Pulmonary Arterial Vascular Smooth Muscle Cells Research Articles

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development.

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells (PASMCs), endothelial dysfunction, and extracellular matrix remodeling. G protein-coupled receptor kinase 2 (GRK2) plays an important role in the maintenance of vascular tone and blood flow. However, the role of GRK2 in the pathogenesis of PAH is unknown. GRK2 levels were detected in lung tissues from healthy people and PAH patients. C57BL/6 mice, vascular smooth muscle cell-specific Grk2 -knockout mice ( Grk2ΔSM22 ), and littermate controls ( Grk2flox/flox ) were grouped into control and hypoxia mice ( n =8). Pulmonary hypertension (PH) was induced by exposure to chronic hypoxia (10%) combined with injection of the SU5416 (cHx/SU). The expression levels of GRK2 and Yes-associated protein (YAP) in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining. The mRNA expression levels of Grk2 and Yes-associated protein ( YAP ) in PASMCs were quantified with real-time polymerase chain reaction (RT-PCR). Wound-healing assay, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and 5-Ethynyl-2'-deoxyuridine (EdU) staining were performed to evaluate the proliferation and migration of PASMCs. Meanwhile, the interaction among proteins was detected by immunoprecipitation assays. The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice. Moreover, cHx/SU-induced PH was attenuated in Grk2ΔSM22 mice compared with littermate controls. The amelioration of PH in Grk2ΔSM22 mice was accompanied by reduced pulmonary vascular remodeling. In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration, whereas this effect was severely intensified by overexpression of GRK2 . We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs, resulting in excessive PASMCs proliferation and migration. Furthermore, GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions. Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH, via regulating YAP expression and nuclear translocation. Therefore, GRK2 serves as a novel therapeutic target for PAH treatment.

Crosstalk between BMP signaling and KCNK3 in phenotypic switching of pulmonary vascular smooth muscle cells

Pulmonary arterial hypertension (PAH) is a progressive and devastating disease whose pathogenesis is associated with a phenotypic switch of pulmonary arterial vascular smooth muscle cells (PASMCs). Bone morphogenetic protein (BMP) signaling and potassium two pore domain channel subfamily K member 3 (KCNK3) play crucial roles in PAH pathogenesis. However, the relationship between BMP signaling and KCNK3 expression in the PASMC phenotypic switching process has not been studied. In this study, we explored the effect of BMPs on KCNK3 expression and the role of KCNK3 in the BMP-mediated PASMC phenotypic switch. Expression levels of BMP receptor 2 (BMPR2) and KCNK3 were downregulated in PASMCs of rats with PAH compared to those in normal controls, implying a possible association between BMP/BMPR2 signaling and KCNK3 expression in the pulmonary vasculature. Treatment with BMP2, BMP4, and BMP7 significantly increased KCNK3 expression in primary human PASMCs (HPASMCs). BMPR2 knockdown and treatment with Smad1/5 signaling inhibitor substantially abrogated the BMP-induced increase in KCNK3 expression, suggesting that KCNK3 expression in HPASMCs is regulated by the canonical BMP-BMPR2-Smad1/5 signaling pathway. Furthermore, KCNK3 knockdown and treatment with a KCNK3 channel blocker completely blocked BMP-mediated anti-proliferation and expression of contractile marker genes in HPAMSCs, suggesting that the expression and functional activity of KCNK3 are required for BMP-mediated acquisition of the quiescent PASMC phenotype. Overall, our findings show a crosstalk between BMP signaling and KCNK3 in regulating the PASMC phenotype, wherein BMPs upregulate KCNK3 expression and KCNK3 then mediates BMP-induced phenotypic switching of PASMCs. Our results indicate that the dysfunction and/or downregulation of BMPR2 and KCNK3 observed in PAH work together to induce aberrant changes in the PASMC phenotype, providing insights into the complex molecular pathogenesis of PAH. [BMB Reports 2022; 55(11): 565-570].

Akt-Dependent Glycolysis-Driven Lipogenesis Supports Proliferation and Survival of Human Pulmonary Arterial Smooth Muscle Cells in Pulmonary Hypertension.

Hyper-proliferation of pulmonary arterial vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Lipogenesis is linked to numerous proliferative diseases, but its role in PAVSMC proliferation in PAH remains to be elucidated. We found that early-passage human PAH PAVSMC had significant up-regulation of key fatty acids synthesis enzymes ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN), and increased unstimulated proliferation compared to control human PAVSMC. Treatment with an allosteric ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) significantly decreased proliferation and induced apoptosis of human PAH PAVSMC. Intracellular lipid content and proliferation of PAH PAVSMC were not reduced by incubation in lipid-depleted media but suppressed by a non-metabolizable analog of glucose 2-Deoxy-D-glucose (2-DG) and partially restored by addition of pyruvate. Protein kinase Akt was upregulated in human PAH PAVSMC in a sirtuin 7 (SIRT7)- and c-Jun N-terminal kinase (JNK)-dependent manner. Pharmacological inhibition of Akt down-regulated ACLY and ACC, significantly reduced intracellular lipid content, inhibited proliferation and induced apoptosis of human PAH PAVSMC. Taken together, these data demonstrate that human PAH PAVSMC have up-regulated lipogenesis, which is supported in an Akt- and glycolysis-dependent manner and is required for increased proliferation and survival. Our data suggest that there is a mechanistic link between glycolysis, lipogenesis, and the proliferation of human PAH PAVSMC and call for further studies to determine the potential attractiveness of a SIRT7/JNK-Akt-lipogenesis axis as a target pathway to inhibit PAVSMC hyper-proliferation in PAH.

Expression of the microRNA‑30 family in pulmonary arterial hypertension and the role of microRNA‑30d‑5p in the regulation of pulmonary arterial smooth muscle cell toxicity and apoptosis

The biological processes of pulmonary artery vascular smooth muscle cells (PA-SMCs) and pulmonary artery endothelial cells in pulmonary arterial hypertension (PAH) are generally abnormal, with increased levels of proliferation and reduced levels of apoptosis. Although microRNAs (miRNAs/miRs) participate in a number of biological processes in a variety of diseases, such as tumors and infections, studies on the association between miRNAs and PAH are limited. In the present study, blood samples were collected from 6 patients with patent ductus arteriosus. The experimental group included 3 patients with severe PAH, while the control group included 3 patients without PAH. Microarray technology was used to detect the presence of any associated miRNAs. Moreover, a rat PAH model was established via left lung resection followed by monocrotaline injection, involving a total of 8 rats in the PAH group and 8 untreated rat in the control group. Reverse transcription-quantitative PCR was performed to verify the expression levels of the miR-30 family in the animal model. miR-30d-5p mimics and anti-miR-30d-5p were transfected into primary cultured PA-SMCs. Levels of cytotoxicity and cell apoptosis were examined, and Notch-3 expression levels were studied using western blotting. The results of the present study demonstrated that miR-30d-5p expression was downregulated in both patient blood and animal models of the PAH group compared with control groups. In primary cultured PA-SMCs, overexpression of miR-30d-5p attenuated the platelet-derived growth factor-induced toxicity of PA-SMCs, while knockdown of miR-30d-5p resulted in the increased toxicity of PA-SMCs compared with control group. The apoptosis rate of PA-SMCs increased with the overexpression of miR-30d-5p compared with control group. Moreover, the expression levels of Notch-3 in the miR-30d-5p group were significantly reduced compared with the anti-miR-30d-5p and miR-NC groups. In total, 10 circulating miRNAs that may be associated with PAH were discovered in the present study. Moreover, the expression of the miR-30 family was verified in animal models in vivo, and seven miRNAs in this family were discovered that may be associated with PAH. Additionally, miR-30d-5p was downregulated in both patients with PAH and animal models compared with control groups. Thus, the results of the present study demonstrated that the regulatory mechanism underlying PA-SMCs may be via the Notch-3 signaling pathway.

Abstract 12968: mitoTEMPO Improves Expression of Pulmonary Artery Soluble Guanylate Cyclase β1 and Lowers Exercise Associated Elevations in Pulmonary Artery Pressures in a Rat Model of Combined Pre- and Post-Capillary Pulmonary Hypertension

Introduction: Exercise induced pulmonary hypertension (EIPH) portends worse prognosis compared to isolated resting PH. Exercise increases mitochondrial reactive oxygen species (mROS) production in peripheral blood mononuclear cells of patients with heart failure. Metabolic syndrome related mROS contribute to EIPH by decreasing the expression of the vasodilatory enzyme soluble guanylate cyclase (sGC) via downregulating an sGC transcription factor, namely nuclear factor Y alpha (NFYA; Satoh et al in press). Hypothesis: mROS scavenging with mitoTEMPO will restore sGC expression and ameliorate EIPH in a rat model of combined pre-and post-capillary pulmonary hypertension (CpcPH). Methods: Obese ZSF1 rats treated with sugen were used to model CpcPH. Half received mitoTEMPO (2mM) for 2 weeks. CpcPH rats treated with vehicle and lean ZSF1 rats served as controls (n=6/group). Hemodynamics were evaluated at rest and during treadmill exercise. Cardiac function was assessed using Doppler-echocardiography. We cultured pulmonary artery vascular smooth muscle cells (PAVSMCs) from lean rats in palmitate, glucose and insulin (PGI) containing media to simulate the metabolic syndrome milieu. Cells were treated with vehicle or mitoTEMPO. Expression of sGCβ1 and NFYA in pulmonary arteries of animals and in cultured cells was quantified by Western Blot. Data were analyzed with repeated measures two-way ANOVA. Results: MitoTEMPO significantly improved EIPH by reducing pulmonary vascular resistance (fig1A), significantly improved right ventricular function (fig1B) and significantly restored NFYA/sGCβ1 expression in pulmonary arteries (fig1C) and PAVSMCs of rats with CpcPH (fig1D). MitoTEMPO significantly blunted PGI-induced downregulation of NFYA and sGCβ1 in PAVSMCs from lean rats (fig1E). Conclusions: Mitochondria targeted antioxidants can improve EIPH in CpcPH by restoring NFYA/sGCβ1 expression, identifying a potential therapeutic target for CpcPH.

Bioactive Compounds From Coptidis Rhizoma Alleviate Pulmonary Arterial Hypertension by Inhibiting Pulmonary Artery Smooth Muscle Cells' Proliferation and Migration.

Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by excessive proliferation and vasoconstriction of small pulmonary artery vascular smooth muscle cells (PASMCs). Coptidis rhizoma (CR) because of the complexity of the components, the underlying pharmacological role and mechanism of it on PAH remains unknown. In this article, the network pharmacological analysis was used to screen the main active constituents of CR and the molecular targets that these constituents act on. Then, we evaluated the importance of berberine and quercetin (biologically active components of CR) on the proliferation and migration of PASMCs and vascular remodeling in experimental models of PAH. Our results showed that berberine and quercetin effectively inhibited the proliferation and migration of hypoxia-induced PASMCs in a manner likely to be mediated by the suppression of MAPK1, NADPH oxidase 4 (NOX4), and cytochrome P450 1B1 (CYP1B1) expression. Furthermore, berberine and quercetin treatment attenuates pulmonary hypertension, reduces right ventricular hypertrophy, and improves pulmonary artery remodeling in monocrotaline-induced pulmonary hypertension in rat models. In conclusion, this research demonstrates CR might be a promising treatment option for PAH, and the network pharmacology approach can be an effective tool to reveal the potential mechanisms of Chinese herbal medicine.

Metabolic syndrome contributes to the pulmonary arterial dysfunction in pulmonary hypertension in heart failure with preserved ejection fraction

Abstract Background Many Heart Failure with preserved Ejection Fraction (HFpEF) patients have metabolic syndrome and develop Exercise Induced Pulmonary Hypertension (EIPH). The pathogenesis of EIPH in HFpEF remains unclear as there is no rodent model. As the SGLT2 inhibitor Empagliflozin improves clinical outcome in patients with type 2 diabetes and cardiovascular risk, we tested its effect on EIPH in a novel rat model of HFpEF. Methods Obese ZSF1 (HFpEF model) with leptin receptor mutation have metabolic syndrome and received the VEGF-inhibitor SU5416 to stimulate PH (Obese + Sugen). Half also received Empagliflozin (0.2 mg/kg/day) in drinking water from 8 to 22 weeks old. Lean ZSF1 lacking the mutation served as controls. During treadmill exercise, right/left ventricle (RV/LV) hemodynamics were evaluated via catheters. Pulmonary artery vascular smooth muscle cells (PAVSMC) prepared from normal or diabetic patients were cultured in standard media, or with Palmitate acid, Glucose and Insulin (PGI) to induce metabolic stress. Flow cytometry was used to evaluate reactive oxygen species (ROS) in mitochondria (Mitosox) or cytoplasm (CellROX). Results Relative to Lean, Obese + Sugen had increased body weight and HgA1C (Fig. 1A). Relative to Lean and at rest, Obese + Sugen showed mildly elevated RVSP and LVEDP. After exercise, LVSP and LVEDP rose similarly in Lean and Obese + Sugen. However, after exercise, Obese + Sugen showed a markedly greater increase in RVSP and exercise intolerance consistent with EIPH (Fig. 1B). In MR imaging of PA, Lean showed dobutamine (5 μg/kg/min)-induced PA dilation, which was not seen in Obese + Sugen (Fig. 1C). Protein levels of sGCβ1 (key regulator of PA relaxation) and its transcription factor (NFYA) both were decreased in PA from Obese + Sugen relative to Lean (Fig. 1D). Obese + Sugen + SGLT2 inhibitor treated rats showed marked improvements metabolic syndrome (body weight, HgA1c), exercise induced increase in RVSP, PA response to dobutamine, and increased NFYA and sGCβ1 expression (Fig. 1A–D). We observed greater ROS-induced DNA damage (8-OHdG staining) (Fig. 1E) and mitochondrial complex I, III, and IV activity in Obese + Sugen PA that was normalized in Obese + Sugen + SGLT2 inhibitor (Fig. 1F), suggesting a role of ROS in EIPH. Control human PAVSMC treated with PGI media showed elevated cytoplasmic and mitochondrial ROS, associated with increased mitochondrial complex I, III, IV and V activity (Fig. 1F, G). PGI media also accelerated the degradation of NFYA RNA and protein level in a manner mimicked by H2O2, and prevented by catalase/SOD (Fig. 1H, I), suggesting PGI-induced ROS enhanced NFYA degradation. Diabetic human PAVSMCs cultured in normal media resembled PGI-treated normal cells with respect to sGCb1 and NFYA expression, and in response to catalase/SOD (Fig. 1H, I). Conclusions In this PH-HFpEF model, metabolic syndrome contributes to PA dysfunction and EIPH through mitochondrial dysfunction and enhanced ROS, which were improved by Empagliflozin treatment. Figure 1 Funding Acknowledgement Type of funding source: None

Notch3 signalling and vascular remodelling in pulmonary arterial hypertension.

Notch signalling is critically involved in vascular morphogenesis and function. Four Notch isoforms (Notch1–4) regulating diverse cellular processes have been identified. Of these, Notch3 is expressed almost exclusively in vascular smooth muscle cells (VSMCs), where it is critically involved in vascular development and differentiation. Under pathological conditions, Notch3 regulates VSMC switching between the contractile and synthetic phenotypes. Abnormal Notch3 signalling plays an important role in vascular remodelling, a hallmark of several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Because of the importance of Notch3 in VSMC (de)differentiation, Notch3 has been implicated in the pathophysiology of pulmonary vascular remodelling in PAH. Here we review the current literature on the role of Notch in VSMC function with a focus on Notch3 signalling in pulmonary artery VSMCs, and discuss potential implications in pulmonary artery remodelling in PAH.

SUMOylation of Vps34 by SUMO1 promotes phenotypic switching of vascular smooth muscle cells by activating autophagy in pulmonary arterial hypertension

IntroductionPulmonary arterial hypertension (PAH) is a life-threatening disease without effective therapies. PAH is associated with a progressive increase in pulmonary vascular resistance and irreversible pulmonary vascular remodeling. SUMO1 (small ubiquitin-related modifier 1) can bind to target proteins and lead to protein SUMOylation, an important post-translational modification with a key role in many diseases. However, the contribution of SUMO1 to PAH remains to be fully characterized. MethodsIn this study, we explored the role of SUMO1 in the dedifferentiation of vascular smooth muscle cells (VSMCs) involved in hypoxia-induced pulmonary vascular remodeling and PAH in vivo and in vitro. ResultsIn a mouse model of hypoxic PAH, SUMO1 expression was significantly increased, which was associated with activation of autophagy (increased LC3b and decreased p62), dedifferentiation of pulmonary arterial VSMCs (reduced α-SMA, SM22 and SM-MHC), and pulmonary vascular remodeling. Similar results were obtained in a MCT-induced PAH model. Overexpression of SUMO1 significantly increased VSMCs proliferation, migration, hypoxia-induced VSMCs dedifferentiation, and autophagy, but these effects were abolished by inhibition of autophagy by 3-MA in aortic VSMCs. Furthermore, SUMO1 knockdown reversed hypoxia-induced proliferation and migration of PASMCs. Mechanistically, SUMO1 promotes Vps34 SUMOylation and the assembly of the Beclin-1-Vps34-Atg14 complex, thereby inducing autophagy, whereas Vps34 mutation K840R reduces Vps34 SUMOylation and inhibits VSMCs dedifferentiation. DiscussionOur data uncovers an important role of SUMO1 in VSMCs proliferation, migration, autophagy, and phenotypic switching (dedifferentiation) involved in pulmonary vascular remodeling and PAH. Targeting of the SUMO1-Vps34-autophagy signaling axis may be exploited to develop therapeutic strategies to treat PAH.

Aloperine suppresses human pulmonary vascular smooth muscle cell proliferation via inhibiting inflammatory response.

Abnormal pulmonary arterial vascular smooth muscle cells (PASMCs) proliferation is critical pathological feature of pulmonary vascular remodeling that acts as driving force in the initiation and development of pulmonary arterial hypertension (PAH), ultimately leading to pulmonary hypertension. Aloperine is a main active alkaloid extracted from the traditional Chinese herbal Sophora alopecuroides and possesses outstanding antioxidation and anti-inflammatory effects. Our group found Aloperine has protective effects on monocroline-induced pulmonary hypertension in rats by inhibiting oxidative stress in previous researches. However, the anti-inflammation effects of Aloperine on PAH remain unclear. Therefore, to further explore whether the beneficial role of Aloperine on PAH was connected with its anti-inflammatory effects, we performed experiments in vitro. Aloperine significantly inhibited the proliferation and DNA synthesis of human pulmonary artery smooth muscle cells (HPASMCs) induced by platelet-derived growth factor-BB, blocked progression through G0/G1to S phase of the cell cycle and promoted total ratio of apoptosis. In summary, these results suggested that Aloperine negatively regulated nuclear factor-κB signaling pathway activity to exert protective effects on PAH and suppressed HPASMCs proliferation therefore has a potential value in the treatment of pulmonary hypertension by negatively modulating pulmonary vascular remodeling.

Inhibitory Antibodies against Activin A and TGF-β Reduce Self-Supported, but Not Soluble Factors-Induced Growth of Human Pulmonary Arterial Vascular Smooth Muscle Cells in Pulmonary Arterial Hypertension.

Increased growth and proliferation of distal pulmonary artery vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary arterial hypertension (PAH). Transforming Growth Factor-β (TGF-β) superfamily plays a critical role in PAH, but relative impacts of self-secreted Activin A, Gremlin1, and TGF-β on PAH PAVSMC growth and proliferation are not studied. Here we report that hyper-proliferative human PAH PAVSMC have elevated secretion of TGF-β1 and, to a lesser extent, Activin A, but not Gremlin 1, and significantly reduced Ser465/467-Smad2 and Ser423/425-Smad3 phosphorylation compared to controls. Media, conditioned by PAH PAVSMC, markedly increased Ser465/467-Smad2, Ser423/425-Smad3, and Ser463/465-Smad1/5 phosphorylation, up-regulated Akt, ERK1/2, and p38 MAPK, and induced significant proliferation of non-diseased PAVSMC. Inhibitory anti-Activin A antibody reduced PAH PAVSMC growth without affecting canonical (Smads) or non-canonical (Akt, ERK1/2, p38 MAPK) effectors. Inhibitory anti-TGF-β antibody significantly reduced P-Smad3, P-ERK1/2 and proliferation of PAH PAVSMC, while anti-Gremlin 1 had no anti-proliferative effect. PDGF-BB diminished inhibitory effects of anti-Activin A and anti-TGF-β antibodies. None of the antibodies affected growth and proliferation of non-diseased PAVSMC induced by PAH PAVSMC-secreted factors. Together, these data demonstrate that human PAH PAVSMC have secretory, proliferative phenotype that could be targeted by anti-Activin A and anti-TGF-β antibodies; potential cross-talk with PDGF-BB should be considered while developing therapeutic interventions.

Mitochondria has a role in regulating hypoxic-dependent cell proliferation in pulmonary cells and vascular remodelling

Introduction Hypoxia is a strong contributor to hyper-proliferation of pulmonary artery vascular smooth muscle cells (PAVMSCs) in pulmonary hypertension.1 The involvement of hypoxia in the PASMC proliferation signalling is poorly understood. The aim of this work was to assess the relationship between hypoxia, PASMC proliferation and vascular remodelling. Methods PAVSMC were isolated from Sprague Dawley rats. Cells were cultured under hypoxic conditions and compared with normoxic cultures. Cell proliferation stimulated with FCS and PDGF was assessed by 3H-thymidine incorporation assay. Proliferative mRNA markers were measured using real-time qPCR and the phosphorylation of MAP kinases; ERK, JNK and P38 were evaluated using western blot. Mitochondrial DRP-1 inhibitor (mdivi-1) was used as a treatment. Results Hypoxia (3%) directly enhanced the proliferative of PAVSMCs to 10% FCS and PDGF compared with normoxic cells. Moreover, cells stimulated with 10% FCS display an increase in ERK and p38 MAPK phosphorylation when compared to normoxic cells. PCR data confirms hypoxia induces proliferative genes; increasing mRNA expression of PCNA, ERK1 and P70s6k when compared to normoxic cultures. Inhibiting mitochondrial fission using Mdivi-1 (10 uM) resulted in a significant reduction of PAVSMC proliferation (p<0.05). Under hypoxic conditions, mitochondria dynamics were shifted to mitochondrial fission through a significant increase in mRNA levels of DRP-1 and P22phox compared to normoxic cells. Conclusion This work confirms hypoxia has directly stimulates PAVSMC proliferation. Significantly mitochondrial function appears to be crucial to hypoxic–dependent PAVSMCs proliferation. Future work will focus on elucidating of the role mitochondria in hypoxic-dependent cell signalling related to PAVSMC and endothelial cell proliferation and migration.

HIPPO-Integrin-linked Kinase Cross-Talk Controls Self-Sustaining Proliferation and Survival in Pulmonary Hypertension.

Enhanced proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiologic components of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). To determine the role and therapeutic relevance of HIPPO signaling in PAVSMC proliferation/apoptosis imbalance in PAH. Primary distal PAVSMCs, lung tissue sections from unused donor (control) and idiopathic PAH lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Immunohistochemical and immunoblot analyses demonstrated that the HIPPO central component large tumor suppressor 1 (LATS1) is inactivated in small remodeled pulmonary arteries (PAs) and distal PAVSMCs in idiopathic PAH. Molecular- and pharmacology-based analyses revealed that LATS1 inactivation and consequent up-regulation of its reciprocal effector Yes-associated protein (Yap) were required for activation of mammalian target of rapamycin (mTOR)-Akt, accumulation of HIF1α, Notch3 intracellular domain and β-catenin, deficiency of proapoptotic Bim, increased proliferation, and survival of human PAH PAVSMCs. LATS1 inactivation and up-regulation of Yap increased production and secretion of fibronectin that up-regulated integrin-linked kinase 1 (ILK1). ILK1 supported LATS1 inactivation, and its inhibition reactivated LATS1, down-regulated Yap, suppressed proliferation, and promoted apoptosis in PAH, but not control PAVSMCs. PAVSM in small remodeled PAs from rats and mice with SU5416/hypoxia-induced PH showed down-regulation of LATS1 and overexpression of ILK1. Treatment of mice with selective ILK inhibitor Cpd22 at Days 22-35 of SU5416/hypoxia exposure restored LATS1 signaling and reduced established pulmonary vascular remodeling and PH. These data report inactivation of HIPPO/LATS1, self-supported via Yap-fibronectin-ILK1 signaling loop, as a novel mechanism of self-sustaining proliferation and apoptosis resistance of PAVSMCs in PAH and suggest a new potential target for therapeutic intervention.

Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: Toward contractile versus synthetic phenotypes.

Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH-SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or -1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH-SMC (fold change 1.5≤ or -1.5≥, p < 0.05). HUASMC expressed increased amount of α-smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH-SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH-SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH-SMC. There was a trend toward reduced proliferation of PAH-SMC with paxillin-si-RNA and increased proliferation with ELAVL1-siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH-SMC proliferation.

Profiling the role of mammalian target of rapamycin in the vascular smooth muscle metabolome in pulmonary arterial hypertension.

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.

A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension.

Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.

Abstract 12: Basigin Promotes Vascular Smooth Muscle Proliferation and Pulmonary Hypertension

Background: Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells (VSMC) by oxidative stress and promotes VSMC proliferation. However, the role of CyPA and its extracellular receptor Basigin (Bsg) in the pathogenesis of pulmonary hypertension (PH) remains to be elucidated. In this study, we tested our hypothesis that CyPA/Bsg signaling promotes the development of PH. Methods and Results: In the pulmonary arteries of PH patients, immunostaining revealed strong expression of CyPA and Bsg (n=5). In cultured human pulmonary arterial VSMC, hypoxia (O 2 2%) significantly increased CyPA secretion and Bsg expression compared with normoxic condition (O 2 21%) (n=4 per group). To determine the role of CyPA/Bsg signaling in PH development, CyPA +/- and Bsg +/- mice were exposed to hypoxia (O 2 10%) for 4 weeks. The pulmonary arteries (PA) of CyPA +/- and Bsg +/- mice exposed to normoxia did not differ in morphology compared with their littermate controls. In contrast, CyPA +/- (n=12) and Bsg +/- mice (n=15) exposed to hypoxia revealed significantly reduced right ventricular systolic pressure (RVSP), PA remodeling and RV hypertrophy compared with their littermate controls (all P&lt;0.01). Importantly, after transplantation of bone marrow, the severity of PH was still exacerbated in Bsg +/+ recipient mice compared with Bsg +/- recipient mice regardless of the source of bone marrow (Bsg +/+ or Bsg +/- ), suggesting the crucial role of Bsg in PA. To further evaluate the role of Bsg, we harvested VSMC from Bsg +/+ and Bsg +/− mice. VSMC proliferation was significantly reduced in Bsg +/− compared with Bsg +/+ in response to 2% FBS, suggesting the crucial role of Bsg in VSMC proliferation. Mechanistic studies demonstrated that Bsg +/- VSMC revealed reduced expression of oxidative stress genes, less secretion of cytokines/chemokines and growth factors. Finally, in the clinical study, plasma CyPA levels in PH patients were increased in accordance with the severity of pulmonary vascular resistance (P&lt;0.001). Furthermore, event-free curve revealed that high plasma CyPA levels predicted poor outcome in PH patients (death or lung transplantation, P&lt;0.001). Conclusions: These results indicate the crucial role of extracellular CyPA and vascular Bsg in the pathogenesis of PH.

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

Enhanced 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. Immunohistochemical 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. These 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.

Abstract 74: Crucial Role of ROCK2 in Vascular Smooth Muscle Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

Background We have previously demonstrated that Rho-kinase (ROCK) pathway in vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of cardiovascular disease including pulmonary arterial hypertension (PAH). Rho-kinase has 2 isoforms; ROCK1 for inflammatory cells and ROCK2 for VSMC. We thus examined the specific role of ROCK2 in VSMC in the pathogenesis of PAH. Methods and Results In patients with PAH as compared with controls, the immunoreactivity of ROCK2 and phosphorylated ERK1/2 co-expressed with αSMA in pulmonary arterial media were markedly increased. Thus, we developed VSMC-specific ROCK2-deficient (ROCK2 +/- ) and VSMC-specific ROCK2-overexpressing (ROCK2-Tg) mice. The extent of hypoxia-induced pulmonary hypertension (10% O 2 for 4 weeks), as evaluated by pulmonary vascular remodeling, right ventricular (RV) systolic pressure and RV hypertrophy, was significantly reduced in ROCK2 +/- mice and was enhanced in ROCK2-Tg mice compared with the littermates (n=10 each, all P&lt;0.05). The expression of ROCK activity and phosphorylated ERK1/2 in lung tissues analyzed by Western blotting and the numbers of Ki67 + proliferating VSMC and CD45 + inflammatory cells in pulmonary arterioles revealed by immunostaining were significantly reduced in the ROCK2 +/- mice and were increased in the ROCK2-Tg mice compared with the littermates (n=4 each, all P&lt;0.05). In cultured murine aortic VSMC, both scratch assay and Boyden chamber assay showed that migration activity was significantly reduced in the ROCK2 +/- VSMC and was increased in the ROCK2-Tg VSMC compared with littermates (n=5 each, both P&lt;0.01). In addition, in primary pulmonary arterial VSMC (PASMC) cultured from patients with PAH, siRNA knockdown (-81%) of ROCK2 expression (ROCK2-siRNA) significantly suppressed the expression of ROCK activity and PDGF-induced phosphorylated ERK1/2 (n=4 each, both P&lt;0.05). Compared with control-siRNA, PASMC transfected with ROCK2-siRNA exhibited significant reduction in both migration activity by scratch assay and proliferation activity by growth curve (n=3 each, both P&lt;0.01). Conclusions ROCK2 in VSMC plays a crucial role in the pathogenesis of PH through enhanced VSMC migration and proliferation in mice in vivo.

The key role of PGC-1α in mitochondrial biogenesis and the proliferation of pulmonary artery vascular smooth muscle cells at an early stage of hypoxic exposure

Peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α) induced by hypoxia regulates mitochondrial biogenesis and oxidative stress. However, the potential role of PGC-1α in hypoxia-promoted proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is completely unknown. In this study, we found that hypoxia significantly induced the expression of PGC-1α in cultured PASMCs and activated mitochondrial biogenesis through upregulation of nuclear respiratory factor-1 and mitochondria transcription factor A in a time-dependent manner. Knockdown of PGC-1α by siRNA abrogated hypoxia-induced PASMCs proliferation via the downregulation of PCNA, cyclinA, and cyclinE. Furthermore, we observed that PI3K/Akt signaling pathway was involved in hypoxia induced PGC-1α expression and PASMCs proliferation. Taken together, these datas reveal PGC-1α as the key regulator to mediate mitochondrial biogenesis and the proliferation of PASMCs at an early stage of hypoxic exposure. This process might bring to light a potential adaptive mechanism for PASMCs to minimize hypoxic damage and our novel findings provide new insight into the development of hypoxic pulmonary hypertension.