Muscle Cells In Pulmonary Hypertension Research Articles

RGS10 inhibits proliferation and migration of pulmonary arterial smooth muscle cell in pulmonary hypertension via AKT/mTORC1 signaling

ABSTRACT Objective: Excessive proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is a core event of pulmonary hypertension (PH). Regulators of G protein signaling 10 (RGS10) can regulate cellular proliferation and cardiopulmonary diseases. We demonstrate whether RGS10 also serves as a regulator of PH. Methods: PASMC was challenged by hypoxia to induce proliferation and migration. Adenovirus carrying Rgs10 gene (Ad-Rgs10) was used for external expression of Rgs10. Hypoxia/SU5416 or MCT was used to induce PH. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were used to validate the establishment of PH model. Results: RGS10 was downregulated in hypoxia-challenged PASMC. Ad-Rgs10 significantly suppressed proliferation and migration of PASMC after hypoxia stimulus, while silencing RGS10 showed contrary effect. Mechanistically, we observed that phosphorylation of S6 and 4E-Binding Protein 1 (4EBP1), the main downstream effectors of mammalian target of rapamycin complex 1 (mTORC1) as well as phosphorylation of AKT, the canonical upstream of mTORC1 in hypoxia-induced PASMC were negatively modulated by RGS10. Both recovering mTORC1 activity and restoring AKT activity abolished these effects of RGS10 on PASMC. More importantly, AKT activation also abolished the inhibitory role of RGS10 in mTORC1 activity in hypoxia-challenged PASMC. Finally, we also observed that overexpression of RGS10 in vivo ameliorated pulmonary vascular wall thickening and reducing RVSP and RVHI in mouse PH model. Conclusion: Our findings reveal the modulatory role of RGS10 in PASMC and PH via AKT/mTORC1 axis. Therefore, targeting RGS10 may serve as a novel potent method for the prevention against PH.”

5-HT2-Receptors and 5-HIAA – Therapeutic Targets for Evaluation of Severity, Progression and Effectiveness of Treatment in Immature Male Rats in a Monocrotalin Model of Pulmonary Hypertension

Suppression of the proliferation of vascular endothelial cells and the interaction of endothelial with smooth muscle cells in pulmonary hypertension (PH) are impaired. Participation of the 5-HT2a-receptor in the mitogenic effect on endothelial, and 5-HT2b-receptor – on vascular smooth muscle cells was revealed. The main organ that metabolizes serotonin is the lung. In the endothelial cells of the vessels of the lungs under the action of the enzyme monoamine oxidase And serotonin is converted to 5-hydroxyindoleacetic acid (5-HIAA), which is subsequently excreted in the urine. Currently, the role of 5-HT2-receptors is not taken into account in the treatment of children with pulmonary hypertension. We have modified the monocrotaline model of pulmonary hypertension for immature rats. A scheme for the administration of a 5-HT2-receptor blocker for the prevention and treatment of pulmonary hypertension in immature rats was developed and tested. A positive correlation was found between the concentration of 5-HIAA in urine and the degree of pulmonary hypertension, which can become a potential marker of pulmonary hypertension. The data obtained indicate the development of pulmonary hypertension in immature rats after a single injection of monocrotaline in the form of replacement of lung tissue with fibrous tissue, the development of pneumosclerosis and bronchiectasis. Also, in animals in this model, changes in the structure of the heart muscle and vascular wall are formed with the development of fibrous tissue, which may indicate the involvement of 5HT2-receptors in the activation of fibroblasts and, accordingly, in the pathogenesis of pulmonary hypertension.

L‐Citrulline Attenuates Effects of Serotonin Signalling During Proliferation of Pulmonary Artery Smooth Muscle Cells in Pulmonary Hypertension

IntroductionPremature infants are at risk for bronchopulmonary dysplasia (BPD), a disorder characterized by abnormal pulmonary development. Inflammation is one major component in causing BPD, which can further lead to abnormal vascular remodelling, causing pulmonary hypertension (PH). During this process, pulmonary artery smooth muscle cell (PASMC) proliferation and hypertrophy contribute to the thickening of the pulmonary artery wall, leading to heightened blood pressures. 5‐hydroxytryptamine (5‐HT) or serotonin has been implicated in PH and is involved in the pathogenesis of PH in adults. Meanwhile, L‐citrulline, a nitric oxide donor, has been effective in reducing PH in acute models of BPD. The role of 5‐HT signalling on PASMC proliferation during early pulmonary vascular development is currently unknown. Thus, the objective of this study is to establish the effect of 5‐HT on PASMC proliferation in a newborn model of early lung development, and to elucidate the interaction of 5‐HT on proliferation of PASMCs in the presence of LPS and L‐citrulline.MethodsSprague Dawley rat pups were grown and euthanized on postnatal day 4, after which the pulmonary arteries (PA) were isolated. Primary cultures of PASMCs were grown from PA explants and treated with varying concentrations of 5‐HT. Proliferation and cell viability assays were performed to measure growth and cell viability after exposure to 5‐HT and L‐citrulline (4mM). Lysate samples were also prepared with the cells to measure protein expression via Western blotting for Cyclin D1, an important protein involved in regulation of the cell cycle, and phosphorylated AKT (p‐AKT), which is involved in cell survival. To investigate the effects of inflammation on 5‐HT signalling, PASMCs were exposed to lipopolysaccharide (LPS, 3 µg/mL) and lysate samples were prepared to measure changes in the expression of the 5‐HT2A receptor.ResultsProliferation assays illustrated a dose‐dependent increase in growth of PASMCs exposed to 5‐HT, reaching a peak 150% growth rate (p < 0.05). Western blots revealed a three‐fold increase in Cyclin D1 expression (p < 0.01) and two‐fold increase in p‐AKT expression (p < 0.05) in PASMCs exposed to 5‐HT compared to protein expression of control PASMCs. Both changes were abrogated in PASMCs exposed to both L‐citrulline and 5‐HT, bringing expression of both proteins to control levels. When exposed to LPS, PASMCs have heightened expression of 5‐HT2A, reaching nearly two times higher than in control PASMCs. This effect is also reversed in PASMCs exposed to LPS and L‐citrulline (p < 0.05).ConclusionOur preliminary data support a role for 5‐HT in pathogenesis on BPD‐PH in the presence and absence of inflammation in a newborn model of lung development, and L‐citrulline may be an attractive safe therapeutic option to reverse or mitigate inflammation induced PH in a newborn model, which could later be translated to human infants.

Zinc-mediated activation of CREB pathway in proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension

BackgroundTranscription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH.MethodsA series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH.ResultsUsing bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation.ConclusionsCREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH.B6vttFt3ef-LGEoxvha7NyVideo abstract

Evidence for Multiple Origins of De Novo Formed Vascular Smooth Muscle Cells in Pulmonary Hypertension: Challenging the Dominant Model of Pre-Existing Smooth Muscle Expansion.

Vascular remodeling is a prominent feature of pulmonary hypertension. This process involves increased muscularization of already muscularized vessels as well as neo-muscularization of non-muscularized vessels. The cell-of-origin of the newly formed vascular smooth muscle cells has been a subject of intense debate in recent years. Identifying these cells may have important clinical implications since it opens the door for attempts to therapeutically target the progenitor cells and/or reverse the differentiation of their progeny. In this context, the dominant model is that these cells derive from pre-existing smooth muscle cells that are activated in response to injury. In this mini review, we present the evidence that is in favor of this model and, at the same time, highlight other studies indicating that there are alternative cellular sources of vascular smooth muscle cells in pulmonary vascular remodeling.

The Role and Regulation of Pulmonary Artery Smooth Muscle Cells in Pulmonary Hypertension.

Pulmonary hypertension (PH) is one of the most devastating cardiovascular diseases worldwide and it draws much attention from numerous scientists. As an indispensable part of pulmonary artery, smooth muscle cells are worthy of being carefully investigated. To elucidate the pathogenesis of PH, several theories focusing on pulmonary artery smooth muscle cells (PASMC), such as hyperproliferation, resistance to apoptosis, and cancer theory, have been proposed and widely studied. Here, we tried to summarize the studies, concentrating on the role of PASMC in the development of PH, feasible molecular basis to intervene, and potential treatment to PH.

Transmission electron microscopic analysis of mitochondria changes in pulmonary arterial hypertension

Mitochondria are dynamic organelles, which play crucial roles in many cellular processes. Their severe damage results in alterations of other ultrastructural components and leads to loss of cellular function. In pulmonary hypertension (PH), increasing of the pulmonary vascular resistance puts the strain on the right ventricle, leading to the demand for ATP production by mitochondria to ensure cardiomyocyte function. The failure of cardiac pump increases the peripheral resistance by inducing the neurohormonal activation and influences the skeletal muscle structure and function, leading to the muscle weakness. In a rat PH model, our previous morphometric study reported changes in the mitochondrial types. Type I–II mitochondria are defined as mitochondria with the preserved ultrastructure and Type III–IV mitochondria are defined as mitochondria with the severe cristae damage that are not able to produce the required amount of ATP for energy‐depended processes in cardiomyocytes. Fischer rats had 100% mortality because of right heart failure in PH. In their RV cardiomyocytes, the types III–IV of mitochondria were predominant, along with a drastic decrease in coefficient of energy efficiency of mitochondria (CEEM), while in LV types II–III of mitochondria were present. Whereas the SD rats that do not die of PH, mostly had mitochondria Type I–II in RV and LV, along with slight decrease in CEEM in RV. In both strains of rats with PH, the treatment with a Bcl‐2 inhibitor, Navitoclax (ABT‐263), lead to severe damage of the mitochondria structure, causing rats to die because of hearth failure. The transmission electron microscopy (TEM) analysis of mitochondria in skeletal muscle cells in PH revealed the decrease in mitochondria volume and the damage to the mitochondria structure. In skeletal muscle cells, Type III mitochondria were present in rats, which correlates with the reduction in aerobic capacities. These results suggest that the ultrastructural defects in mitochondria in both cardiac and skeletal muscle in PH impact the development of the hearth failure and death, thus PH can be described as a systemic disease.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LRP1 promotes synthetic phenotype of pulmonary artery smooth muscle cells in pulmonary hypertension

Pulmonary hypertension (PH) is characterized by a thickening of the distal pulmonary arteries caused by medial hypertrophy, intimal proliferation and vascular fibrosis. Low density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis by mediating endocytosis of numerous ligands and by initiating and regulating signaling pathways.Here, we demonstrate the increased levels of LRP1 protein in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline-treated rats. Platelet-derived growth factor (PDGF)-BB upregulated LRP1 expression in pulmonary artery smooth muscle cells (PASMC). This effect was reversed by the PDGF-BB neutralizing antibody or the PDGF receptor antagonist. Depletion of LRP1 decreased proliferation of donor and IPAH PASMC in a β1-integrin-dependent manner. Furthermore, LRP1 silencing attenuated the expression of fibronectin and collagen I and increased the levels of α-smooth muscle actin and myocardin in donor, but not in IPAH, PASMC. In addition, smooth muscle cell (SMC)-specific LRP1 knockout augmented α-SMA expression in pulmonary vessels and reduced SMC proliferation in 3D ex vivo murine lung tissue cultures.In conclusion, our results indicate that LRP1 promotes the dedifferentiation of PASMC from a contractile to a synthetic phenotype thus suggesting its contribution to vascular remodeling in PH.

Dynamic and diverse changes in the functional properties of vascular smooth muscle cells in pulmonary hypertension.

Pulmonary hypertension (PH) is the end result of interaction between pulmonary vascular tone and a complex series of cellular and molecular events termed 'vascular remodelling'. The remodelling process, which can involve the entirety of pulmonary arterial vasculature, almost universally involves medial thickening, driven by increased numbers and hypertrophy of its principal cellular constituent, smooth muscle cells (SMCs). It is noted, however that SMCs comprise heterogeneous populations of cells, which can exhibit markedly different proliferative, inflammatory, and extracellular matrix production changes during remodelling. We further consider that these functional changes in SMCs of different phenotype and their role in PH are dynamic and may undergo significant changes over time (which we will refer to as cellular plasticity); no single property can account for the complexity of the contribution of SMC to pulmonary vascular remodelling. Thus, the approaches used to pharmacologically manipulate PH by targeting the SMC phenotype(s) must take into account processes that underlie dominant phenotypes that drive the disease. We present evidence for time- and location-specific changes in SMC proliferation in various animal models of PH; we highlight the transient nature (rather than continuous) of SMC proliferation, emphasizing that the heterogenic SMC populations that reside in different locations along the pulmonary vascular tree exhibit distinct responses to the stresses associated with the development of PH. We also consider that cells that have often been termed 'SMCs' may arise from many origins, including endothelial cells, fibroblasts and resident or circulating progenitors, and thus may contribute via distinct signalling pathways to the remodelling process. Ultimately, PH is characterized by long-lived, apoptosis-resistant SMC. In line with this key pathogenic characteristic, we address the acquisition of a pro-inflammatory phenotype by SMC that is essential to the development of PH. We present evidence that metabolic alterations akin to those observed in cancer cells (cytoplasmic and mitochondrial) directly contribute to the phenotype of the SM and SM-like cells involved in PH. Finally, we raise the possibility that SMCs transition from a proliferative to a senescent, pro-inflammatory and metabolically active phenotype over time.

Role of Macrophage Migration Inhibitory Factor in the Proliferation of Smooth Muscle Cell in Pulmonary Hypertension

Pulmonary hypertension (PH) contributes to the mortality of patients with lung and heart diseases. However, the underlying mechanism has not been completely elucidated. Accumulating evidence suggests that inflammatory response may be involved in the pathogenesis of PH. Macrophage migration inhibitory factor (MIF) is a critical upstream inflammatory mediator which promotes a broad range of pathophysiological processes. The aim of the study was to investigate the role of MIF in the pulmonary vascular remodeling of hypoxia-induced PH. We found that MIF mRNA and protein expression was increased in the lung tissues from hypoxic pulmonary hypertensive rats. Intensive immunoreactivity for MIF was observed in smooth muscle cells of large pulmonary arteries (PAs), endothelial cells of small PAs, and inflammatory cells of hypoxic lungs. MIF participated in the hypoxia-induced PASMCs proliferation, and it could directly stimulate proliferation of these cells. MIF-induced enhanced growth of PASMCs was attenuated by MEK and JNK inhibitor. Besides, MIF antagonist ISO-1 suppressed the ERK1/2 and JNK phosphorylation induced by MIF. In conclusion, the current finding suggested that MIF may act on the proliferation of PASMCs through the activation of the ERK1/2 and JNK pathways, which contributes to hypoxic pulmonary hypertension.

The effect of acidosis on pulmonary vascular smooth muscle cell metabolic response to hypoxia: implications for Pulmonary Hypertension

Background A glycolytic shift is key to apoptotic resistance in cancer cells and pulmonary vascular smooth muscle cells in pulmonary hypertension (PH). Its reversal may lead to apoptosis and reverse pulmonary vascular remodeling. We found that acidosis reversed remodeling in experimental PH and sought to investigate the metabolic response of pulmonary artery smooth muscle cells (PASMCs) to hypoxia. Materials and Methods We exposed rats to hypoxia to develop PH; acidosis was induced by ammonium chloride (NH4Cl) in drinking water. Primary PASMCs were exposed to acidosis (pH 6.8), physiologic pH (7.4) or hypoxia. Glucose Transporter-1 (Glut-1) mRNA and protein were assessed in lungs and PASMCs. Mitochondrial membrane potential (MMP) and apoptosis were assessed by JC-1 and caspase-3 cleavage, respectively. Results Glut-1 mRNA increased in lungs of hypoxic animals compared to normoxic with a decrease in hypoxic animals treated with NH4Cl. Isolated PASMCs showed higher Glut-1 mRNA in hypoxia than normoxia at physiologic and acidic pH. Acidic pH decreased Glut-1 protein in normoxia and hypoxia. Exposure to acidic pH decreased MMP and increased apoptosis by hydrogen peroxide. Conclusions Acidosis results in decreased Glut-1 in lungs and PASMCs, a decreased MMP and increased propensity to apoptosis. We speculate that acidosis reverses the glycolytic phenotype in hypoxia, thus promoting an apoptotic phenotype. Supported by NIH grant: 5T32HD007466

Evidence for Inflammatory Signaling in Idiopathic Pulmonary Artery Hypertension

Knowledge of molecular mechanisms underlying pulmonary arterial hypertension (PAH) continues to increase with the emerging theme that PAH is a heterogeneous disease involving multiple molecular abnormalities. Mutations in several genes have been identified in subsets of patients with PAH, and multiple signaling systems that influence vascular tone, function, and remodeling have been associated with PAH.1,2,3 In addition to mutations in BMPR2 , serotonin ( 5-HT ) and polymorphisms in its transporter ( SERT ) play a critical role in the pulmonary vascular smooth muscle hyperplasia and vascular remodeling found in PAH.4,5 Other genes and signals thought to contribute to the development of idiopathic pulmonary arterial hypertension (IPAH) include somatic mutations of BAX ,5 upregulation of Angiopoietin 1 ,6 transforming growth factor β1 polymorphisms,7 ALK1 mutation,8 SMAD8 mutation,9 and increased hyaluronic acid content associated with increased Hyaluronan Synthase 1 and decreased Hyaluronoglucosaminidase 1 gene expression.10 A recent observation also suggests that the noncanonical Wnt pathway is activated in IPAH.11 Article see p 2313 Several transient receptor potential canonical (TRPC) family …

Cross Talk Between Endothelial and Smooth Muscle Cells in Pulmonary Hypertension

The mechanism of pulmonary artery smooth muscle cell (PA-SMC) hyperplasia in idiopathic pulmonary artery hypertension (iPH) may involve both an inherent characteristic of PA-SMCs and abnormal control by external stimuli. We investigated the role of pulmonary microvascular endothelial cells (P-ECs) in controlling PA-SMC growth. Serum-free medium of quiescent P-ECs elicited marked PA-SMC proliferation, and this effect was greater with P-ECs from patients with iPH than from control subjects and greater with PA-SMCs from these patients than from control subjects. Fluoxetine, which inhibits serotonin-induced mitogenesis by blocking the serotonin transporter, and p-chlorophenylalanine, which inhibits serotonin synthesis by blocking tryptophan hydroxylase (TPH), caused a similar 60% reduction in the growth-promoting effect of P-EC media, whereas endothelin receptor blockers had no effect. Assays of TPH activity in P-EC medium based on p-chlorophenylalanine-sensitive 5-hydroxytryptophan accumulation or serotonin determination indicated serotonin synthesis by P-ECs and an increase in this TPH-dependent process in iPH. Expression of the tph1 gene encoding the peripheral form of the TPH enzyme was increased in lungs and P-ECs from patients with iPH. Lung TPH1 immunostaining was confined to the pulmonary vessel intima. P-ECs produce paracrine factors governing PA-SMC growth. Serotonin, the main P-EC-derived growth factor, is overproduced in iPH and contributes to PA-SMC hyperplasia.