Pulmonary Artery Smooth Muscle Cells Migration Research Articles

Role of platelet-derived growth factor-BB (PDGF-BB) in human pulmonary artery smooth muscle cell proliferation

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and suppressed apoptosis. Platelet-derived growth factor (PDGF) is a potent mitogen involved in cell proliferation and migration. PDGF-BB induces the proliferation and migration of PASMCs and has been proposed to be a key mediator in the progression of PAH. Previous studies have shown that PDGF and its receptor are substantially elevated in lung tissues and PASMCs isolated from patients and animals with PAH, but the underlying mechanisms are still poorly manifested. MAP kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH2-terminal kinase1/2 (JNK1/2), and p38 are the key intracellular signals for stimuli-induced cell proliferation, survival, and apoptosis. Therefore, the purpose of this study is to determine whether PDGF-BB on cell proliferation process is mediated through the MAP kinases pathway in human PASMCs (HPASMCs). Our results showed PDGF-BB-induced proliferating cell nuclear antigen (PCNA), Cyclin A and Cyclin E expression in a concentration-dependent manner. The expression levels of phosphorylated JNK (p-JNK) was upregulated with 20 ng/ml PDGF-BB treatment, while PDGF-BB could not increase phosphorylated ERK1/2 (p-ERK1/2) and p-38 (p-p38) expression. The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of antagonist of the JNK pathway or si-JNK. In addition, PDGF-BB protected against the loss of mitochondrial membrane potentials evoked by serum deprivation (SD) in a JNK-dependent manner. These results suggest that PDGF-BB promotes HPASMCs proliferation and survival, which is likely to be mediated via the JNK pathway.

Hypoxia-Inducible Factor-1α in Pulmonary Arterial Smooth Muscle Cells and Hypoxia-induced Pulmonary Hypertension

Hypoxia-Inducible Factor-1α in Pulmonary Arterial Smooth Muscle Cells and Hypoxia-induced Pulmonary Hypertension

Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mTOR signal and the inhibition of autophagy under hypoxia

Apelin is highly expressed in the lungs, especially in the pulmonary vasculature, but the functional role of apelin under pathological conditions is still undefined. Hypoxic pulmonary hypertension is the most common cause of acute right heart failure, which may involve the remodeling of artery and regulation of autophagy. In this study, we determined whether treatment with apelin regulated the proliferation and migration of rat pulmonary arterial smooth muscle cells (SMCs) under hypoxia, and investigated the underlying mechanism and the relationship with autophagy. Our data showed that hypoxia activated autophagy significantly at 24 hrs. The addition of exogenous apelin decreased the level of autophagy and further inhibited pulmonary arterial SMC (PASMC) proliferation via activating downstream phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/the mammalian target of Rapamycin (mTOR) signal pathways. The inhibition of the apelin receptor (APJ) system by siRNA abolished the inhibitory effect of apelin in PASMCs under hypoxia. This study provides the evidence that exogenous apelin treatment contributes to inhibit the proliferation and migration of PASMCs by regulating the level of autophagy.

B-type natriuretic peptide inhibits angiotensin II-induced proliferation and migration of pulmonary arterial smooth muscle cells

Pulmonary vascular remodeling, characterized by disordered proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a pathognomonic feature of pulmonary arterial hypertension. Thus, pharmacologic strategy targeting on anti-proliferation and anti-migration of PASMCs may have therapeutic implications for PAH. Here we investigated the effects and underlying mechanisms of B-type natriuretic peptide (BNP) on angiotensin II (Ang II)-induced proliferation and migration of PASMCs. Proliferation and migration of PASMCs cultured from Wistar rats were induced by Ang II, with or without BNP treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) overload, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and the cGMP/PKG pathway were examined. We found that BNP inhibited Ang II-induced PASMCs proliferation and migration dose dependently. BNP could also arrest the cell cycle progression in the G0/G1-phase. In addition, BNP attenuated intracellular calcium overload caused by Ang II. Moreover, Ang II-induced ROS production was mitigated by BNP, with associated down-regulation of NAD(P)H oxidase 1 (Nox1) and reduced mitochondrial ROS production. Finally, Ang II-activated MAPKs and Akt were also counteracted by BNP. Of note, all these effects of BNP were abolished by a PKG inhibitor (Rp-8-Br-PET-cGMPS). In conclusion, BNP inhibits Ang II-induced PASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production by Nox1 and mitochondria, and down-regulation of MAPK and Akt signal transduction, through the cGMP/PKG pathway. Therefore, this study implicates that BNP may have a therapeutic role in pulmonary vascular remodeling.

Sildenafil Inhibits Hypoxia-Induced Transient Receptor Potential Canonical Protein Expression in Pulmonary Arterial Smooth Muscle via cGMP-PKG-PPARγ Axis

Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% O2 for 21 d) and rat distal PASMCs (4% O2 for 60 h) were taken as models to mimic CHPH. Real-time PCR, Western blotting, and Fura-2-based fluorescent microscopy were performed for mRNA, protein, and Ca(2+) measurements, respectively. The cellular cyclic guanosine monophosphate (cGMP) analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt (CPT-cGMP) (100 μM) inhibited TRPC1 and TRPC6 expression, store-operated Ca(2+) entry (SOCE), and the proliferation and migration of PASMCs exposed to prolonged hypoxia. The inhibition of CPT-cGMP on TRPC1 and TRPC6 expression in PASMCs was relieved by either the inhibition or knockdown of cGMP-dependent protein kinase (PKG) and peroxisome proliferator-activated receptor γ (PPARγ) expression. Under hypoxic conditions, CPT-cGMP increased PPARγ expression. This increase was abolished by the PKG antagonists Rp8 or KT5823. PPARγ agonist GW1929 significantly decreased TRPC1 and TRPC6 expression in PASMCs. Moreover, hypoxia exposure decreased, whereas sildenafil treatment increased, PKG and PPARγ expression in PASMCs ex vivo, and in rat distal PAs in vivo. The suppressive effects of sildenafil on TRPC1 and TRPC6 in rat distal PAs and on the hemodynamic parameters of CHPH were inhibited by treatment with the PPARγ antagonist T0070907. We conclude that sildenafil inhibits TRPC1 and TRPC6 expression in PASMCs via cGMP-PKG-PPARγ-dependent signaling during CHPH.

Function of NADPH Oxidase 1 in Pulmonary Arterial Smooth Muscle Cells After Monocrotaline-Induced Pulmonary Vascular Remodeling

Chronic hypoxia induces pulmonary hypertension (PH) that is concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this. Recent findings suggest that ROS production by NADPH oxidase 4 (Nox4) is important in this remodeling. We investigated whether ROS production by Nox is also important in an inflammatory model of monocrotaline (MCT)-induced PH. We examined ROS production, their possible sources, and their impact on the function of pulmonary arterial smooth muscle cells (PASMC) isolated from MCT-treated and healthy rats. MCT-PASMC showed increased intracellular superoxide production, migration, and proliferation compared with healthy controls due to increased Nox1 expression. A comparison of PASMC from MCT- and nontreated rats revealed an up-regulation of Sod2, Nrf2, cyclin D1, and matrix metalloproteinase-9 (MMP-9) as well as an increased phosphorylation of cofilin and extracellular signal-regulated kinases (Erk). Expression of Sod2, Nrf2, and cyclin D1 and phosphorylation of cofilin and Erk were Nox1 dependent. The role of ROS in PH is not fully understood. Mitochondria and Nox have been suggested as sources of altered ROS generation in PH, yet it remains unclear whether increased or decreased ROS contributes to the development of PH. Our studies provide evidence that for different triggers of PH, different Nox isoforms regulate proliferation and migration of PASMC. In contrast to hypoxia-induced PH, Nox1 but not Nox4 is responsible for pathophysiological proliferation and migration of PASMC in an inflammatory model of MCT-induced PH via increased superoxide production. Thus, different Nox isoforms may be targeted in different forms of PH.

Targeting cell motility in pulmonary arterial hypertension

Pulmonary artery smooth muscle cells (PASMC), in pulmonary arterial hypertension (PAH), contribute to obliterative vascular remodelling and are characterised by enhanced proliferation, suppressed apoptosis and, a much less studied, increased migration potential. One of the major proteins that regulate cell migration is focal adhesion kinase (FAK), but its role in PAH is not fully understood. We hypothesised that targeting cell migration by FAK inhibition may be a new therapeutic strategy in PAH. In vivo, inhalation of FAK-siRNA (n=5) or oral delivery of PF-228 (FAK inhibitor PF-573 228; n=5) inhibited rat monocrotaline induced PAH, improving the haemodynamics, vascular remodelling (media thickness), and right ventricular hypertrophy. In vitro, FAK was activated in PAH human lungs (n=8) or PASMC when compared to those form healthy subjects (Western blot, n=5), in a Src-dependent manner, as it was reversed by the specific Src inhibitor PP2. The degree of FAK phosphorylation at Y576 correlated positively with pulmonary vascular resistance in PAH patients. FAK inhibition (siRNA, PF-228 and PP2) in PAH-PASMCs induced a fivefold increase in apoptosis (percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling), a 2.5-fold decrease in proliferation (%Ki67), an 18% decrease in cell migration (colorimetric assay) and a 50% decrease in cell invasion (wound healing). Suppressing PASMC migration by FAK inhibition inhibits PAH progression and may open a new therapeutic window in PAH.

EETs and CYP2J2 inhibit TNF-α-induced apoptosis in pulmonary artery endothelial cells and TGF-β1-induced migration in pulmonary artery smooth muscle cells

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) have multiple biological functions in cardiovascular homeostasis. The antiinflammatory, anti-migratory and pro-proliferative effects of EETs suggest a possible beneficial role for EETs in the apoptosis, proliferation and migration of pulmonary vascular cells. In this study, we investigated the effects of exogenous EETs and cytochrome P450 2J2 (CYP2J2) overexpression on tumor necrosis factor-α (TNF-α)-induced pulmonary artery endothelial cell (PAEC) apoptosis, and transforming growth factor-β1 (TGF-β1)-induced pulmonary artery smooth muscle cell (PASMC) proliferation and migration. PAECs and PASMCs were cultured from porcine pulmonary arteries. Our findings indicated that EETs or CYP2J2 overexpression significantly protected the PAECs from TNF-α-induced apoptosis, as evaluated by cell viability and flow cytometry. Two mechanisms were found to be involved in these important protective effects: firstly, EETs and CYP2J2 overexpression inhibited the decrease in the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xL, as well as the increase in the expression of the pro-apoptotic protein, Bax, mediated by TNF-α; secondly, they activated the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways. We also found that 11,12-EET and 14,15-EET significantly inhibited TGF-β1-stimulated PASMC migration. However, EETs did not suppress TGF-β1-induced PASMC proliferation in vitro. These data may represent a novel approach to mitigate pulmonary vascular remodeling in diseases, such as pulmonary arterial hypertension.

The C‐terminal tail of Aquaporin 1(AQP1) is Required for Migration and Proliferation of Pulmonary Arterial Smooth Muscle Cells (PASMCs)

Both PASMC proliferation and migration are important contributors to the vascular remodeling that occurs during the development of pulmonary hypertension. AQP1 is a member of the water channel family of proteins. We previously demonstrated that AQP1 is expressed in and mediates migration and proliferation of PASMCs; however, the mechanism by which AQP1 controls these responses is unclear. The C‐terminal tail of AQP1 contains putative calcium and protein binding sites. Thus, we wanted to explore whether the C‐terminal tail of AQP1 was required for migration and proliferation. Rat PASMCs were harvested from distal pulmonary arteries enzymatically and plated onto culture dishes. Proliferation and migration were measured using BrdU incorporation and transwell filters. PASMCs were treated with 50 ifu/cell for 4 h with an adenoviral construct expressing green fluorescent protein (AdGFP), the control for infection, wild‐type AQP1 (AdAQP1) or AQP1 lacking the entire cytoplasmic C‐terminal tail (AdAQP1CT). Compared to AdGFP cells, infection with AdAQP1 or AdAQP1CT increased total and surface AQP1 protein levels and the rate of change in cell volume in response to hypotonic challenge to a similar level. Infection with AdAQP1, but not AdAQP1CT, increased migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs via a mechanism that includes the C‐terminal tail but is independent of water transport.

Sodium butyrate inhibits platelet‐derived growth factor‐induced proliferation and migration in pulmonary artery smooth muscle cells through Akt inhibition

Sodium butyrate (BU) is a molecule that acts as a histone deacetylase inhibitor. As compared with its well-known antineoplastic/antiproliferative effects, little is known about BU action on vascular cell dynamics. An imbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is essential in the onset and progression of pulmonary arterial hypertension (PAH), a disease that is characterized by vascular lung derangement and that frequently has an unfavorable outcome. Here, we show that, in PASMCs of PAH rats, BU counteracted platelet-derived growth factor (PDGF)-induced Ki67 expression, and arrested the cell cycle, mainly at G0 /G1 . BU decreased proliferating cell nuclear antigen, c-Myc and cyclin D1 transcription and protein expression, while increasing p21 expression. BU reduced the transcription of PDGF receptor-β, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing, migration and pulmonary artery ring assays indicated that BU inhibited PDGF-induced PASMC migration. BU strongly inhibited PDGF-induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculin A. BU-treated cells showed a remarkable increase in acetylated Akt, indicating an inverse relationship between the levels of acetylated Akt and phospho-Akt. These findings may provide novel perspectives on the use of histone deacetylase inhibitors in PAH.

Bone morphogenetic protein 2 decreases TRPC expression, store-operated Ca2+ entry, and basal [Ca2+]i in rat distal pulmonary arterial smooth muscle cells

Recent studies indicate that multiple bone morphogenetic protein (BMP) family ligands and receptors are involved in the development of pulmonary arterial hypertension, yet the underlying mechanisms are incompletely understood. Although BMP2 and BMP4 share high homology in amino acid sequence, they appear to exert divergent effects on chronic hypoxic pulmonary hypertension (CHPH). While BMP4 promotes vascular remodeling, BMP2 prevents CHPH. We previously demonstrated that BMP4 upregulates the expression of canonical transient receptor potential channel (TRPC) proteins and, thereby, enhances store-operated Ca(2+) entry (SOCE) and elevates intracellular Ca(2+) concentration ([Ca(2+)]i) in pulmonary arterial smooth muscle cells (PASMCs). In this study, we investigated the effects of BMP2 on these variables in rat distal PASMCs. We found that treatment with BMP2 (50 ng/ml, 60 h) inhibited TRPC1, TRPC4, and TRPC6 mRNA and protein expression. Moreover, BMP2 treatment led to reduced SOCE and decreased basal [Ca(2+)]i in PASMCs. These alterations were associated with decreased PASMC proliferation and migration. Conversely, knockdown of BMP2 with specific small interference RNA resulted in increased cellular levels of TRPC1, TRPC4, and TRPC6 mRNA and protein, enhanced SOCE, elevated basal [Ca(2+)]i, and increased proliferation and migration of PASMCs. Together, these results indicate that BMP2 participates in regulating Ca(2+) signaling in PASMCs by inhibiting TRPC1, TRPC4, and TRPC6 expression, thus leading to reduced SOCE and basal [Ca(2+)]i and inhibition of cell proliferation and migration.

Abstract 19603: Disruption of βPDGF Receptor-Dependent PI3k and Plcγ Signaling Protects from Hypoxia-Induced Pulmonary Hypertension

Background: Vascular remodeling is critical during the pathogenesis of pulmonary hypertension (PH). Growth factors, such as platelet-derived growth factor (PDGF), appear to be involved in these processes. With pharmacological inhibitors, we demonstrated previously that phosphatidyl inositol-3 kinase (PI3K) and phospholipase Cγ (PLCγ) are the critical mediators of βPDGF receptor (βPDGFR)-dependent proliferation and migration in pulmonary arterial smooth muscle cells (PASMC). Whereas expression data and data with the tyrosine kinase inhibitor imatinib suggest a role for PDGF in PH, data from genetic models is lacking. Methods and results: We utilized βPDGFR F3/F3 (“F3”) mice that were generated by introducing point mutations of the PI3K and PLCγ binding sites into the βPDGFR locus by gene targeting. We isolated PASMC from F3 and wild-type (WT) mice. Co-immunoprecipitation experiments after PDGF treatment showed that PI3K and PLCγ did not bind to the activated F3 receptor, whereas overall phosphorylation and binding of RasGap and SHP-2 were unaffected. In line with these observations, PDGF-mediated phosphorylation of Akt and Erk 1/2 was inhibited in F3-PASMC compared to WT. To address, if cellular responses were altered, we analyzed βPDGFR-dependent proliferation (as BrdU incorporation) and migration (modified Boyden chamber). These experiments revealed that these responses were essentially completely blocked in F3-PASMC compared to WT. To analyze the development and severity of PH in vivo, we exposed F3 and WT mice to hypoxia for 3 weeks. Following this treatment, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and right ventricular (RV) hypertrophy was assessed as RV weight/left ventricular + septal weight (RV/LV+S). These analyses revealed that RVSP and RV/LV+S were significantly reduced in F3 compared to WT mice. Conclusions: These data show that disruption of βPDGFR-dependent PI3K and PLCγ signaling blocks PDGF-dependent proliferation and migration of PASMC in vitro and protects from the development of hypoxia-induced PH in vivo. Consequently, these data support a critical role of PDGF during the pathogenesis of PH.

Sodium Tanshinone IIA Sulfonate Inhibits Canonical Transient Receptor Potential Expression in Pulmonary Arterial Smooth Muscle from Pulmonary Hypertensive Rats

Danshen, the dried root of Salvia miltiorrhiza, is widely used in clinics in China for treating various diseases, including cardiovascular diseases. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA isolated as the major active component from Danshen, was recently reported to be effective in attenuating the characteristic pulmonary vascular changes associated with chronically hypoxic pulmonary hypertension (CHPH); however, the underlying detailed mechanisms are poorly understood. In this study, we investigated the effects of STS on basal intracellular Ca(2+) concentration ([Ca(2+)](i)) and store-operated Ca(2+) entry (SOCE) in distal pulmonary arterial smooth muscle cells (PASMCs) exposed to prolonged hypoxia or isolated from CHPH rats. SOCE measured by Mn(2+) quenching of Fura-2 fluorescence in PASMCs from rats exposed to chronic hypoxia (10% O(2), 21 d) was increased by 59%, and basal [Ca(2+)](i) was increased by 119%; this effect was inhibited by intraperitoneal injection of STS. These inhibitory effects of STS on hypoxic increases of SOCE and basal [Ca(2+)](i) were associated with reduced expression of canonical transient receptor potential (TRPC)1 and TRPC6 in distal pulmonary arterial smooth muscle and decreases on right ventricular pressure, right ventricular hypertrophy, and peripheral pulmonary vessel thickening. In ex vivo cultured distal PASMCs from normoxic rats, STS (0-25 μM) dose-dependently inhibited hypoxia-induced cell proliferation and migration, paralleled with attenuation in increases of basal [Ca(2+)](i), SOCE, mRNA, and protein expression of TRPC1 and TRPC6. STS also relieved right ventricular systolic pressure, right ventricular hypertrophy, and TRPC1 and TRPC6 protein expression in distal pulmonary arteries in a monocrotaline-induced rat model of pulmonary arterial hypertension. These results indicate that STS prevents pulmonary arterial hypertension development likely by inhibiting TRPC1 and TRPC6 expression, resulting in normalized basal [Ca(2+)](i) and attenuated proliferation and migration of PASMCs.

Pathogenic Role of Store-Operated and Receptor-Operated Ca2+ Channels in Pulmonary Arterial Hypertension

Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.

Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells

Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.

BIX‐01294 treatment blocks cell proliferation, migration and contractility in ovine foetal pulmonary arterial smooth muscle cells

Recent studies have indicated a role of epigenetic phenomena in pathogenesis of pulmonary hypertension, but in foetal pulmonary artery smooth muscle cell (PASMC) proliferation this is still largely unknown. G9a is a key enzyme for histone H3 dimethylation at position lysine-9. In this study, we have investigated the function of G9a in ovine foetal PASMC proliferation, migration and contractility. Cell proliferation was measured by cell counting and BrdU incorporation assay and cell cycle analysis was performed by flow cytometry. Expression of cell cycle-related genes was determined by real-time PCR and the wound-healing scratch assay was used to measure cell migration. A gel contraction assay was used to determine contractility of foetal PASMCs. Global DNA methylation was measured by liquid chromatography-mass spectroscopy. Inhibition of G9a by its inhibitor BIX-01294 reduced proliferation of foetal PASMCs and induced cell cycle arrest in G(1) phase. This was accompanied by increased p21 expression, but not p53 and other cell cycle-related genes. Treatment of foetal PASMCs with BIX-01294 inhibited platelet-derived growth factor-induced cell proliferation and migration. Contractility of foetal PASMCs was also markedly inhibited by BIX-01294. Expression of calponin and ROCK-II proteins was reduced by BIX-01294 in a dose-dependent manner and BIX-01294 significantly increased global methylation level in the foetal PASMCs. Our results demonstrate for the first time that histone lysine methylation is involved in cell proliferation, migration, contractility and global DNA methylation in foetal PASMCs. Further understanding of this mechanism may provide insight into proliferative vascular disease in the lungs.

Function of NADPH oxidase 1 in pulmonary arterial smooth muscle cells from monocrotaline-induced pulmonary hypertension

Chronic alveolar hypoxia induces pulmonary hypertension (PH) and pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this process. Recent findings suggested that ROS production by NADPH oxidase 4 (Nox4) is plays an important role for PH development in hypoxia-induced PH in mice and human idiopathic pulmonary arterial hypertension (IPAH). Against this background we investigated whether NADPH oxidases are also active in PH development induced by monocrotaline (MCT) in rats. ROS production, their possible sources and their impact on the function of pulmonary arterial smooth muscle cells (PASMC) was observed. MCT-PASMC showed an increased intracellular superoxide production, migration and proliferation as compared to healthy controls due to increased Nox1 expression. Comparing PASMC from MCT-treated and non-treated rats revealed an upregulation of Sod2, Nrf2 and cyclin D1, as well as an increased phosphorylation of cofilin. However, only Nrf2 and cyclin D1 turned out to be Nox1 dependent. In conclusion, we showed that in contrast to hypoxia-induced PH not Nox4 but Nox1 is a key regulator of proliferation and migration in PASMC. Our data provided evidence that an upregulation of superoxide derived from increased Nox1 expression enhances proliferation and migration of PASMC. Proliferation is most probably increased via cyclin D1. Thus, different Nox isoforms maybe contribute to PH development in in PH of different origin.

Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

IntroductionPulmonary Arterial Hypertension (PAH) is a progressively devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. The proliferation of cancer cells is mediated by increased expression of Enhancer of Zeste Homolog 2 (EZH2), a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes. However, the role of EZH2 in PAH has not been studied. In this study, it is hypothesized that EZH2 could play a role in the proliferation of PASMCs.MethodsIn the present study, the expression patterns of EZH2 were investigated in normal and hypertensive mouse PASMCs. The effects of EZH2 overexpression on the proliferation of human PASMCs were tested. PASMCs were transfected with EZH2 or GFP using nucleofector system. After transfection, the cells were incubated for 48 hours at 37°C. Proliferation and cell cycle analysis were performed using flow cytometry. Apoptosis of PASMCs was determined using annexin V staining and cell migration was tested by wound healing assay.ResultsEZH2 protein expression in mouse PASMCs were correlated with an increase in right ventricular systolic pressure and Right Ventricular Hypertrophy (RVH). The overexpression of EZH2 in human PASMCs enhances proliferation, migration, and decrease in the rate of apoptosis when compared to GFP-transfected cells. In the G2/M phase of the EZH2 transfected cells, there was a 3.5 fold increase in proliferation, while there was a significant decrease in the rate of apoptosis of PASMCs, when compared to control.ConclusionThese findings suggest that EZH2 plays a role in the migration and proliferation of PASMCs, which is a major hallmark in PAH. It also suggests that EZH2 could play a role in the development of PAH and can serve as a potential target for new therapies for PAH.

Endogenous and pharmacological inhibition of calpain decreases migration of hypoxic pulmonary arterial smooth muscle cells (PASMCs)

Prolonged exposure to hypoxia causes pulmonary hypertension due to contraction, proliferation and migration of PASMCs, which result from hypoxia‐induced elevations in basal intracellular calcium concentration ([Ca2+]i). Calpain, a Ca2+‐dependent cysteine protease, has two major isoforms, μ‐ and m‐calpain, that require μM and mM [Ca2+]i, respectively, for activation. Calpain is known to participate in fibroblast and endothelial cell migration and proliferation, and PASMCs exhibit increased calpain levels during hypoxia (Ma et al, JCI 2011). In this study, we hypothesized that reducing calpain activity through in vivo overexpression of calpastatin (CAST), an endogenous calpain inhibitor, or in vitro inhibition with the pharmacological agent, calpeptin, would impair migration of PASMCs. For in vivo experiments, wild type (WT) and CAST overexpressing mice were exposed to normoxia or chronic hypoxia (10% O2; 21 d). For in vitro experiments, PASMCs were isolated from WT or CAST mice before exposure to normoxia or hypoxia (4% O2; 24 h). PASMC migration was measured using a modified Boyden chamber transwell assay. For both in vitro and in vivo experiments, WT, but not CAST, PASMCs exhibited increased migration in response to hypoxia. In WT PASMCs, calpeptin prevented hypoxia‐induced migration. These results suggest that calpain activity is necessary to facilitate the migration of hypoxic PASMCs.

PAR-2 Inhibition Reverses Experimental Pulmonary Hypertension

A hallmark of the vascular remodeling process underlying pulmonary hypertension (PH) is the aberrant proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). Accumulating evidence suggests that mast cell mediators play a role in the pathogenesis of PH. In the present study we investigated the importance of protease-activated receptor (PAR)-2 and its ligand mast cell tryptase in the development of PH. Our results revealed strong increase in PAR-2 and tryptase expression in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline (MCT)-treated rats. Elevated tryptase levels were also detected in plasma samples from IPAH patients. Hypoxia and platelet-derived growth factor (PDGF)-BB upregulated PAR-2 expression in PASMC. This effect was reversed by HIF (hypoxia inducible factor)-1α depletion, PDGF-BB neutralizing antibody, or the PDGF-BB receptor antagonist Imatinib. Attenuation of PAR-2 expression was also observed in smooth muscle cells of pulmonary vessels of mice exposed to hypoxia and rats challenged with MCT in response to Imatinib treatment. Tryptase induced PASMC proliferation and migration as well as enhanced synthesis of fibronectin and matrix metalloproteinase-2 in a PAR-2- and ERK1/2-dependent manner, suggesting that PAR-2-dependent signaling contributes to vascular remodeling by various mechanisms. Furthermore, PAR-2(-/-) mice were protected against hypoxia-induced PH, and PAR-2 antagonist application reversed established PH in the hypoxia mouse model. Our study identified a novel role of PAR-2 in vascular remodeling in the lung. Interference with this pathway may offer novel therapeutic options for the treatment of PH.