, and PPARγ. Meanwhile, the influence of miR-27a in the formation and development of PAH was discussed. Our results demonstrated that during the pathophysiology of PAH, miR-27a, PPARγ, and ET-1 were cross-inhibited, which indicated that the miR-27a/PPARγ/ET-1 signaling pathway was dysregulated; in addition, BST could competitively bind to ET-1 receptors and inhibit the miR-27a/PPARγ/ET-1 signaling pathway, thereby delaying the proliferation of PASMCs and affecting the development of PAH. Our results give a new understanding of the pathogenesis and treatment of PAH and provide more reliable evidence for the application of BST in the treatment of PAH in the clinic.

The m6A methyltransferase METTL3 promotes hypoxic pulmonary arterial hypertension

AimsN6-methyladenosine (m6A) is the most prevalent internal chemical RNA modification in mammal mRNAs. Accumulating evidence has shown the critical role of m6A in cardiovascular diseases including cardiac hypertrophy, heart failure, ischemic heart disease, vascular calcification, restenosis, and aortic aneurysm. However, whether m6A participates in the occurrence and development of hypoxic pulmonary hypertension (HPH) remains largely unknown. The present study aims to explore the role of key transferase METTL3, in the development of HPH. Materials and methodsPulmonary artery smooth muscle cells (PASMCs) and hypoxic rat models were used to research the METTL3-mediated m6A in HPH. EdU, transwell and TUNEL were performed to evaluate the proliferation, migration and apoptosis rates. m6A RNA Methylation Quantification Kit and m6A-qPCR were utilized to measure the total m6A level and m6A level of PTEN mRNA. RNA immunoprecipitation was used to detect the interaction between METTL3 and PTEN mRNA. Key findingsBoth METTL3 mRNA and protein were found abnormally upregulated in vivo and in vitro. Furthermore, downregulation of METTL3 attenuated PASMCs proliferation and migration. In addition, m6A binding protein YTHDF2 was found significantly increased in PASMCs under hypoxia. YTHDF2 recognized METTL3 mediated m6A modified PTEN mRNA and promoted the degradation of PTEN. Decreased PTEN led to over-proliferation of PASMCs through activation of PI3K/Akt signaling pathway. SignificanceMETTL3/YTHDF2/PTEN axis exerts a significant role in hypoxia induced PASMCs proliferation, providing a novel therapeutic target for HPH.

Galectin-3 participates in PASMC migration and proliferation by interacting with TGF-β1

Pulmonary vascular remodelling is one of the most important factors for pulmonary hypertension (PH). Galectin-3 (Gal-3) is a β-galactoside-binding lectin. In the latest literature, Gal-3 has been reported to be involved in pulmonary vascular remodelling, and its underlying mechanism is unclear. Our research aims to prove the effect of Gal-3 on the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMC) induced by transforming growth factor β1 (TGF-β1) and to study its mechanism. In vivo experiment: In Sprague-Dawley (SD) rats, monocrotaline was injected intraperitoneally to establish a PH model, and the Gal-3 inhibitor (modified citrus pectin, MCP) 28 Ds was administered in the stomach. The results indicate that Gal-3 and TGF-β1 may be involved in the occurrence and development of PH, which may be related to the Smad2/3 signalling pathway. In vitro experiment: Human pulmonary artery smooth muscle cells were pretreated with the Gal-3 inhibitor (MCP) for 24 h, then TGF-β1 or Gal-3 was administered to the cells for 24 h. The results show that exogenous TGF-β1 and Gal-3 can activate the downstream Smad2/3 signalling pathway, and increase the proliferation and migration ability of HPASMC. However, the Gal-3 inhibitor (MCP) inhibited these effects. Further results display that TGF-β1 and Gal-3 could mutually regulate the protein and mRNA expression levels. In summary, the results of this study indicate that Gal-3 regulates the Smad2/3 signalling pathway through protein interaction with TGF-β1, in turn regulates the proliferation and migration of HPASMC, thereby regulating the occurrence and development of PH.

Substantial involvement of TRPM7 inhibition in the therapeutic effect of Ophiocordyceps sinensis on pulmonary hypertension

Ophiocordyceps sinensis (OCS), an entomopathogenic fungus, is known to exert antiproliferative and antitissue remodeling effects. Vascular remodeling and vasoconstriction play critical roles in the development of pulmonary hypertension (PH). The therapeutic potential of OCS for PH was investigated using rodent PH models, and cultured pulmonary artery endothelial and smooth muscle cells (PAECs and PASMCs), with a focus on the involvement of TRPM7. OCS ameliorated the development of PH, right ventricular hypertrophy and dysfunction in the monocrotaline-induced PH rats. The genetic knockout of TRPM7 attenuated the development of PH in mice with monocrotaline pyrrole-induced PH. TRPM7 was associated with medial hypertrophy and the plexiform lesions in rats and humans with PH. OCS suppressed proliferation of PASMCs derived from the PH patients. Ethanol extracts of OCS inhibited TRPM7-like current, TGF-β2-induced endothelial-mesenchymal transition, IL-6-induced STAT3 phosphorylation, and PDGF-induced Akt phosphorylation in PAECs or PASMCs. These inhibitory effects were recapitulated by either siRNA-mediated TRPM7 knockdown or treatment with TRPM7 antagonist FTY-720. OCS and FTY-720 induced vasorelaxation in the isolated normal human pulmonary artery. As a result, the present study proposes the therapeutic potential of OCS for the treatment of PH. The inhibition of TRPM7 is suggested to underlie the therapeutic effect of OCS.

Hypoxia-induced mitochondrial reactive oxygen species (mtROS) differentially regulates smooth muscle cell (SMC) proliferation of pulmonary and systemic vasculature

BackgroundVascular remodeling plays a pivotal role in regulation of hypoxia-mediated pulmonary and systemic hypertension via the phenotypic modulation of smooth muscle cells (SMCs) of pulmonary and systemic arteries, respectively. Mitochondria serve as putative oxygen (O2) sensors, and consequently, adaptations to hypoxia are mediated via HIF (hypoxia-inducible factors) activation, which impinges on mitochondrial function by suppressing the mitochondrial activity. Therefore, we explored the implication of hypoxia-mediated mitochondrial stress in pulmonary and systemic arterial remodeling. MethodsThe hypoxic (10% O2) effect on human pulmonary artery and aortic SMCs was examined in vitro by cell viability assay, proliferation index, autophagy, and comet assays. Mitochondrial ROS (mtROS), membrane potential (MMP), and mitochondrial morphology were assessed using mitochondrial-selective fluorescent probes. Further, the cell cycle distribution was analyzed by flow cytometry using propidium iodide staining. ResultsOur data indicate no significant alterations in cell viability and active proliferation of hypoxic PASMCs; however, an excessive rise in mtROS production and disrupted MMP, accompanied by enhanced DNA damage and reduced autophagy was observed, highlighting the ‘apoptosis resistance’ phenotype in these cells. Conversely, in hypoxia-treated hASMCs, a modest rise in mtROS levels was associated with reduced DNA damage; followed by upregulated autophagy; increased S–phase DNA content and cell viability, depicting the cytoprotective effect of hypoxia-induced autophagy against mitochondrial damage in hASMCs. ConclusionOur findings suggest that differential impact of mtROS on proliferative capacity may contribute to the variable hypoxic responses in pulmonary and systemic vasculature. Therefore, targeting mtROS may serve as an effective therapeutic strategy to prevent hypoxia-induced hypertension.

Abstract 15259: Breast Cancer and Heritable Pah Due to Bmpr2 Mutation: An Unexpected Discovery

Introduction: BMPR2 mutation is reported in 80% of familial PAH and 20% of idiopathic cases. Recently, a rat model of hereditary Pulmonary Arterial Hypertension with a BMPR2 loss-of-function mutation has been generated. Similarly to human, only around 20% of the rats spontaneously developed mild PAH. Unexpectedly, we observed that 20% (95%CI:12-28) of the 82 Bmpr2+/Δ71 rats (93% of females) versus 4% (95%CI:0-9)of the 84 age-matched wild type littermates spontaneously developed mammary masses requiring euthanasia. Interestingly, rats with tumor had elevated PA and clearly exhibit PAH signs. Hypothesis: We hypothesized that mammary tumor exacerbates the penetrance of PAH in the BMPR2 mutated female rats. Methods/Results: BMPR2 mutated and wild-type female young rats were treated with the breast cancer inducer 7, 12- diméthylbenz(a)anthracene (DMBA) or Vehicle. DMBA causes on average more tumors development in BMPR2 mutated rats versus WT rats (6,2 vs. 4) P=0,0275. Compare to both vehicle-treated and DMBA rats (without tumor) BMPR2 mutated rats with DMBA-induced tumors had a significant increase in mean PA pressure (p<0,0001); total pulmonary resistance (p<0,0001) and a significant decrease in cardiac output (P=0,0293). Histologically, these changes were associated with an increase of pulmonary artery remodelling assessed by immunofluorescence. Mechanistically, development of PAH in the BMPR2 mutated rats with tumor was associated with a significant upregulation of BRD4 (western blot) (p=0,0149); IL-1β (P=0,0052), BRD3 (P=0,0791) and BIRC5 (P=0,0166) mRNA expression levels. Co-culture experiments with breast cancer cells and PASMC from both WT and BMPR2 mutated rats are ongoing to identify whether cancer cells are realising factors responsible for BRD4 activation and thus PASMC proliferation and resistance to apoptosis. Conclusions: We demonstrated for the first time that breast cancer exacerbates PAH development in BMPR2 mutated rats. Increased BRDs driven pro-inflammatory signalling is associated with PAH development in BMPR2 mutated rats. We are currently exploring whether BMPR2 mutated patients are more prone to breast cancer development or not and whether PAH development occurs more frequently in breast cancer patients.

Role of smooth muscle Rac1 in the vascular remodeling associated with pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signaling play a major role in PAH. We previously described the major role of smooth muscle cell (SMC) Rac1 in NO-induced dilation of systemic arteries. Here, our aim was to analyze the role of SMC Rac1 in pulmonary circulation and in PAH. Rac1 activity was assessed by immunofluorescence in lung biopsies. PAH has been induced by exposure of wild-type (WT) and SMC Rac1-deficient mice (SM-Rac1-KO mice) to chronic hypoxia (10% O2, 4 weeks). In vitro, the proliferation of PASMC in hypoxic condition was measured by EdU staining and contractility of PA rings was analyzed by myography. Oxidative stress was assessed using the DHE probe and by electron paramagnetic resonance (EPR). A 3-fold increase of Rac-GTP in PA was observed in PAH patients compared to control ( n = 4-6). To determine whether Rac1 activation plays a causal role in PAH, WT and SM-Rac1-KO mice were exposed to hypoxia. As expected, exposure to chronic hypoxia induced an increase in systolic pressure in RV and RV hypertrophy which were reduced by 32.5% and 60% respectively, in SM-Rac1-KO mice compared to WT ( n = 5–7). PA remodeling was also significantly reduced in hypoxic SM-Rac1-KO compared to WT mice, while PA contractility was not affected by SMC Rac1 deletion. In vitro, exposure of primary PASMC to hypoxia stimulated proliferation and ROS production that were decreased by 50% and 66%, respectively, by the Rac1 inhibitor, EHT1864. Our results show that SMC Rac1 activity is increased in PA of PAH patients and plays a causal role in hypoxia-induced PAH in mice by stimulating ROS-dependent PA remodeling. These data suggest that pharmacological Rac1 inhibition might have a beneficial effect in the treatment of PAH.

Calcium Homeostasis Modulator (CALHM1/2) and Pulmonary Arterial Hypertension

RATIONALEConcentric pulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH). Excessive PASMC proliferation and inhibited PASMC apoptosis have been implicated in the development and progression of pulmonary vascular wall thickening in PAH. Transition from the contractile phenotype to the synthetic or proliferative phenotype of PASMC is thus an important pathogenic process that promotes vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is not only a trigger for PASMC contraction (and pulmonary vasoconstriction), but also an important stimulus for PASMC proliferation. Calcium homeostasis modulators (CALHM), CALHM1/2, are membrane Ca2+ channels that are allosterically regulated by voltage and extracellular Ca2+. CALHM1/2 are closed at the resting membrane potential but can be opened by strong membrane depolarization. Reduction of extracellular [Ca2+] increases CALHM open probability, which allows the channel to be activated at negative potential. We aimed to study whether CALHM1/2 are involved in the phenotypical transition of PASMC and whether CALHM1/2 are upregulated in highly proliferative PASMC from patients with PAH.METHODSFreshly isolated pulmonary artery with the endothelium removed (as contractile PASMC) and primary cultured PASMC (as synthetic or proliferative PASMC) from rats were used for this study. Western blot analysis was used to compare protein expression levels of CALHM1 and CALHM2 in fresh PA tissue and cultured PASMC derived from the same rats. In addition, we compared protein expression levels of CALHM1/2 in PASMC from normal subjects and PAH patients.RESULTSThe protein expression level of CALHM2 in proliferative PASMC (the primary cultured PASMC in growth factor‐containing medium) was significantly higher than in contractile PASMC (the freshly isolated PA tissue). The increased CALHM2 in proliferative PASMC was indeed associated with a significantly increased expression of PCNA, a cell proliferation marker, and a significantly decreased expression of MHC and TGLN4, markers for contractile phenotype of SMC. Furthermore, we also observed that mRNA expression level of CALHM2 was significantly higher in PASMC isolated from PAH patients than in PASMC from normal subjects.CONCLUSIONUpregulation of CALHM2 may play an important role in the phenotypical transition of PASMC from the contractile phenotype to the synthetic or proliferative phenotype. Ca2+ influx through voltage‐ and extracellular Ca2+‐regulated CALHM2 in the plasma membrane of PASMC is an important trigger for PASMC proliferation and may play an important role in stimulating PASMC proliferation in patients with PAH.Support or Funding InformationNIH

A20 attenuates hypoxia-induced pulmonary arterial hypertension by inhibiting NF-κB activation and pulmonary artery smooth muscle cell proliferation

PAH is a progressive disease characterized by uncontrolled proliferation of PASMCs. Zinc finger protein A20 is a negative feedback regulatory protein of NF-κB activity. The aim of this study was to evaluate zinc finger protein A20 can alleviate PAH in hypoxia exposed mice. C57BL/6 mice received a tail vein injection of adenovirus-mediated ad-A20 and ad-A20 shRNA were exposed to hypoxia. PASMCs isolated from rat pulmonary arteries were cultured in hypoxia, and were transfection of A20 adenovirus. Pulmonary hemodynamic parameters were measured by right heart catheterization. Pulmonary vascular morphological changes were analyzed by HE and α-SMA staining. The expression changes of A20, NF-κB and its downstream protein were detected. The expression of phospho-p65 was increased with the prolongation of hypoxia time. The expression of A20 in lung tissue of chronic hypoxia group decreased with the prolongation of hypoxia time. Adenovirus-mediated A20 (ad-A20) overexpression significantly attenuated the abnormally increased RVSP, RV/(LV + S) ratio, WT%, WA%, α-SMA and the pulmonary vessel muscularization. Ad-A20 treatment markedly attenuated the degradation of phospho-p65 and inhibited the induction of phospho-IκBα induced by hypoxia treatment. Furthermore, silencing A20 abolished the protection by anti-inflammatory activity and the inhibitory effect on cell proliferation. We showed that Zinc finger protein A20 can block NF-κB signaling pathway, alleviates the hypoxia-induced abnormal elevation of pulmonary arterial pressure, hyperproliferation of PASMCs and the pulmonary vascular remodeling.

Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

ObjectivesHypoxia is an important risk factor for pulmonary arterial remodelling in pulmonary arterial hypertension (PAH), and the Janus kinase 2 (JAK2) is believed to be involved in this process. In the present report, we aimed to investigate the role of JAK2 in vascular smooth muscle cells during the course of PAH.MethodsSmooth muscle cell (SMC)‐specific Jak2 deficient mice and their littermate controls were subjected to normobaric normoxic or hypoxic (10% O2) challenges for 28 days to monitor the development of PAH, respectively. To further elucidate the potential mechanisms whereby JAK2 influences pulmonary vascular remodelling, a selective JAK2 inhibitor was applied to pre‐treat human pulmonary arterial smooth muscle cells (HPASMCs) for 1 hour followed by 24‐hour hypoxic exposure.ResultsMice with hypoxia‐induced PAH were characterized by the altered JAK2/STAT3 activity in pulmonary artery smooth muscle cells. Therefore, induction of Jak2 deficiency in SMCs protected mice from hypoxia‐induced increase of right ventricular systolic pressure (RVSP), right ventricular hypertrophy and pulmonary vascular remodelling. Particularly, loss of Jak2 significantly attenuated chronic hypoxia‐induced PASMC proliferation in the lungs. Similarly, blockade of JAK2 by its inhibitor, TG‐101348, suppressed hypoxia‐induced human PASMC proliferation. Upon hypoxia‐induced activation, JAK2 phosphorylated signal transducer and activator of transcription 3 (STAT3), which then bound to the CCNA2 promoter to transcribe cyclin A2 expression, thereby promoting PASMC proliferation.ConclusionsOur studies support that JAK2 could be a culprit contributing to the pulmonary vascular remodelling, and therefore, it could be a viable target for prevention and treatment of PAH in clinical settings.

P4146The six-transmembrane protein Stamp2 protects from hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension via actions in mononuclear cells and CXCL12

Abstract Background The six-transmembrane protein of prostate (Stamp2) is a potent anti-inflammatory player in adipocytes and also in macrophages. Stamp2's actions in these cells protects from diet-induced diabetes and from atherosclerosis mice. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PH), we sought to investigate the role of Stamp2 in PH. Methods and results Morphometric analyses of small pulmonary arteries after 3 weeks of chronic hypoxia (10% O2) showed aggravated pulmonary vascular remodeling in Stamp2−/− mice as compared to WT, demonstrated by a significantly reduced number of non-muscularized vessels and higher extent of fully-muscularized vessels. Consequently, right ventricular systolic pressure (RVSP, Millar catheter via right jugular vein) was significantly higher in Stamp2−/− mice (33.4±0.7 mmHg vs. 30.3±1.4, p<0.05). As endothelial (EC) and smooth muscle cells (PASMC) are critical for remodeling processes in PH, the role of Stamp2 in these cells was explored. However, siRNA-mediated knock-down of Stamp2 in human microvascular EC had no effect on apoptotic susceptibility (CellDeath Detection ELISA), or release of IL-6 (qPCR). Furthermore, Stamp2-deficiency in isolated primary PASMC had no effect on proliferation (BrdU incorporation) and chemotaxis (modified Boyden chamber). As Stamp2 deficiency promotes higher expression of inflammatory cytokines (IL6, IL1b, MCP1, TNFa, CXCL12, qPCR) and increased numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages are potentially driving vascular remodeling in PH. To test this hypothesis, PASMC proliferation and chemotaxis were assessed in response to treatment with supernatants from primary thioglycolate-elicited peritoneal Stamp2−/− or WT-macrophages. These experiments revealed that supernatants from Stamp2−/− macrophages induced PASMC proliferation and chemotaxis significantly stronger, thus providing a link between inflammatory actions in Stamp2 deficiency and vascular remodeling. To gain further insights, a cytokine array was performed with supernatants from Stamp2−/− and WT-macrophages, revealing CXCL12 as the most relevant candidate. Experiments with neutralizing antibodies confirmed the role of CXCL12 in driving Stamp2's actions on vascular remodelling processes in PASMC. Importantly, Stamp2 expression (qPCR, western blot analyses) was significantly lower in the lung of humans with idiopathic PAH (IPAH), as well as in experimental PH in rats (monocrotalin, sugen/hypoxia) and in mice (hypoxia). Conclusions Stamp2 deficiency aggravates hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension in mice. On the cellular level, actions of Stamp2 in macrophages drive vascular remodelling processes in smooth muscle cells via secreted factors such as CXCL12. The finding of decreased expression of Stamp2 in human and various experimental forms of PH points towards a general protective role of Stamp2.

Apigenin Attenuates Pulmonary Hypertension by Inducing PASMCs Mitochondria-Dependent Apoptosis Via Inhibiting Hypoxia Inducible Factor 1α-KV1.5 Channel Pathway

Background: To assess the therapeutic effect of apigenin on pulmonary hypertension (PH) and explore the underlying mechanisms of apigenin in PASMCs proliferation and apoptosis. Methods: Hypoxic PH model was established in Sprague-Dawley rats and administration with apigenin. RV systolic pressure (RVSP), RV weight (RV/LV+S) %, mean systolic blood pressure were measured and pulmonary vascular remolding calculated. PASMCs apoptosis and proliferation were respectively detected by TUNEL and PCNA immunohistochemistry. Immunoblot analysis, mitochondrial membrane potential (Δψm) were detected in vivo and in vitro. Also, KV1.5 inhibitor diphenyl phosphine oxide-1(DPO-1) and HIF-1α overexpression lentiviral vector were used to search functional studies. Findings: Administration of apigenin prevented the development of PH, hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodeling, and also prevented progression of established PH. Meanwhile, apigenin exhibits an inducing effects of mitochondria-dependent apoptosis on hypoxic PASMCs. In addition, apigenin could reverse the KV1.5 expression which inhibited by hypoxia both in vivo and in vitro. However, KV1.5 inhibitor, diphenyl phosphine oxide-1(DPO-1), can offset apigenin-induced mitochondria-dependent apoptosis. Moreover, functional studies revealed that apigenin activated mitochondria-dependent apoptosis by modulation of hypoxia-induced factor 1α (HIF-1α) signaling. Interpretation: Our studies revealed that apigenin attenuates PH via inhibiting HIF-1α-KV1.5 channel pathway to induce PASMCs mitochondria-dependent apoptosis. Funding: The National Natural Science Foundation of China (No. 81700051, 81670048, and 81700052), the National Key Technology R&D Program of the 12th National Five-year Development Plan (No. 2012BAI05B01), and the National Key Research and Development Pro-grams of China (No. 2016YFC1304500, 2016-YFC0903600). Declaration of Interest: The authors declare that they have no conflicts of interest concerning this article. Ethical Approval: All of the rats studies were approved by the Huazhong University of Science and Technology Committee and the Tongji Medical College Ethics Committee at Tongji Hospital and were performed to conform to NIH guidelines (Guide for the Care and Use of Laboratory Animals).

Reactive Oxygen Species-Dependent Calpain Activation Contributes to Airway and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease.

Aims: Airway and pulmonary vascular remodeling is an important pathological feature in the pathogenesis of chronic obstructive pulmonary disease (COPD). Tobacco smoke (TS) induces the production of large amounts of reactive oxygen species (ROS) in COPD lungs. We investigated how ROS lead to airway and pulmonary vascular remodeling in COPD. Results: We used in vitro bronchial and pulmonary artery smooth muscle cells (BSMCs and PASMCs), in vivo TS-induced COPD rodent models, and lung tissues of COPD patients. We found that H2O2 and TS extract (TSE) induced calpain activation in BSMCs and PASMCs. Calpain activation was elevated in smooth muscle of bronchi and pulmonary arterioles in COPD patients and TS-induced COPD rodent models. Calpain inhibition attenuated H2O2- and TSE-induced collagen synthesis and proliferation of BSMCs and PASMCs. Exposure to TS causes increases in airway resistance, right ventricular systolic pressure (RVSP), and thickening of bronchi and pulmonary arteries. Calpain inhibition by smooth muscle-specific knockout of calpain and the calpain inhibitor MDL28170 attenuated increases in airway resistance, RVSP, and thickening of bronchi and pulmonary arteries. Moreover, smooth muscle-specific knockout of calpain did not reduce TS-induced emphysema in the mouse model, but MDL28170 did reduce TS-induced emphysema in the rat model. Innovation: This study provides the first evidence that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling in TS-induced COPD. Calpain might be a novel therapeutic target for the treatment of COPD. Conclusion: These results indicate that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling and pulmonary hypertension in COPD.

LRP1 Deficiency in Vascular SMC Leads to Pulmonary Arterial Hypertension That Is Reversed by PPARγ Activation.

Arterial remodeling-a hallmark of many cardiovascular pathologies including pulmonary arterial hypertension (PAH)-is regulated by TGFβ1 (transforming growth factor-β1)-TGFβ receptors and the antagonistic, vasoprotective BMPR2 (bone morphogenetic protein receptor 2)-PPARγ (peroxisome proliferator-activated receptor-γ) axis. However, it is unclear which factors drive detrimental TGFβ1 pathways in the hypertensive pulmonary vasculature. We hypothesized that LRP1 (low-density lipoprotein receptor-related protein 1) expression is decreased in PAH, leading to enhancement (disinhibition) of TGFβ1 signals and that the PPARγ agonist pioglitazone can restore vascular homeostasis and prevent PAH resulting from LRP1 deletion in vascular smooth muscle cells (SMCs). Targeted deletion of LRP1 in vascular SMC (smLRP1-/-) in mice disinhibited TGFβ1-CTGF (connective tissue growth factor) signaling, leading to spontaneous PAH and distal pulmonary arterial muscularization as assessed by closed-chest cardiac catheterization and anti-αSMA staining. Pioglitazone inhibited the canonical TGFβ1-CTGF axis in human pulmonary artery SMC and smLRP1-/- main pulmonary artery (CTGF and NOX4) and reversed PAH in smLRP1-/- mice. TGFβ1 boosted pSmad3 in PASMC from smLRP1-/- mice versus controls. Pioglitazone-activated PPARγ binds to Smad3 in human pulmonary artery SMC (coimmunoprecipitation), thereby blocking its phosphorylation and overriding LRP1 deficiency. Finally, mRNA and protein expression of LRP1 was decreased in pulmonary plexiform lesions of patients with end-stage idiopathic PAH (laser capture microdissection, qPCR, and immunohistochemistry). Downregulation of LRP1 protein was also demonstrated in explanted PASMC from patients with PAH and accompanied by enhanced TGFβ1-pSmad3-CTGF signaling and increased TGFβ1-induced PASMC proliferation that was prevented by pioglitazone. Here, we identify LRP1 as an integrator of TGFβ1-mediated mechanisms that regulate vascular remodeling in mice and clinical PAH and PPARγ as a therapeutic target that controls canonical TGFβ1 pathways. Hence, pharmacologic PPARγ activation represents a promising new therapy for patients with PAH who lack the vasoprotective LRP1 in vascular SMC.