Apoptosis Of Pulmonary Artery Smooth Muscle Cells Research Articles

MiR-200c regulates endothelin-1 induced PASMCs abnormal proliferation and apoptosis.

miR-200c is an antioncogene in multiple tumors. However, its function in the pathogenesis of pulmonary arterial hypertension (PAH) has not been thoroughly investigated nor understood. In this study, we discovered that miR-200c was able to substantially upregulate in pulmonary arterial smooth muscle cells (PASMCs) treated with endothelin-1 (ET-1). miR-200c also induced cell proliferation and suppressed cell apoptosis in PASMCs in vitro. However, miR-200c had no effect on G1/S/G2 transitions during the cell cycle. Furthermore, we identified miR-200c as a new regulator of the microtubule associated protein 2 (MAP-2) and zinc finger E-box binding homeobox1 (ZEB-1) in PASMCs. miR-200c inhibited MAP-2 and ZEB-1 expression by directly binding to their 3'-untranslated regions(3'UTR) according to luciferase assay results. Our findings provide novel insights into the mechanisms of PAH pathogenesis and potential molecular biomarkers for PAH diagnosis and treatment. © 2017 IUBMB Life, 69(11):877-886, 2017.

Inhibition of ARC promoting the apoptosis of rat pulmonary arterial smooth muscle cells after serum deprivation invitro.

Apoptosis has important pathophysiological consequences contributing to pulmonary arterial hypertension (PAH). However, the mechanism underlying apoptosis in PAH remains unknown. Apoptosis repressor with caspase recruitment domain (ARC) is an essential factor in cell apoptosis, and regulates intrinsic and extrinsic apoptosis signaling pathways. It is hypothesized that ARC may be involved in the apoptotic responses of pulmonary arterial smooth muscle cells (PASMCs) following mild chronic injury. In the present study, serum deprivation (SD) was used to induce apoptosis of PASMCs. It was demonstrated that the expression of ARC in PASMCs was significantly increased following SD stimulation within 24 h, and ARC downregulation using small interfering RNA significantly enhanced the apoptosis of PASMCs following SD stimulation. In addition, the results demonstrated that ARC downregulation significantly increased the expression of proapoptotic factors and the level of reactive oxygen species (ROS), and decreased the mitochondrial membrane potential following SD exposure, suggesting ARC regulates the apoptosis of PASMCs via modulating mitochondrial function and ROS accumulation. The results of the present study revealed that ARC inhibition promotes the apoptosis of PASMCs following SD stimulation, and that ARC expression increases in the early stages of SD injury.

Mechanisms of N‑acetylcysteine in reducing monocrotaline‑induced pulmonary hypertension in rats: Inhibiting the expression of Nox1 in pulmonary vascular smooth muscle cells.

The aim of the present study was to investigate the impact of N‑acetylcysteine (NAC) on the expression of reduced nicotinamide adenine dinucleotide phosphate oxidase1 (Nox1), and the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) in rats exhibiting monocrotaline (MCT)‑induced pulmonary hypertension, and to investigate the possible mechanisms and treatment roles of NAC in pulmonary vascular remodeling (PVR). A total of 18 Wistar rats were randomly divided into three groups: The control (C) group; the MCT (M) group; and the NAC (N) group. The right ventricular hypertrophy index (RVHI) and other indicators were recorded 6weeks subsequently. Groups C and M were divided into two subgroups: Groups C1 and M1 (control); and group C2 and M2 group (treated with ML171). Group N was not sub‑divided. PASMCs were isolated, and the vascular remodeling and Nox1 positioning were observed. The expression of Nox mRNA in each group, and the proliferation, apoptosis, and superoxide dismutase (SOD) activity of PASMCs, prior to and following the ML171 treatment, were measured. NAC was able to decrease RVHI and other indicators (P<0.001). The mRNA expression of Nox1 and Nox4 in group M was significantly increased compared with group C (P<0.05), and NAC was able to significantly decrease the expression of these two factors in lung tissue (P<0.001). MCT‑PASMCs exhibited differences in Nox1 mRNA expression (P<0.001), and the total SOD activity was Nox1‑dependently increased (r=0.949; P<0.001). NAC was able to decrease Nox1‑derived reactive oxygen species in PASMCs, thereby improving PVR. Nox1 was able to increase SOD activity, thereby demonstrating its positive effect on the proliferation of MCT‑PASMCs.

Upregulation of SRF Is Associated With Hypoxic Pulmonary Hypertension by Promoting Viability of Smooth Muscle Cells via Increasing Expression of Bcl-2.

The aim of study was to investigate the involvement of hypoxia-induced upregulation of serum response factor (SRF) and its downstream effector, B cell leukemia-2 (Bcl-2), in hypoxia-induced pulmonary hypertension (PH). Immunohistochemistry analysis and western blot analysis were used to detect the levels of SRF and Bcl-2 in rats exposed to hypoxia. Furthermore, the regulatory relationship between SRF and Bcl-2 was investigated in PASMCs using real-time PCR and western-blot analysis. We found that mPAP (mean pulmonary arterial pressure) and WA (the ratio of vascular wall area to external diameter) were increased after exposure to hypoxia, while LA (the ratio of vascular lumen area to total area) decreased after exposure to hypoxia. The immunohistochemistry analysis displayed a substantial increase in SRF and Bcl-2 in pulmonary arterial walls after 14 days of hypoxia. And the western blotting showed that SRF and Bcl-2 protein levels were much higher after 7 days of hypoxia and then remained at a high level. And then the levels of SRF and Bcl-2 in pulmonary artery smooth muscle cells (PASMCs) exposed to hypoxia were substantially suppressed following introduction of SRF siRNA, and the level of Bcl-2 was remarkably inhibited by Bcl-2 siRNA, while Bcl-2 siRNA had no effect on SRF level. Finally, SRF siRNA, and Bcl-2 siRNA significantly reduced viability of PASMCs exposed to hypoxia, and enhanced apoptosis of PASMCs exposed to hypoxia. These data validated that SRF responded to hypoxia, which subsequently was involved in pulmonary hypertension by abnormally promoting viability of PASMCs via modulating expression of Bcl-2. J. Cell. Biochem. 118: 2731-2738, 2017. © 2017 Wiley Periodicals, Inc.

HMGB1 down-regulation mediates terameprocol vascular anti-proliferative effect in experimental pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis. Pulmonary artery smooth muscle cells (PASMCs) play a crucial role in PAH pathophysiology, displaying a hyperproliferative, and apoptotic-resistant phenotype. In the present study, we evaluated the potential therapeutic role of terameprocol (TMP), an inhibitor of cellular proliferation and promoter of apoptosis, in a well-established pre-clinical model of PAH induced by monocrotaline (MCT) and studied the biological pathways modulated by TMP in PASMCs. Wistar rats injected with MCT or saline (SHAM group) were treated with TMP or vehicle. On day 21 after injection, we assessed bi-ventricular hemodynamics and cardiac and pulmonary morphometry. The effects of TMP on PASMCs were studied in a primary culture isolated from SHAM and MCT-treated rats, using an iTRAQ-based proteomic approach to investigate the molecular pathways modulated by this drug. In vivo, TMP significantly reduced pulmonary and cardiac remodeling and improved cardiac function in PAH. In vitro, TMP inhibited proliferation and induced apoptosis of PASMCs. A total of 65 proteins were differentially expressed in PASMCs from MCT rats treated with TMP, some of which involved in the modulation of transforming growth factor beta pathway and DNA transcription. Anti-proliferative effect of TMP seems to be explained, at least in part, by the down-regulation of the transcription factor HMGB1. Our findings support the beneficial role of TMP in PAH and suggest that it may be an effective therapeutic option to be considered in the clinical management of PAH.

The regulation of MAPK signaling pathway on cell proliferation and apoptosis in hypoxic PASMCs of rats

To explore the relationship between hypoxic pulmonary arterial smooth muscle cells(PASMCs)proliferation, apop-tosis and mitogen-activated protein kinases(MAPK) signal pathway in rats. PASMCs were obtained from male SD rats by the enzyme digestion method and primarily cultured; PASMCs were identified through two methods:immunofluorescence staining and light microscopy; the 4~6th generation PASMCs of logarithmic growth state of good growth period were selected, and randomly divided into 7 groups:normoxic con-trol group (N), hypoxia group (H), DMSO group (D), extracellular signal-regulated kinase1/2(ERK1/2) inhibitor-U0126 group (U) and p38MAPK inhibitor-SB203580 group (S), the p38MAPK activator-Anisomycin group (A), the ERK1/2 activator-Staurosporine Aglycone group (SA). When all the models were completed, the all groups joined the CCK-8 to measure cell proliferation; cell apoptosis of each group was detected by TUNEL kit after the modeling. Compared with N group, the expression of OD value in H group was up-regulated (0.990 ±0.041 vs 1.143 ±0.033,P < 0.01). There was no statistical significance on PASMCs apoptosis index(AI) in H group (4.913 ±0.451 vs 5.452 ±0.557, P > 0.05); Compared With H group, there were no statistical significance on the expression of PASMCs OD value and apoptosis index(AI)in D group (1.143 ±0.033 vs 1.142 ±0.049,5.452 ±0.557 vs 5.402 ±0.651,P > 0.05); the expression of OD value in U group was down-regulated, and the expression of AI was up-regulated (1.143 ±0.033 vs 0.985 ±0.078, 5.452 ±0.557 vs 10.145 ±2.545, P < 0.01); the expression of OD value in S group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 vs 1.295 ±0.039, 5.452 ±0.557 vs 3.093 ±0.409, P < 0.01); the expression of OD value in A group was down-regulated, and the expres-sion of AI was up-regulated (1.143 ±0.033 vs 0.347 ±0.067, 5.452 ±0.557 vs 25.753 ±1.262, P < 0.01); the expression of OD value in SA group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 vs 1.685 ±0.100, 5.452 ±0.557 vs 1.700 ±0.095, P < 0.01). The regulation of PASMCs' proliferation and apoptosis under hypoxia condition have a relationship with the participation of MAPK signal pathway.

Let-7a-transfected mesenchymal stem cells ameliorate monocrotaline-induced pulmonary hypertension by suppressing pulmonary artery smooth muscle cell growth through STAT3-BMPR2 signaling

BackgroundCell-based gene therapy has become a subject of interest for the treatment of pulmonary arterial hypertension (PAH), a devastating disease characterized by pulmonary artery smooth muscle cell (PASMC) hyperplasia. Mesenchymal stem cells (MSCs) have been recently acknowledged as a potential cell vector for gene therapy. Here, we investigated the effect of MSC-based let-7a for PAH.MethodsAfter isolation and identification of MSCs from rat bone marrow, cells were infected with recombinant adenovirus vector Ad-let-7a. Lewis rats were subcutaneously injected with monocrotaline (MCT) to induce PAH, followed by the administration of MSCs, MSCs-NC (miR-control), or MSC-let-7a, respectively. Then, right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling were evaluated. Rat pulmonary artery smooth muscle cells (rPASMCs) under hypoxia were co-cultured with MSCs or MSC-let-7a. Cell proliferation and apoptosis were separately determined by 3H thymidine incorporation and flow cytometry analysis. The underlying mechanism was also investigated.ResultsMSC transplantation enhanced let-7a levels in MCT-induced PAH rats. After injection with MSC-let-7a, RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling were notably ameliorated, indicating a protective effect of MSC-let-7a against PAH. When co-cultured with MSC-let-7a, hypoxia-triggered PASMC proliferation was obviously attenuated, concomitant with the decrease in cell proliferation-associated proteins. Simultaneously, the resistance of PASMCs to apoptosis was remarkably abrogated by MSC-let-7a administration. A mechanism assay revealed that MSC-let-7a restrained the activation of signal transducers and activators of transcription 3 (STAT3) and increased its downstream bone morphogenetic protein receptor 2 (BMPR2) expression. Importantly, preconditioning with BMPR2 siRNA dramatically abated the suppressive effects of MSC-let-7a on PASMC proliferation and apoptosis resistance.ConclusionsCollectively, this study suggests that MSCs modified with let-7a may ameliorate the progression of PAH by inhibiting PASMC growth through the STAT3-BMPR2 signaling, supporting a promising therapeutic strategy for PAH patients.

Resveratrol Protects Against Pulmonary Arterial Hypertension in Rats via Activation of Silent Information Regulator 1

Background/Objectives: The polyphenol resveratrol (Rev) has been found to exhibit various beneficial effects including prevention of pulmonary arterial hypertension (PAH). The present study was designed to investigate the action and potential mechanism of Rev on PAH, focusing on the role of SIRT1 (Silent Information Regulator 1) in apoptosis of pulmonary artery smooth muscle cells (PASMCs). Methods: PAH rats were established by exposure to hypoxia for 21 days. Rev and SRT1720 (a selective SIRT1 activator) were used to reverse PAH by gavaging rats. PASMCs were confronted with hypoxia for 24 h or 48 h and were then treated with Rev or SRT1720 in vitro. Western blot was performed to detect the protein expression of SIRT1. CCK-8 and scratch wound experiments were carried out to verify cell proliferation. In addition, the TUNEL positive assay and flow cytometry assay were used to measure PASMC apoptosis. Mitochondrial permeability transition (mPT) was identified by confocal microscopy. Right ventricular systolic pressure (RVSP) was determined with a Gould pressure transducer, and right ventricular hypertrophy (RVH) was determined by weighing the cardiac muscle. Results: We demonstrated that Rev could reverse the remodelling of the pulmonary vasculature, thus contributing to alleviating the severity of PAH. Down-regulation of SIRT1 was observed in PAH, but administration of Rev had no obvious effect on the protein expression of SIRT1. In addition, Rev could induce mitochondrial swelling and nuclear pyknosis, leading to small, dense, and dysmorphic mitochondria in rats exposed to hypoxia alone. Rev treatment inhibited PASMC proliferation in a dose-dependent manner in vitro. Incubation with SRT1720, a specific activator of SIRT1, significantly retarded PASMC proliferation and promoted PASMC apoptosis in vitro. The mechanism could be associated with inducing mPT damage in PASMCs. Rev and SRT1720 treatment mitigated RVSP and reduced RVH. Conclusion: Rev produced a beneficial effect partially by enhancing the activation of SIRT1, thus improving RVSP and reducing RVH. SIRT1 activation increased PASMC apoptosis by inducing mPT dysfunction, which might be a novel future strategy for the treatment of PAH.

Schisandrin B displays a protective role against primary pulmonary hypertension by targeting transforming growth factor β1

Pulmonary arterial smooth muscle cells (PASMCs) in the medial layer of the vessel wall are involved in vessel homeostasis, but also for pathologic vascular remodeling in diverse diseases, such as pulmonary arterial hypertension (PAH). Pulmonary vascular remodeling in PAH results in vascular disorders, but its underlying molecular mechanisms are still not to be fully disclosed. In this study, we investigated the expression and function of the transforming growth factor (TGF)-β1 in human PASMC cultured under the condition of hypoxia and elucidated the effect of schisandra chinensis and its active ingredients on proliferation, migration, and apoptosis in human PASMCs. We demonstrated that schisandrin B (Sch.B) alleviated the severity of PAH in PASMCs cultured under the condition of hypoxia. Significant upregulation of TGF-β1 was observed in hypoxia-induced human PASMCs. Interestingly, administration of Sch.B substantially attenuated TGF-β1 level in these PASMCs. In order to elucidate Sch.B function, the hypoxia-induced human PASMC was stimulated with Sch.B or cotreatment with TGF-β1 in vitro. In agreement with its TGF-β1-reducing effect, Sch B relieved human PASMCs migration and promoted the apoptosis of human PASMCs, by activation of TGF-β1 downstream signal pathways in PASMCs. In contrast, co-treatment with TGF-β1 promoted human PASMC proliferation and migration and inhibited the apoptosis of human PASMC, which can attenuate the protective role of Sch.B in human PASMC. Taken collectively, these findings suggest that the vascular relaxation evoked by Sch.B was mediated by direct effect on vascular smooth muscle cell via TGF-β1 downstream signal pathways.

Iptakalim induces mitochondria-dependent apoptosis in hypoxic rat pulmonary arterial smooth muscle cells.

Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (HPH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Iptakalim (Ipt), an ATP sensitive potassium channel opener, ameliorates HPH in animal models. Here we investigated the effects of Ipt on proliferation and apoptosis of hypoxic rat PASMCs, and to determine the possible underlying mechanisms. Primary rat PASMCs were isolated and cultured. PASMCs were cultured for 24h in normoxia or hypoxia (5% O2) conditions with and without Ipt. Cell proliferation and cycle were determined by MTT assay and flow cytometry, respectively. Mitochondrial membrane potential (Δym) was detected by fluorescence microscope Western blot assays were used to examine the expression of cyclin D, CDK4, endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), platelet-derived growth factor-BB (PDGF-BB), Bax, Bcl-2, cytochrome c (Cyt c), caspase-9, and caspase-3 in PASMCs. We found that hypoxia significantly stimulated proliferation and rendered resistance to apoptosis in PASMCs. Ipt suppressed proliferation and induced cell cycle arrest in hypoxia PASMCs. Ipt decreased the expression of cyclin D, CDK4, HIF-1, ET-1, and PDGF-BB in hypoxia PASMCs. It reversed the depolarization of Δψm in hypoxia PASMCs too. Ipt significantly upregulated Bax expression and downregulated Bcl-2 expression, and promoted the release of Cyt c from mitochondria to cytoplasm in hypoxia PASMCs. Furthermore, Ipt significantly activated the caspase cascades evidenced by increased expression of caspase-9 and caspase-3 in hypoxia PASMCs. Ipt could inhibit cell proliferation and induce apoptosis associated with cell cycle arrest, decreased ET-1, HIF-1, cyclin D, CDK4, PDGF-BB and Δψm, increased Bax/Bcl-2 ratio, enhanced Cyt c release, and activation of caspases in PASMCs under hypoxia status. Our data indicated that Ipt could be a therapeutic candidate for treatment of HPH.

Axis inhibition protein 2 deficiency leads to hypoxic pulmonary hypertension through β-catenin signaling pathway

Pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, altered vasoreactivity and vascular remodeling induced by smooth muscle cell proliferation. Similarities exist between cancer and PAH. Aberrant expression of the tumor suppressor protein is closely associated with PAH. Here, we tested the hypothesis that a tumor suppressor-axis inhibition protein 2 (Axin2) deficiency leads to PAH. We measured right ventricular systolic pressure in Axin2 knockout mice and assessed the expression of Axin2 in patients. We found that Axin2 expression level was decreased in both mice exposed to chronic hypoxia and patients with PAH in remodeled pulmonary arterioles. Axin2 knockout mice showed elevated mean right ventricular systolic pressure and enhanced contraction in response to phenylephrine. An increase in the cross-sectional area of the vessels was occupied by the vessel wall, indicating pulmonary vascular remodeling. Furthermore, knocking down Axin2 with small interfering RNA inhibited apoptosis of pulmonary arterial smooth muscle cells (PASMCs). This inhibition was significantly abolished by β-catenin inhibitors, indicating that Axin2 through β-catenin increased vascular wall by inhibiting the apoptosis of PASMCs. Importantly, overexpression of Axin2 attenuates the development of hypoxia-induced PAH in mice. Taken together, our study, for the first time, established that Axin2 plays a key role in the progression of PAH. We identified Axin2 as a novel mediator of pulmonary vasoconstriction and PASMC growth in hypoxia-mediated PAH. Our results suggest that downregulation of Axin2 in the pulmonary vasculature may be an underlying mechanism in the development of hypoxia-induced PAH.

Calcineurin ‐NFAT Signaling Molecules Regulate Proliferation, Migration and Apoptosis in PASMCs of Pulmonary Hypertensive Rats induced by Monocrotaline

Pulmonary arterial hypertension (PAH) is characterized by increased resting intracellular free Ca2+ concentration of pulmonary arterial smooth muscle cells (PASMCs), angiogenesis and vascular remodeling. Previous studies of PAH show that canonical transient receptor potential (TRPC) genes are upregulated and store‐operated Ca2+ entry is augmented in PASMCs of monocrotaline (MCT)‐treated rats. While increased intracellular Ca2+ can further activate calcineurin (CaN) and make nuclear factor of activated T cells (NFAT) dephosphorylation and nuclear translocation. Since CaN and NFAT may functionally regulate the transcription of a variety of cytokines, we sought to examine the effects of CaN‐NFAT signaling molecules on proliferation, apoptosis and migration in PASMCs of control and MCT‐induced rats. Rats developed severe PAH and right ventricular hypertrophy within three weeks after single intraperitoneal injection of MCT at a dose of 60 mg/kg. MCT treatment can upregulated mRNA and protein expression of STIM1/2‐ORAI1/2‐CaNBβ‐NFATc2/3/4 on PAs, as well as developed proliferation and migration and decreased apoptosis on PASMCs. Pretreatment with Cyclosporine A (CsA) inhibited proliferation and migration on PASMCs of control (CON) and MCT rats, in a dose dependence manner (15, 30 60μM), but inhibited apoptosis only on high concentration (60μM). Proliferation, migration and apoptosis resistance of PASMCs in CON and MCT rats were obviously inhibit by siRNA of NFATc2 and NFATc4. Pretreatment with siRNA of NFATc3 were no effect to proliferation, migration and apoptosis resistance of PASMCs in CON rats, but can obviously suppressed proliferation and migration, promoted apoptosis of PASMCs in MCT rats. Our results hence show that MCT treatment caused significant alterations in the expression of STIM1/2‐ORAI1/2‐CaNβ‐NFATc2/3/4 signaling molecules. NFATc2/4 are essential factor for PASMCs growth, however, NFATc2/34 may contribute to pulmonary vascular remodeling in PAH induced by MCT.Support or Funding InformationThis work is supported by NSFC31171104, NSFC 31371165, NSFC 31571179 and FJMU09ZD010

Reactive oxygen species effect PASMCs apoptosis via regulation of dynamin-related protein 1 in hypoxic pulmonary hypertension.

The high level of reactive oxygen species and up-regulation of mitochondrial fission protein dynamin-related protein-1, both of which involved in pulmonary artery smooth muscle cells (PASMCs) apoptosis, have been detected in the lungs of rodent pulmonary arterial hypertension models. However, the regulatory mechanisms between ROS and DRP1 are poorly understood. In this study, ROS inhibitor, hypoxic rodent PAH models, small interfering RNA, polymerase chain reaction, Western blot, flow cytometry, immunohistochemistry and immunofluorescence were used. We determined that ROS, mainly derive from mitochondria, mediate mitochondria fission of PASMCs contributing to pulmonary vascular remodeling. Meanwhile, we also observed that hypoxia-induced DRP1 expression depends on ROS generation, especially mitochondrial ROS (mROS). Moreover, the levels of ROS and mROS evoked by hypoxia were regulated by DRP1. Furthermore, we verified the apoptosis suppression of PASMCs under hypoxia due to the interaction between ROS/mROS and DRP1. Our study reveals a novel mechanism of hypoxia-induced pulmonary vascular remodeling, suggesting a new therapeutic strategy which is targeting on the positive feedback of ROS/mROS-DRP1 for the treatment of PAH.

Involvement of CapG in proliferation and apoptosis of pulmonary arterial smooth muscle cells and in hypoxia-induced pulmonary hypertension rat model

ABSTRACTPurpose: Actin-binding protein capping protein gelsolin-like (CapG) was preferentially expressed in human pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, and reduced CapG expression was accompanied by impaired migration ability in vitro. We intended to investigate the effects of CapG on rat PASMCs and hypoxia-induced pulmonary hypertension (HPH) rat model. Materials and Methods: We investigated the effect of RNA interference-medicated down-regulation of CapG expression in rat PASMCs as well as in HPH rat model. The proliferation, apoptosis and cell cycle of PASMCs were evaluated. The HPH rat model was established by intratracheal instillation of lentiviral vector and subsequent hypoxia exposure for four weeks. Right ventricular systolic pressure, right ventricular hypertrophy and the percentage of medial wall thickness were measured to evaluate the development of HPH. Results: Knock-down CapG in PASMCs resulted in decreased proliferation, increased apoptosis and induced cell cycle inhibition. Down-regulation of CapG expression locally could attenuate pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy in HPH rat model. Conclusions: Our study indicated that CapG participated in the pathogenesis of pulmonary vascular remodeling in HPH rats, which was probably mediated by promoting the proliferation and inhibiting the apoptosis of PASMCs. We proposed CapG modulating protective effects of pulmonary hypertension.

Mitofusin 2 Downregulation Triggers Pulmonary Artery Smooth Muscle Cell Proliferation and Apoptosis Imbalance in Rats With Hypoxic Pulmonary Hypertension Via the PI3K/Akt and Mitochondrial Apoptosis Pathways.

During hypoxia-induced pulmonary hypertension (HPH), pulmonary artery smooth muscle cells (PASMCs) proliferate as part of the characteristic pulmonary vascular remodeling. We investigated the expression of mitofusin 2(Mfn2) and its role in maintaining the balance between PASMC proliferation and apoptosis during hypoxia. In an experimental model of HPH, we exposed rats to hypoxia (10% ± 0.5% O2) or room air for 4 weeks. We found that Mfn2 messenger RNA and protein levels were reduced and that proliferating cell nuclear antigen protein expression was upregulated in HPH rat lung tissues. We also exposed primary cultured PASMCs from rat pulmonary arterioles to normoxia (21% O2/5% CO2) or hypoxia (2.5% O2/5% CO2) for 24 hours. We found that PASMC proliferation increased under hypoxic conditions and that more hypoxic cells than normoxic cells entered the S + G2/M phase. Additionally, phosphorylated Akt and proliferating cell nuclear antigen expression increased, whereas Mfn2 expression, cleaved caspase 9 expression, and the ratio of mitochondrial to cytosolic cytochrome C expression each decreased. These hypoxia-induced effects were reversed in PASMCs by Mfn2 overexpression and by phosphatidylinositide 3-kinases (PI3K) inhibition. Our results indicate that downregulation of Mfn2 in HPH may activate the PI3K/Akt pathway, thereby causing more cells to enter the S + G2/M phase of the cell cycle and inhibiting the mitochondrial apoptosis pathway.

Carvacrol induces the apoptosis of pulmonary artery smooth muscle cells under hypoxia

The abnormal apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important pathophysiological process in pulmonary vascular remodeling and pulmonary arterial hypertension (PAH). Carvacrol, an essential oil compound from oregano and thyme, has displayed antimicrobial, antitumor, and antioxidant properties. Although carvacrol has pro-apoptosis properties in tumor cells, the underlying mechanisms of carvacrol in PASMC apoptosis remain unclear. Thus, in this study, we aim to investigate the role of carvacrol in pulmonary vascular remodeling and PASMC apoptosis in hypoxia. Right Ventricular Hypertrophy Measurements and pulmonary pathomorphology data show that the ratio of the heart weight/tibia length (HW/TL), the right ventricle/left ventricle plus septum (RV/LV+S) and the medial width of the pulmonary artery increased in chronic hypoxia and were reversed by carvacrol treatment under hypoxia. Additionally, carvacrol inhibited PASMC viability, attenuated oxidative stress, induced mitochondria membrane depolarization, increased the percentage of apoptotic cells, suppressed Bcl-2 expression, decreased procaspase-3 expression, promoted caspase-3 activation, and inhibited the ERK1/2 and PI3K/Akt pathway. Taken together, these findings suggest that carvacrol attenuates the pulmonary vascular remodeling and promotes PASMC apoptosis by acting on, at least in part, the intrinsic apoptotic pathway. This process might provide us new insight into the development of hypoxic pulmonary hypertension.

Abstract 13972: Dexmedetomidine Ameliorates Monocrotaline Induced Pulmonary Arterial Hypertension in Rats

[Introduction] Pulmonary arterial hypertension (PAH) is characterized by increased proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Dexmedetomidine (DEX) is a selective α2-aderenergic receptor agonist that is used for sedation in clinical practice. It has been reported that DEX inhibits inflammatory responses through cytokines, such as TNF-alpha, IL-6. Furthermore other reports show that G-protein-coupled receptors (GPCRs) are regulated by β-arrestins, which are also involved with inflammation. [Hypothesis] DEX ameliorates monocrotaline (MCT)-induced PAH in rats by its anti-inflammatory effect. [Methods] We treated 6 weeks-old male Sprague-Dawley rats with a single 60mg/kg intraperitoneal injection of MCT. After 14 days of injection, one group of rats was started to administer dexmedetomidine (dose: 2μg/kg/hour, MCT+DEX group) continuously using osmotic pumps, the other group was not treated with DEX (MCT group). We performed physiological examination and cardiac catheterization to measure right ventricular systolic pressure (RVSP) at day 23. [Results] Both RVSP and survival rate of rats in MCT+DEX group markedly improved compared with those in MCT group (RVSP; 38mmHg±11mmHg vs 91mmHg±6mmHg, survival rate; 42% vs 0% at day 30). In histological analysis, DEX reduced the medial hypertrophy of pulmonary arterioles, and decreased phosphorylated-NF-kB p65 (p-p65) positive PASMCs in MCT+DEX group compared with those of MCT group. In addition, DEX suppressed PASMCs proliferation with PCNA staining, and induced apoptosis of PASMCs with TUNEL assay. Then we examined the involvement of β-arrestins in PAH. It showed that βarrestin1 expressions reduced in MCT group compared with that of MCT+DEX group with western blotting and immunohistochemistry. However β-arrestin2 expressions had no significant difference between the two groups. [Conclusions] DEX ameliorates MCT-induced PAH in rats, one of the mechanism of which may be NF-kB inhibition through β-arrestin1. DEX can be a new therapeutic tool for PAH.

Quercetin reverses experimental pulmonary arterial hypertension by modulating the TrkA pathway

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation, resistance to apoptosis, and increased migration of pulmonary artery smooth muscle cells (PASMCs). We hypothesized that quercetin exerts protective effects against this disease; thus, a chronic hypoxia model of PAH was generated using male Sprague-Dawley rats, which were treated with quercetin. In this model, quercetin prevented the development of PAH, right ventricular hypertrophy, and vascular remodeling after exposure to hypoxia. Quercetin inhibited PASMC proliferation and increased the apoptosis of PASMCs in vivo. In vitro, quercetin significantly inhibited hypoxia-induced PASMC proliferation, arrested cells in G1/G0 and inhibited cell migration in a dose-dependent manner. Moreover, our results showed that quercetin increased cyclin D1 protein levels and decreased the protein expression of cyclin B1 and Cdc2. Additionally, quercetin altered the Bax/Bcl-2 ratio and reduced MMP2, MMP9, CXCR4, integrin β1, and integrin α5 expression. Using genome-wide microarray analysis, we found that factors regulating proliferation, apoptosis, cell cycle, and migration were related to the tyrosine receptor kinase A (TrkA) pathway. In addition, activation of the TrkA/AKT signaling cascade during hypoxia was inhibited by quercetin in a dose-dependent manner. Moreover, quercetin alone inhibited the TrkA/AKT signaling pathway, resulting in decreased PASMC migration, cell cycle arrest and the induction of apoptosis. Our data suggest that quercetin is a potential candidate for the treatment of hypoxia-induced PAH.

Dexamethasone induces apoptosis in pulmonary arterial smooth muscle cells.

BackgroundDexamethasone suppressed inflammation and haemodynamic changes in an animal model of pulmonary arterial hypertension (PAH). A major target for dexamethasone actions is NF-κB, which is activated in pulmonary vascular cells and perivascular inflammatory cells in PAH. Reverse remodelling is an important concept in PAH disease therapy, and further to its anti-proliferative effects, we sought to explore whether dexamethasone augments pulmonary arterial smooth muscle cell (PASMC) apoptosis.MethodsAnalysis of apoptosis markers (caspase 3, in-situ DNA fragmentation) and NF-κB (p65 and phospho-IKK-α/β) activation was performed on lung tissue from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH), before and after day 14–28 treatment with dexamethasone (5 mg/kg/day). PASMC were cultured from this rat PH model and from normal human lung following lung cancer surgery. Following stimulation with TNF-α (10 ng/ml), the effects of dexamethasone (10−8–10−6 M) and IKK2 (NF-κB) inhibition (AS602868, 0–3 μM (0-3×10−6 M) on IL-6 and CXCL8 release and apoptosis was determined by ELISA and by Hoechst staining. NF-κB activation was measured by TransAm assay.ResultsDexamethasone treatment of rats with MCT-induced PH in vivo led to PASMC apoptosis as displayed by increased caspase 3 expression and DNA fragmentation. A similar effect was seen in vitro using TNF-α-simulated human and rat PASMC following both dexamethasone and IKK2 inhibition. Increased apoptosis was associated with a reduction in NF-κB activation and in IL-6 and CXCL8 release from PASMC.ConclusionsDexamethasone exerted reverse-remodelling effects by augmenting apoptosis and reversing inflammation in PASMC possibly via inhibition of NF-κB. Future PAH therapies may involve targeting these important inflammatory pathways.

Effect of puerarin on PI3K/AKT pathway-mediated apoptosis of PASMCs

To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether the extracellular signal PI3K/AKT pathway was involved in the Pue-induced PASMC apoptosis. With the serum starvation group (SD group) as the control group, the MTT colorimetry method, Annexin V-FITC apoptosis detection kit and Western blot were used to detect Pue's effect on apoptosis of rat PASMCs. The protein immunoblot assay was used to detect whether PI3K/AKT pathway was involved in the inhibition of hypoxia-induced PASMC apoptosis process. The results show that under normoxic conditions, Pue had no effect on PASMC apoptosis; Under hypoxia conditions, Pue can inhibit PASMC apoptosis; Under normoxic and hypoxic conditions, Pue had no effect on TNF-α expression. Pue can reverse hypoxia-induced Bcl-2 (P <0.01), up-regulate it and down-regulated Bax (P <0.01). Under normoxic conditions, Pue had no effect on P-AKT expression. Both LY294002 and Pue can inhibit hypoxia-induced Bcl-2, up-regulation of P-AKT expression and down-regulation of Bax expression. Compared with the hypoxia + Pue group or the hypoxia + LY294002 group, the hypoxia + Pue + LY294002 group showed more significantly changes in Bcl-2, Bax, P-AKT expressions. The results show that, Pue can inhibit the hypoxic-induced PASMC apoptosis, which may be regulated through PI3K/AKT pathway.