Migration Of Human Pulmonary Artery Smooth Muscle Cells Research Articles

In Vitro Experimental Approach for Studying Human Pulmonary Artery Smooth Muscle Cells and Endothelial Cells Proliferation and Migration.

Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs.

Inhibition of KIR2.1 decreases pulmonary artery smooth muscle cell proliferation and migration

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the in vivo experiment, a PH model was established by intraperitoneally injecting Sprague-Dawley rats monocrotaline (MCT). Hematoxylin and eosin staining revealed evidence of PVR in the rats with PH. Immunofluorescence staining and western blot analysis revealed increased levels of the KIR2.1, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) proteins in pulmonary blood vessels and lung tissues following exposure to MCT, and the TGF-β1/SMAD2/3 signaling pathway was activated. For the in vitro experiments, the KIR2.1 inhibitor, ML133, or the TGF-β1/SMAD2/3 signaling pathway blocker, SB431542, were used to pre-treat human PASMCs (HPASMCs) for 24 h, and the cells were then treated with platelet-derived growth factor (PDGF)-BB for 24 h. Scratch and Transwell assays revealed that PDGF-BB promoted cell proliferation and migration. Immunofluorescence staining and western blot analysis demonstrated that PDGF-BB upregulated OPN and PCNA expression, and activated the TGF-β1/SMAD2/3 signaling pathway. ML133 reversed the proliferation and migration induced by PDGF-BB, inhibited the expression of OPN and PCNA, inhibited the TGF-β1/SMAD2/3 signaling pathway, and reduced the proliferation and migration of HPASMCs. SB431542 pre-treatment also reduced cell proliferation and migration; however, it did not affect KIR2.1 expression. On the whole, the results of the present study demonstrate that KIR2.1 regulates the TGF-β1/SMAD2/3 signaling pathway and the expression of OPN and PCNA proteins, thereby regulating the proliferation and migration of PASMCs and participating in PVR.

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.

LncRNA HOXA-AS3 Promotes the Progression of Pulmonary Arterial Hypertension through Mediation of miR-675-3p/PDE5A Axis.

Pulmonary arterial hypertension (PAH) seriously threatens the elder people. Long non-coding RNAs (lncRNAs) are involved in multiple diseases. However, the study of the lncRNAs in the occurrence of PAH is just beginning. For this, we sought to explore the biological function of lncRNA HOXA cluster antisense RNA 3 (HOXA-AS3) in PAH. Hypoxia (HYP) was used to mimic in vitro model of PAH. Gene and protein expressions in cells were detected by q-PCR and Western blotting, respectively. In addition, cell proliferation and viability were tested by CCK-8 and MTT assay. Cell apoptosis was measured by flow cytometry. Wound healing was used to detect cell migration. Furthermore, the connection of HOXA-AS3, miR-675-3p, and phosphodiesterase 5A (PDE5A) was verified by dual-luciferase report assay. HOXA-AS3 and PDE5A were upregulated in human pulmonary artery smooth muscle cells (HPASMCs) in the presence of HYP, while miR-675-3p was downregulated. Moreover, knockdown of HOXA-AS3 suppressed the growth and migration of HPASMCs, but induced the apoptosis. Overexpression of miR-675-3p achieved the same effect. MiR-675-3p inhibitor or overexpression of PDE5A notably reversed the inhibitory effect of HOXA-AS3 knockdown on PAH. Finally, HOXA-AS3 could sponge miR-675-3p, and PDE5A was directly targeted by miR-675-3p. HOXA-AS3 increased the development of PAH via regulation of miR-675-3p/PDE5 axis, which could be the potential biomarker for treatment of PAH.

Targeting Jak-Stat Signaling in Experimental Pulmonary Hypertension.

In pulmonary arterial hypertension (PAH), progressive structural remodeling accounts for the pulmonary vasculopathy including the obliteration of the lung vasculature that causes an increase in vascular resistance and mean blood pressure in the pulmonary arteries ultimately leading to right heart failure-mediated death. Deciphering the molecular details of aberrant signaling of pulmonary vascular cells in PAH is fundamental for the development of new therapeutic strategies. We aimed to identify kinases as new potential drug targets that are dysregulated in PAH by means of a peptide-based kinase activity assay. We performed a tyrosine kinase-dependent phosphorylation assay using 144 selected microarrayed substrate peptides. The differential signature of phosphopeptides was used to predict alterations in tyrosine kinase activities in human pulmonary arterial smooth muscle cells (HPASMCs) from patients with idiopathic PAH (IPAH) compared with healthy control cells. Thereby, we observed an overactivation and an increased expression of Jak2 (Janus kinase 2) in HPASMCs from patients with IPAH as compared with controls. In vitro, IL-6-induced proliferation and migration of HPASMCs from healthy individuals as well as from patients with IPAH were reduced in a dose-dependent manner by the U.S. Food and Drug Administration-approved Jak1 and Jak2 inhibitor ruxolitinib. In vivo, ruxolitinib therapy in two experimental models of pulmonary arterial hypertension dose-dependently attenuated the elevation in pulmonary arterial pressure, partially reduced right ventricular hypertrophy, and almost completely restored cardiac index without signs of adverse events on cardiac function. Therefore, we propose that ruxolitinib may present a novel therapeutic option for patients with PAH by reducing pulmonary vascular remodeling through effectively blocking Jak2-Stat3 (signal transducer of activators of transcription)-mediated signaling pathways.

Levels of aryl hydrocarbon receptor nuclear translocator in lung tissue of children with congenital heart disease pulmonary hypertension and its effect on proliferation and migration of pulmonary artery smooth muscle cells

Objective To observe the levels of aryl hydrocarbon receptor nuclear translocator (ARNT) in lung tissue of children with congenital heart disease (CHD) pulmonary artery hypertension (PAH) and its effect on proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs), and to investigate the action mechanism of ARNT in the pathogenesis of CHD-PAH. Methods Sixty patients with CHD ventricular septal defect-associated PAH were selected as CHD-PAH group and 60 children with CHD ventricular septal defect without PAH were selected as CHD group (CHD group) in our hospital. There were no significant difference in age and gender between the two groups. HPASMCs were divided into normoxia group and hypoxia group, which were induced by normoxia and hypoxia respectively. HPASMCs were divided into blank control group (BC group), negative control group (NC group) and siR-ARNT group. The cells in the siR-ARNT group were transfected with ARNT-shRNA lentivirus, and those in the NC group were transfected with negative control lentivirus. The cells in the BC group were not transfected. The expression levels of ARNT protein and mRNA in lung tissue and cells were detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The cell proliferation ability was determined by the cell counting kit-8 (CCK-8) method. Transwell and scratch assays were used to measure cell migration ability. SPSS 20.0 software was used for analysis. The test methods were t test and analysis of variance. Results The expression levels of ARNT protein and mRNA in lung tissue of CHD-PAH group (0.53±0.06 and 2.36±0.17) were significantly higher than those in CHD group (0.09±0.02, 1.00±0.12, t=53.889 and 50.626, P 0.05). Conclusion The ARNT levels in lung tissue of children with CHD-PAH are elevated. ARNT may participate in the development of CHD-PAH by affecting the proliferation and migration of HPASMCs. Key words: Congenital heart disease; Pulmonary hypertension; Aryl hydrocarbon receptor nuclear translocation protein; Pulmonary artery smooth muscle cells; Proliferation; Migration

MiR-19a modulates hypoxia-mediated cell proliferation and migration via repressing PTEN in human pulmonary arterial smooth muscle

AimThe dysfunction of human pulmonary arterial smooth muscle cells (HPASMCs) has been suggested to participate in the pathophysiology of pulmonary arterial hypertension (PAH). This study determined miR-19a expression in hypoxia-induced HPASMCs and explored the mechanistic actions of miR-19a in hypoxia-induced HPASMC proliferation and migration. MethodsQRT-PCR and western blot assays respectively determined the mRNA and protein expression of miR-19a, phosphatase and tensin homolog (PTEN) and hypoxia-inducible factor-1 alpha (HIF-1α). In vitro functional assays determined HPASMC proliferation and migration, respectively. Luciferase reporter assay determined interaction between miR-19a and PTEN. The knockdown effects of miR-19a on PAH were confirmed in in vivo mice model. ResultsHypoxia treatment time-dependently up-regulated miR-19a expression and enhanced cell proliferation in HPASMCs. MiR-19a overexpression increased cell proliferation and migration of HPASMCs, while repression of miR-19a reduced cell proliferative and migratory potentials of hypoxia-treated HPASMCs. Bioinformatics analysis and luciferase reporter assay showed that PTEN 3’ untranslated region was targeted by miR-19a, and miR-19a repressed the mRNA and protein expression of PTEN in HPASMCs. Further rescue studies revealed that miR-19a regulated proliferative and migratory potentials of hypoxia-treated HPASMCs via suppressing PTEN expression. In addition, HIF-1α was identified as one of the mediators for the hypoxia-induced aberrant expression levels of miR-19a and PTEN. MiR-19a overexpression enhanced PI3K/AKT signaling, which was attenuated by enforced expression of PTEN in HPASMCs. More importantly, knockdown of miR-19 attenuated the chronic hypoxia-induced PAH in in vivo mice model. ConclusionThis study presented a novel mechanistic action of miR-19a-mediated cell proliferation and migration of HPASMCs.

Long non‑coding RNA MALAT1 sponges miR‑124‑3p.1/KLF5 to promote pulmonary vascular remodeling and cell cycle progression of pulmonary artery hypertension.

Previous studies have demonstrated that long non-coding RNA (lncRNA) is involved in vascular remodeling. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA is associated with the proliferation and migration of vascular smooth muscle and endothelial cells; however, its biological role in pulmonary artery hypertension (PAH) is currently unclear. The aim of the present study was to investigate the post-transcriptional regulation of MALAT1 in human pulmonary artery smooth muscle cells (HPASMCs). The results revealed that MALAT1 expression levels were significantly upregulated in the pulmonary arteries (PAs) and HPASMCs obtained from patients with PAH compared with adjacent normal PA tissues and HPASMCs. Knockdown of MALAT1 suppressed the viability and proliferation of HPASMCs and prevented cells entering the G0/G1 cell cycle phase. MALAT1 overexpression exerted the opposite effects. Bioinformatics analysis predicted complementary binding of hsa-microRNA (miR)-124-3p.1 with the 3′-untranslated region of MALAT1. Luciferase reporter assays and RNA immunoprecipitation experiments demonstrated molecular binding between MALAT1 and hsa-miR-124-3p.1. This resulted in the formation of an RNA-induced silencing complex. In addition, Kruppel-like factor 5 (KLF5) was confirmed to be a target gene of MALAT1/hsa-miR-124-3p.1. MALAT1 silencing did not inhibit the proliferation and migration of HPASMCs following knockdown of hsa-miR-124-3p.1. In addition, MALAT1 knockdown was demonstrated to attenuate the expression of KLF5. Following MALAT1 knockdown, the expression level of KLF5 was rescued by inhibition of hsa-miR-124-3p.1 expression. The results of the current study indicate that the MALAT1/hsa-miR-124-3p.1/KLF5 axis may serve a key role in HPASMCs. In addition, the results contribute to what is known regarding the role of MALAT1 in PAH development and provide a novel theoretical basis for the development of new therapeutic interventions for patients with PAH.

Targeting IL-17 attenuates hypoxia-induced pulmonary hypertension through downregulation of β-catenin

BackgroundThe role of interleukin 17 (IL-17) in hypoxic pulmonary hypertension (HPH) remains unclear. This study is designed to explore whether IL-17 is a potential target for HPH treatment.MethodsClinic samples from...

Aldehyde dehydrogenase 2 protects against oxidative stress associated with pulmonary arterial hypertension

The cardioprotective benefits of aldehyde dehydrogenase 2 (ALDH2) are well established, although the regulatory role of ALDH2 in vascular remodeling in pulmonary arterial hypertension (PAH) is largely unknown. ALDH2 potently regulates the metabolism of aldehydes such as 4-hydroxynonenal (4-HNE), the endogenous product of lipid peroxidation. Thus, we hypothesized that ALDH2 ameliorates the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs) by inhibiting 4-HNE accumulation and regulating downstream signaling pathways, thereby ameliorating pulmonary vascular remodeling. We found that low concentrations of 4-HNE (0.1 and 1μM) stimulated cell proliferation by enhancing cyclin D1 and c-Myc expression in primary HPASMCs. Low 4-HNE concentrations also enhanced cell migration by activating the nuclear factor kappa B (NF-κB) signaling pathway, thereby regulating matrix metalloprotein (MMP)-9 and MMP2 expression in vitro. In vivo, Alda-1, an ALDH2 agonist, significantly stimulated ALDH2 activity, reducing elevated 4-HNE and malondialdehyde levels and right ventricular systolic pressure in a monocrotaline-induced PAH animal model to the level of control animals. Our findings indicate that 4-HNE plays an important role in the abnormal proliferation and migration of HPASMCs, and that ALDH2 activation can attenuate 4-HNE-induced PASMC proliferation and migration, possibly by regulating NF-κB activation, in turn ameliorating vascular remodeling in PAH. This mechanism might reflect a new molecular target for treating PAH.

MiR-20a regulates the PRKG1 gene by targeting its coding region in pulmonary arterial smooth muscle cells

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with de-differentiation of pulmonary artery smooth muscle cells (PASMC). Here, we show that miR-20a expression is up-regulated in response to hypoxia in both mouse and human PASMC. We also observed that miR-20a represses the protein kinase, cGMP-dependent, type I (PRKG1) gene and we identified two crucial miR-20a binding sites within the coding region of PRKG1. Functional studies showed that miR-20a promotes the proliferation and migration of human PASMC, whereas it inhibits their differentiation. In summary, we provided a possible mechanism by which hypoxia results in decreased PRKG1 expression and in the phenotypic switching of PASMC.

Silencing of Sodium–Hydrogen Exchanger 1 Attenuates the Proliferation, Hypertrophy, and Migration of Pulmonary Artery Smooth Muscle Cells via E2F1

We previously found that deficiency of the sodium-hydrogen exchanger 1 (NHE1) gene prevented hypoxia-induced pulmonary hypertension and vascular remodeling in mice, which were accompanied by a significantly reduced proliferation of pulmonary artery smooth muscle cells (PASMCs), and which decreased the medial-wall thickness of pulmonary arteries. That finding indicated the involvement of NHE1 in the proliferation and hypertrophy of PASMCs, but the underlying mechanism was not fully understood. To define the mechanism by which the inhibition of NHE1 decreases hypoxic pulmonary hypertension and vascular remodeling, we investigated the role of E2F1, a nuclear transcription factor, in silencing the NHE1 gene-induced inhibition of the proliferation, hypertrophy, and migration of human PASMCs. We found that: (1) silencing of NHE1 by short, interfering RNA (siRNA) significantly inhibited PASMC proliferation and cell cycle progression, decreased hypoxia-induced hypertrophy (in terms of cell size and protein/DNA ratio) and migration (in terms of the wound-healing and migration chamber assays); (2) hypoxia induced the expression of E2F1, which was reversed by NHE1 siRNA; and (3) the overexpression of E2F1 blocked the inhibitory effect of NHE1 siRNA on the proliferation, hypertrophy, and migration of PASMCs. The present study determined that silencing the NHE1 gene significantly inhibited the hypoxia-induced proliferation, hypertrophy, and migration of human PASMCs via repression of the nuclear transcription factor E2F1. This study revealed a novel mechanism underlying the regulation of hypoxic pulmonary hypertension and vascular remodeling via NHE1.

Abstract 376: A Potential Role for TRAIL in the Pathogenesis of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a life threatening disease characterised by the narrowing and occlusion of small pulmonary arteries. Endothelial cell (EC) loss, and increased proliferation and migration of pulmonary artery smooth muscle cells (PA-SMC) are strongly implicated in disease pathogenesis. Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) induces EC apoptosis and SMC migration, consistent with an important role in vascular biology. TRAIL is a type II TNF family transmembrane protein, whose extracellular domain can be proteolytically cleaved from the cell surface to act as a soluble cytokine (sTRAIL) capable of binding to five TRAIL receptors, (TRAIL-R1– 4) and osteoprotegerin (OPG). We hypothesised that TRAIL expression is increased in PAH and that this contributes to the excessive migration of PA-SMC that characterises this condition. TRAIL gene expression was studied in microarrays of PA-SMC RNA isolated from idiopathic PAH (IPAH) patients, and from the lungs of monocrotaline-treated (Mct) rats. Immunohistochemistry was performed to localise TRAIL in both human and rat lung sections. To investigate the functional effects of TRAIL in PA-SMC, human PA-SMC were incubated with recombinant human TRAIL (1–50 ng/ml) for 5h and migration assessed by transwell assay. TRAIL gene expression was 10-fold higher in PA-SMC from IPAH patients compared to controls. Moreover, strong TRAIL immunoreactivity was observed in pulmonary vascular lesions from IPAH patients but not in vessels from patients undergoing lobectomy for lung carcinoma. In the Mct-rat model of PAH we found a 20-fold increase in TRAIL gene expression and a 2-fold increase in protein from whole lung extracts. Immunohistochemistry revealed strong TRAIL immunoreactivity within the muscularised pulmonary arteries of Mct-treated but not saline-treated control rats. Recombinant TRAIL was found to induce a dose dependent (maximum 3 fold) increase in migration of human PA-SMC compared with cells incubated with 0.1% serum alone. These data suggest that TRAIL may be an important molecule in the pathogenesis of PAH. Further studies to elucidate the regulation and function of this molecule in PAH are warranted.