Phenotypic Diversity of Vascular Smooth Muscle Cells in Pulmonary Arterial Hypertension: Implications for Therapy.

Pulmonary arterial hypertension (PAH) is a progressive incurable condition that is characterized by extensive remodeling of the pulmonary circulation, leading to severe right-sided heart failure and death. Similar to other vascular contractile cells, pulmonary arterial smooth muscle cells play central roles in physiological and pathologic vascular remodeling because of their remarkable ability to dynamically modulate their phenotype to ensure contractile and synthetic functions. The dysfunction and molecular mechanisms underlying their contribution to the various pulmonary vascular lesions associated with PAH have been a major focus of research. The aim of this review is to describe the medial and nonmedial origins of contractile cells in the pulmonary vascular wall and present evidence of how they contribute to the onset and progression of PAH. We also highlight specific potential target molecules and discuss future directions that are being explored to widen the therapeutic options for the treatment of PAH

Rodent models of cardiopulmonary disease: their potential applicability in studies of air pollutant susceptibility.

The mechanisms by which increased mortality and morbidity occur in individuals with preexistent cardiopulmonary disease following acute episodes of air pollution are unknown. Studies involving air pollution effects on animal models of human cardiopulmonary diseases are both infrequent and difficult to interpret. Such models are, however, extensively used in studies of disease pathogenesis. Primarily they comprise those developed by genetic, pharmacologic, or surgical manipulations of the cardiopulmonary system. This review attempts a comprehensive description of rodent cardiopulmonary disease models in the context of their potential application to susceptibility studies of air pollutants regardless of whether the models have been previously used for such studies. The pulmonary disease models include bronchitis, emphysema, asthma/allergy, chronic obstructive pulmonary disease, interstitial fibrosis, and infection. The models of systemic hypertension and congestive heart failure include: those derived by genetics (spontaneously hypertensive, Dahl S. renin transgenic, and other rodent models); congestive heart failure models derived by surgical manipulations; viral myocarditis; and cardiomyopathy induced by adriamycin. The characteristic pathogenic features critical to understanding the susceptibility to inhaled toxicants are described. It is anticipated that this review will provide a ready reference for the selection of appropriate rodent models of cardiopulmonary diseases and identify not only their pathobiologic similarities and/or differences to humans but also their potential usefulness in susceptibility studies

The contribution of mesothelial cells to lung development

Thesis (Ph.D.)--Boston UniversityMesothelium-derived progenitors have been demonstrated to contribute to differentiated mesenchymal components of the heart, liver, and gut during organogenesis. The precise contribution of the mesothelium to lung development, however, has not been fully clarified and the key signals regulating mesothelial cell entry have not been identified. To rigorously address this issue, we employed mice with an inducible Cre expressed from the Wilm's tumor-1 (WT1) locus for high fidelity lineage tracing after confirming that Cre-recombinase was mesothelial-specific and faithfully recapitulated endogenous WT1 gene expression. We visualized WT1+ mesothelial cell entry into the fetal lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells, and desmin+ fibroblasts by lineage tagging. In view of the role of Sonic Hedgehog (Hh) signaling in regulating mesenchymal cell differentiation and epithelial-mesenchymal transition, we hypothesized that this pathway regulates events associated with migration of mesothelial cells into the developing lung. To examine for this, we first used two independent reporter mice to show that Hh signaling is active within the lung mesothelium at time points coinciding with the appearance of mesothelium-derived cells in the lung parenchyma. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted loss-of hedgehog function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of Hh signaling. In order to examine whether WT1 interacts with Hh pathway, we conducted ChIP assays on fetal lung mesothelial cells, and found that WT1 directly binds and regulates promoter elements of downstream targets of Hh pathway. Consistent with this observation, Hh pathway gene expression was down-regulated in isolated WT1 deficient fetal lung mesothelial cells. Taken together, these findings lend further support to a paradigm in which mesothelial cells are an important source of progenitors for mesenchymal structures. Our findings also reveal a role for Hh pathway in the early events associated with mesothelial cell entry and indicate that WT1 likely acts upstream of Hh signaling

Hypoxia-mediated human pulmonary arterial fibroblast proliferation is dependent on p38 mitogen-activated protein kinase activity.

Abstract Background: Pulmonary hypertension (PH) is a rare condition that can occur as a primary disease process, Idiopathic Pulmonary Hypertension (IPH) or secondary to other disorders. In Familial IPH mutations have been identified in the bone morphogenetic protein receptor II gene (BMPRII) (chromosome 2q32-31) a member of the Transforming Growth Factor (TGF) (Lane et al, 2000). Despite the mutation being present in all cells, vascular wall remodelling is only seen in the pulmonary circulation with marked thickening of the intima and neointimal formation, muscularisation of small-generation resistance vessels and thickening of the adventitial layer together with increased ECM deposition. Similar appearances are noted in the pulmonary circulation’s response to hypoxia. for this projectProlonged exposure of the pulmonary circulation to hypoxia results in vasoconstriction and subsequent vascular wall remodelling. The hypothesis of this work is that the pulmonary circulation’s response to hypoxia may be partially explained by the existence of differences exist in cell signalling pathways in between adventitial fibroblasts from pulmonary and systemic arteries in HPAF. Studies from the Scottish Pulmonary Vascular (SPVU) Laboratory have shown that pulmonary arterial fibroblasts (PAFB) in bovine and rat models of acute hypoxic exposure preferentially proliferate to hypoxia, whereas systemic arterial fibroblasts (SAFB) do not , that the stress mitogen activated protein kinase p38 MAPK is consistently activated in PAFB exposed to acute hypoxia, and is constitutively upregulated in PAFB cultured from rats exposed to chronic hypoxia (Welsh et al, 1998; Welsh et al; 2001). This response to hypoxic exposure has been shown to be dependent on p38 MAPK activity, as use of SB203580 can block the hypoxia-mediated proliferative response to acute hypoxia (Scott et al, 1998; Welsh et al, 2001). Aims and methods: We wished to establish whether the pro-proliferative response of PAFB to acute hypoxic exposure previously noted in bovine and rat models could also be demonstrated in a human model. We wished to establish a role for both classic MAPK and stress MAPKs in hypoxia-mediated PAFB proliferation. We also wished to examine the role of hypoxia inducible factor 1 (HIF1) in human arterial fibroblast responses to acute hypoxia. There is a body of literature that documents cross talk between p38 MAPK and the Bone Morphogenetic Protein (BMPR) signalling pathways. We wished to establish whether Smad proteins (involved in the downstream signalling cascade from BMPR) might play a role in human pulmonary and systemic arterial fibroblast proliferation to acute hypoxia. Following approval from the local Ethics Committee, PAFB were harvested from patients undergoing lobectomy for the treatment of lung cancer. Left internal mammary arteries (SAFB) were harvested from patients undergoing coronary artery bypass grafting. Cells from systemic and pulmonary arterial fibroblasts were grown in conditions of normoxia or acute hypoxia (PO2 35 mmHg ~ 5% O2). Cellular proliferation was assessed using [3H]Thymidine uptake as a surrogate. p38, p44/p42 - ERK1/2 and JNK MAPKs and Smad protein activity was assessed using Western Blotting Techniques with the use of appropriate primary and secondary antibodies and Chemiluminescence to detect the presence of protein. p38 MAPK isoform activity was assessed using Catch and Release® immunophoresis techniques. Findings and conclusions: We demonstrated that acute hypoxic exposure results in human PAFB proliferation, associated with increased p44/p42 – ERK 1/2 MAPK activity, but dependent on p38 MAPK activity. We also found that the p38 MAPK isoform was expressed in human PAFB following hypoxic exposure but this did not appear to be involved in the hypoxia-mediated proliferative response. p38 MAPK activity appeared to occur in a bi-phasic pattern with peaks of activity at t = 6 and 16 hours, the second peak was found to be responsible for the hypoxia-mediated proliferation seen in these cells in agreement with previous work from the SPVU laboratory (Scott et al, 1998; Welsh et al., 2001). The second peak in p38 MAPK activity was synchronous with peak HIF1 activity (between t = 8 –16 hours). We demonstrated that HIF1 activity can be abrogated by pre-incubation of human PAFB with SB203580 suggesting a mechanistic link between p38 MAPK activation and HIF1 in a human model of acute hypoxic exposure. We have also demonstrated that that BMPR2-associated Smad 1, 5 and 8 activation is increased in hypoxic human SAFB, suggestive of the activation of an anti-proliferative pathway in these cells that is not associated with p38 MAPK activity. To our knowledge this is the first demonstration of an active response in SAFB to acute hypoxic exposure that involves the active upregulation of an anti-proliferative pathway in these cells. In addition we have demonstrated that in hypoxic pulmonary arterial fibroblasts phospho Smad 1, 5 and 8 expression is reduced (suggestive of the down-regulation of an anti-proliferative pathway) and can be further abrogated by pre-incubation with SB203580. This suggests that in SAFB Smad 1, 5 and 8 activation occurs independent of p38 MAPK activation while in PAFB, p38 MAPK activity augments Smad 1, 5 and 8 activation

Modifying factors in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a debilitating disease of small pulmonary resistance arteries with vasoconstriction and vascular remodelling contributing to the disease pathology. A genetic basis for the disease was linked to heterozygous loss of function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene. The mutation is found in the majority of familial PAH cases and a significant number of apparently sporadic cases. The low penetrance of the disease in families carrying BMPR2 mutations and the absence of mutations in the majority of idiopathic patients indicates that BMPR2 deficiency alone is insufficient to induce PAH. It is generally accepted PAH has a multi-factorial pathology with endogenous and environmental factors acting in concert with genetic pre-disposition to create the disease phenotype. Enhancement of the serotonin (5-HT) system has been implicated in PAH with the 5-HT transporter (5-HTT) receiving the most attention as a modifying gene in the development of PAH and there is compelling animal and human data implicating a role for increased expression of the 5-HTT as a modulating factor. The aim of this study was to investigate if genetic pre-disposition interacts with other additional modifying factors to create the symptoms of PAH. Transgenic mice overexpressing the 5-HTT (5-HTT+), deficient in BMPR2 (BMPR2+/-) or a double transgenic (5-HTT+/BMPR2+/-) were employed in addition to mice lacking tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme for the synthesis of 5-HT, and therefore lacking peripheral 5-HT (Tph1-/-). Additional known or suspected modifying factors assessed in these genetic models were hypoxia, dexfenfluramine (Dfen) and its major metabolite nordexfenfluramine (NDfen), 5-HT, bone morphogenetic protein-2 (BMP-2), KCNQ channels and the role of gender. Mice were examined in vivo for evidence of a pulmonary hypertensive phenotype following exposure to hypoxia and Dfen. Female 5-HTT+ mice were the only group tohave a rise in two indices of PAH - namely right ventricular pressure (RVP) and vascular remodelling - in room air. Female 5-HTT+ mice also had an exaggerated pulmonary hypertensive phenotype in hypoxia. BMPR2+/- mice, were, unexpectedly least susceptible to hypoxic induced increases in RVP although female mice deficient in BMPR2 (both BMPR2+/- and 5-HTT+/BMPR2+/-) had more extensive vascular remodelling under hypoxia compared with WT and 5-HTT+ mice. Male mice did not express the phenotypic changes just outlined. No synergistic effect was observed between 5-HTT+ and BMPR2+/- that resulted in a more pronounced pulmonary hypertensive phenotype. WT and BMPR2+/- mice were chronically oral-dosed with Dfen. Female mice from both genotypes developed similar degrees of PAH. Male mice did not develop elevated RVP but BMPR2+/- males did have evidence of vascular remodelling, although at a lower level than the females. Female Tph1-/- mice did not develop PAH following Dfen indicating Dfen associated PAH is dependent on peripheral 5-HT synthesis. The presence of intact 5-HT synthesis was also associated with an increased vasoconstrictor response to 5-HT in isolated intralobar pulmonary arteries (IPAs), a situation not paralleled with the other serotonergic vasoconstrictors, Dfen and NDfen, indicating differing mechanisms of action underlying the respective vasoconstrictor responses. The vasoconstrictor action of 5-HT, Dfen, NDfen and the KCNQ potassium channel blocker linopirdine were all assessed in IPAs. Pulmonary arteries from BMPR2+/- mice showed enhanced vasoconstriction to 5-HT and NDfen. 5-HTT+ and 5-HTT+/BMPR2+/- mice showed enhanced vasoconstriction to NDfen but decreased vasoconstriction to 5-HT. Female 5-HTT+/BMPR2+/- mice were the only group tested to show significantly greater vasoconstriction to Dfen compared with WT. The vasoconstrictor response to linopirdine was significantly reduced in BMPR2+/- mice but neither linopirdine nor BMP-2 affected 5- HT induced vasoconstriction. Female gender is an established risk factor for PAH. To investigate possible events that may underlie this risk, male (testosterone) and female (estradiol and 2-methoxyestradiol (2-ME)) sex hormones were assessed for their vasoactive properties in IPAs. All three hormones relaxed pre-constricted vessels but only at supraphysiological (>0.1 µM) concentrations. Each hormone also reduced the vasoconstriction exerted by 5-HT at 10-5 M in male mice but not in females. No such effect, however, was observed in either gender at a physiological (10-9 M) concentration. NDfen induced vasoconstriction was also unaffected by 10-9 M estradiol. Finally, male and female mouse lungs were assessed for protein expression of 5-HT and BMPR2 signalling compounds (p-Smad1/5/8, p-ERK1/2 and p-p38 MAPK). Female mouse lungs displayed higher expression of the mitogenic mediator p-ERK1/2 than male mouse lungs with the other proteins unchanged. In conclusion, this study confirms overexpression of the 5-HTT as a trigger for elevated RVP and vascular remodelling in mice and a cause of more severe hypoxic PAH. BMPR2+/- mice are phenotypically normal in room air and show divergent pulmonary effects following hypoxia with loss of BMPR2 seemingly attenuating hypoxic induced increases in RVP but causing a simultaneous worsening of vascular remodelling, this latter effect consistent with the important role BMPR2 has in maintaining vascular integrity. Dfen induced PAH in mice was found to be dependent on peripheral 5-HT synthesis with BMPR2 mutation not acting as a risk factor. Loss of BMPR2 can enhance vasoconstriction to serotonergic agonists and when combined with overexpression of the 5-HTT, leads to a dramatic increase in sensitivity to Dfen induced vasoconstriction. Evidence was also found for altered KCNQ channel function in transgenic animals. Unexpectedly, female gender emerged as the most crucial risk factor for PAH in this thesis

Multimodality imaging to quantify the pulmonary vascular tree in COPD

Chronic obstructive pulmonary disease (COPD) is a progressive and debilitating disease resulting in chronic cough, shortness of breath, activity limitation and decreased pulmonary function. Developments in imaging technology have provided sensitive and reliable modalities for evaluating regional lung function and disease progression, and there is a growing interest in the role of imaging the vasculature in COPD. The ability to predict whether a patient is at risk of accelerated decline is important to disease management strategies. We hypothesize that CT blood vessel volume measurements are significantly different in ex-smokers without COPD than in those with this disease and will be related to disease severity. 90 participants completed both baseline and follow-up visits: 41 ex-smokers without COPD (71±10yrs) and 49 participants with COPD (71±8yrs). From baseline to follow-up, RA950 increased significantly for ex-smokers and GOLD II participants, while PV1 decreased significantly for GOLD I. There were no differences in VDP when grouped according to change in FEV1. Participants whose FEV1 increased by more than 20mL/year experienced a significantly smaller change in RA950 compared to those whose FEV1 decreased by more than 40mL. Independent samples t-tests indicate a significant difference in the rate of PV1 progression between COPD groups with and without emphysema, but not VDP or RA950. Emphysema, or COPD phenotype, is related to vascular structure within the lung and the progression of vascular remodelling. Future work should include investigations of sex-differences in airways disease, and the use of machine learning to predict disease progression with optimized CT imaging parameters

THE ROLE OF THE MECHANICAL ENVIRONMENT ON CD117+ ENDOTHELIAL CELL ANGIOGENESIS

Angiogenesis is a complex process coordinating cell migration, proliferation, and lumen formation. Changes to the microenvironment regulate angiogenesis through mechanotransduction and cytokine signals. In pulmonary hypertension, something in the process becomes abnormal, resulting in changes to the microenvironment and the formation of a glomerulus of dysfunctional capillaries, called a plexiform lesion. Endothelial cells, expressing CD117 (CD117+ EC clones) increase in the plexiform lesions of pulmonary hypertension, independent of pro-angiogenic VEGF signaling. We hypothesize that the mechanical environment and the macromolecular composition of the extracellular matrix, both, contribute to the aberrant angiogenesis. When we changed the mechanical environment, we changed the angiogenic potential and cellular phenotype of CD117+ Endothelial cell clones. Turbulent flow, pathologic substrate stiffness, and pathologic stretch increased Endothelial-to-mesenchymal markers, such as acta2, cnn1, snail, and slug in CD117+ EC clones while CD117- ECs showed minimal change. We perturbed the mechanical environment of CD117+ EC clones and identified changes in Bone Morphogenic Protein-2, an often overlooked pro-angiogenic cytokine. We coupled changes in the mechanical environment to Rho GTPase intracellular signaling, to predict how changes to the mechanotransduction would affect angiogenesis through a computational model. In our model of angiogenesis, we found vessel synchronicity to depend on both which cell undergoes mitosis, and also at which phase of GTPase cycling the cell undergoes mitosis. We believe changes to the GTPase cycling may be the mechanism linking mechanotransduction to the abnormal vessels found in pulmonary hypertension. We are the first group to look at the role of the ECM composition, independent of stiffness. Our results show diseased ECM composition alone leads to phenotypic changes indicative of PH progression. In conclusion, these results provide a possible cytokine implicated in the mechanotransduction of PH, established a computational model of angiogenesis which provides a mechanotransduction mechanism of disease progression, and established that the ECM composition alone is capable of phenotypic changes leading to disease progression

Current Basic and Pathological Approaches to the Function of Muscle Cells and Tissues

This volume contains 17 short review articles classified into 3 parts. Part I consists of 7 articles dealing with basic aspects of contractile mechanism in skeletal and smooth muscle cells and also function of melanocytes having many properties common to those of smooth muscles. Part II and Part III contain articles dealing with pathological aspects of cardiac and smooth muscle cell functions, and dealing with factors influencing structure and function of cardiac and smooth muscle cells and tissues. The Editor believes that these articles are stimulating and informative for readers interested in basic, pathological and clinical aspects of muscle cells and tissues

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH