The mouse through the looking glass: a new door into the pathophysiology of pulmonary hypertension.

Pulmonary hypertension is a pathophysiologic process characterized by progressive elevation of pulmonary vascular resistance and right heart failure, which is a common complication of many diseases. Pulmonary hypertension with no apparent causes (unknown etiology) is termed primary pulmonary hypertension or, more recently, idiopathic pulmonary arterial hypertension (IPAH). Before the availability of disease-specific (targeted) therapy (through the mid-1980s) the median life expectancy from the time of diagnosis in patients with this disease was 2.8 years.1-3 Modern treatment has markedly improved physical function and has extended survival, and the 5-year mortality is 50%.1 Although there is already more than 100 years of research history, the mechanisms of this disease are still not very clear.2 Recently, with the development of cell biology and molecular genetics, further research into the mechanisms responsible for pulmonary hypertension have been possible, which has helped in its diagnosis and treatment. It is believed that the mechanisms of pulmonary hypertension can not only be described by pathophysiology but involve multiple factors (pathways) like cellular, humoral and molecular genetics, etc. The increased contraction and remodeling of blood vessels and thrombosis are the major pathophysiological basis for the development of pulmonary hypertension.3 Endothelial cells, smooth muscle cells, fibroblasts and platelets all play important roles in the development of pulmonary hypertension. In addition, vasoconstrictive and vasodilating factors, proliferative stimulating and inhibiting factors, coagulating and anti-coagulating substances and different vasoactive substances are involved in its development. Currently, there is extensive work being done on the role of genetic factors in the development of pulmonary hypertension.4 CELLULAR MECHANISMS AND PULMONARY HYPERTENSION The pulmonary vascular remodeling is the major pathological basis of pulmonary hypertension. The structural changes in all three layers (inner, medial and external) of the pulmonary vascular wall have significant meaning in the incidence, development and recovery of the pulmonary hypertension. Endothelial cells An intact vascular endothelium, under normal physiological conditions, plays a very significant role in maintaining the phenotype of smooth muscle cells and the structure of blood vessels. Some abnormal conditions like hypoxia, mechanical injuries, inflammations, drugs and toxins, etc. affect the structure, function and metabolism of endothelial cells, which are the initial (primary) causes of pulmonary hypertension.5 The damage to the endothelium affects its barrier function and damages its link to smooth muscle cells. It also disturbs the equilibrium of the vasoactive substances produced by the endothelium and pulmonary circulation and their regulation of smooth muscle cells, which in turn promote the proliferation of smooth muscle cells and finally modulate the structure of pulmonary vessels. Endothelial damages not only causes disturbances in proliferation and apoptosis but also affect the coagulation of blood.6 In 90% of the lesions, endothelial cells do not express transforming growth factor (TGF)-β2 receptors, which suggests that tumor inhibiting genes are responsible in the development of IPAH.7 Smooth muscle cells During pulmonary artery hypertension, the static tunica media of smooth muscle cells are transformed to a proliferative synthetic state. The smooth muscle cells proliferate and enlarge resulting in hypertrophy of the tunica media. Moreover, the precursor cells of smooth muscle (like intermediate cells and pericytes), which do not differentiate under normal condition, differentiate into new smooth muscle cells. The partial muscular arteries and non muscular arteries get muscularisation which forms new muscular arteries.8 Our previous studies, as well as others, showed that the proliferative index (PI), the apoptotic indexes (AI) and the ratio of PI and AI of the pulmonary artery smooth muscle cells in rats with pulmonary hypertension were higher than those of normal rats. This indicates that hyperplasia and apoptosis of smooth muscle cells of the pulmonary artery occurs during the pulmonary vascular structural remodeling and the imbalance of hyperplasia and apoptosis also take part in the process. Moreover, caspases-3, bcl-2 and NF-κB genes take part in the regulatory mechanism for the smooth muscle cell apoptosis.9,10 Pulmonary smooth muscle cells also synthesize and secrete different vasoactive substances which regulate the pulmonary vascular structural remodeling and the pulmonary hypertension. Fibroblast The proliferation of the fibroblasts in the outer layer of blood vessels, and the abnormal deposition of connective tissues and the changes in extracellular matrix (ECM) are important components for pulmonary vascular structural remodeling. ECM includes collagen, elastin, etc. Our research team recently found that the expression of pulmonary artery collagen I, collagen III, procollagen I mRNA and procollagen III mRNA in rats with pulmonary hypertension were significantly elevated compared with the normal rats. The positive signals were mainly located in the media and adventitia of median and small pulmonary arteries. The expression of collagen degradation regulatory enzymes, tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA, metalloproteinase-1 (MMP-1) mRNA and the ratio of TIMP-1 and MMP-1 in the pulmonary artery were elevated in rats with pulmonary hypertension. These results suggested that with the development of pulmonary hypertension, collagen, as an important component of extracellular matrix, accumulated resulting from increased synthesis and decreased degradation of collagen.11 Our research also showed that elastin, another important ECM component, was also increased in hypoxic pulmonary hypertensive rats. Additionally, after 1 week of hypoxia the inner elastin lamina of the pulmonary artery of rats became thinned and the thickness of the inner elastin lamina was inversely proportional to the pulmonary artery pressure,12 which indicated that the changes in elastin also participated in the development of pulmonary hypertension. Platelets and thrombosis Functional disturbance of platelets and thrombosis play an important role in the development of IPAH. The damage to the endothelium of pulmonary blood vessels induces the activation and agglutination of platelets. The abnormality in the thrombomoduling system and fibinolysis system induce in situ thrombosis in the pulmonary artery.13 Platelets not only have anti-coagulant function but also produce constriction and remodulating active substances which cause remodulating of pulmonary blood vessels. Inflammatory cells The level of inflammatory cell factors like anti-nuclear antibody (ANA), interleukin (IL)-1 and IL-6 are found to be increased in some IPAH patients. Histology showed infiltration of macrophages and lymphocytes which indicated inflammatory cells might take part in the development of IPAH.14 Moreover, an inflammatory reaction plays a role, to some extent, in the development of pulmonary hypertension induced by connective tissue diseases and HIV infection. It is found that the immunosuppressive treatment is beneficial in some cases of lupus induced-pulmonary hypertension MOLECULAR MECHANISMS OF PULMONARY HYPERTENSION Endothelial cells, smooth muscle cells, fibroblasts, platelets and macrophages associated with blood vessels can produce different vasoactive substances. Under normal condition these substances exhibit a dynamic equilibrium; maintain the normal physiological structure and function of pulmonary vessels. The external stimuli (like high pulmonary blood flow, hypoxia and toxins) disturb the equilibrium and induce thrombosis, constriction and pulmonary vascular structural remodeling, which is an important mechanism in the development of pulmonary hypertension. Nitric oxide (NO) NO is closely related to the development of pulmonary hypertension. There exist different opinions regarding the role of NO in pulmonary hypertension. While not all conclusions are accordant. Most researchers believe that the expression of NO synthase (NOS) decreases during pulmonary hypertension resulting in a decrease in NO synthesis and, according to etiology and disease severity, the changes in NO levels also differ accordingly.15 Our research team, as well as others, found that long-term inhalation of NO or use of NO precursor L-arginine (L-Arg) or NO donor nitroglycerin alleviated pulmonary hypertension as well as pulmonary vascular structural remodeling in the hypoxic rats along with an increase in plasma NO. In contrast, NOS inhibitor significantly aggravated pulmonary vascular structural remodeling with downregulation of the endogenous NO/NOS pathway, which indicated NO played an important regulatory role in the development of pulmonary hypertension and pulmonary vascular structural remodeling.16,17 Recently, many hospitals reported the uses of NO inhalation, NO donors and precursor in the treatment of pulmonary hypertension and the effective treatment. Carbon monoxide (CO) Endogenous CO is mainly produced by the degradation of heme in the presence of heme oxygenase (HO). It relaxes the blood vessels and inhibits the proliferation of smooth muscle cells of blood vessels. Recent studies showed that there was the expression of HO in the smooth muscle cells and endothelial cells of pulmonary vessels, which indicated pulmonary circulation was one of the important places for production and release of endogenous CO. Our research team found that the CO/HO-1 system was increased in a time-dependent double-peak manner in hypoxic pulmonary hypertensive rats.18 For example, the content of CO in lung homogenates of rats from hypoxia day 1 and hypoxia day 3 groups was markedly increased compared with that of normal controls, while the content of CO in lung homogenates in the rats of hypoxia day 7 decreased to the baseline. Administration of zinc protoporphyrin (ZnPP), an inhibitor of HO-1, in the hypoxic rats decreased the content of CO in lung homogenate, decreased the apoptosis of the pulmonary artery smooth muscle cells and enhanced the proliferation of pulmonary artery smooth muscle cells and thus worsened hypoxic pulmonary hypertension and pulmonary vascular structural remodeling. Meanwhile, exogenous supply of CO had an adverse action.19 These results show that up-regulation of the CO/HO pathway plays an important role in the regulation of hypoxic pulmonary hypertension. Hydrogen sulfide (H2S) After the discovery of the role of NO and CO, H2S, known as toxic gas for a long time, is also described as a biologically active substance. It can be endogenously generated from cysteine in a reaction catalyzed by cystathionine β-synthesis (CBS) and cystathionine γ-lyase (CSE). The data from our research team, as well as others, demonstrated that H2S exhibited similar functions as NO and CO in the body, such as relaxation of blood vessels, inhibition of the proliferation of smooth muscle cells20 and promotion of the apoptosis of smooth muscle cells, etc. We also reported that H2S played a important role in pathophysiological processes of cardiovascular diseases, such as hypertension, pulmonary hypertension, shock and myocardial injury, etc. Our research team discovered that both the gene expression and activity of CSE were suppressed in lung tissues, and the plasma level of H2S was decreased under hypoxia. Furthermore, supplement of H2S donor molecules opposed the elevation of pulmonary arterial pressure and lessened the pulmonary vascular structure remodeling during hypoxic pulmonary hypertension, while DL-propargylglycine (PPG), a CSE inhibitor, aggravated hypoxic pulmonary hypertension. This indicates that the H2S/CSE pathway plays a significant role in the regulation of hypoxic pulmonary hypertension.21,22 NO, CO and H2S are three gaseous signaling molecules that have complicate interrelations in the regulation of pulmonary hypertension.23,24 Since 1987, NO, CO and more recently H2S, the endogenous gas molecules produced from metabolic pathway, have been recognized respectively as signal molecules involved in the regulation of homeostasis and to play important roles under physiological and pathophysiological conditions. The research into these endogenous gas signaling molecules opened a new avenue in life science. The biological regulatory effects of other endogenous gas molecules, which have previously been regarded as metabolic waste, are now a field of investigation in the life sciences and medicine. In recent years, we began to pay attention to the effects of endogenous sulfur dioxide (SO2) and its derivatives on mammalian and human physiology. SO2 can be produced endogenously from normal metabolism of sulfur containing amino acids. L-cysteine is oxidized via cysteine dioxygenase to L-cysteinesulfinate and the latter can proceed through transamination by glutamate oxaloacetate transaminase (GOT) to β-sulfinylpyruvate which decomposes spontaneously to pyruvate and SO2. During oxidative stress in mammals activated neutrophils can convert H2S to sulfite through a reduced form of a nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent process. Recently, our research team showed that SO2 could be endogenously generated in cardiovascular tissues and could exert important cardiovascular effects, such as vasorelaxant and negative inotropic effects. Moreover, SO2 might play a considerable role in the regulation of systemic circulatory pressure, pulmonary circulatory pressure and vascular structural remodeling in the pathogenesis of hypertension and hypoxic pulmonary hypertension. More studies of the significance of endogenous SO2 in the cardiovascular system under physiological and pathophysiological conditions need to be conducted.25-27 Vasoactive peptides and others vasoactive substances The arachidonic acid metabolic products include prostaglandin E1 (PGE1), PGE2, PGI2 and thromboxane. Among them, PGE2 and thromboxane constrict blood vessels whereas PGE1 and PGI2 dilate blood vessels. PGI2 has strong vasorelaxative effects. It inhibits proliferation of smooth muscle cells and agglutination of platelets. Disturbance in the metabolism of arachidonic acid and reduction in the expression of PGI2 synthesis enzymes occur in pulmonary hypertensive patients.28 Currently, PGI2 and similar functioning substances are successfully used in the treatment of pulmonary hypertension29 and in many countries it is recommended for treatment. In 1993, a new vasoactive polypeptide in chromaffin hemangioma called adrenomedullin (ADM) was found. It has vasorelaxative and hypotensive effects and also inhibits migration and proliferation of smooth muscles of blood vessels. There are different kinds of ADM receptors expressed in pulmonary tissues that combine with specific high affinity binding sites on ADM. The expression of ADM and its receptors increase in hypoxic hypertensive rat lung tissue. Serum ADM levels are also elevated. Our research team and others found that chronic infusion of ADM significantly decreased mean pulmonary artery pressure in hypoxic rats, lessened the muscularization of small pulmonary vessels, attenuated relative medial thickness and relative medial area of pulmonary arteries and alleviated the ultrastructural changes in pulmonary arteries of hypoxic rats. ADM inhibited the proliferation of pulmonary artery smooth muscle cells. Meanwhile, plasma proadrenomedullin N-terminal 20-peptide (PAMP) concentration and the expression of PAMP protein and mRNA in pulmonary arteries in rats with hypoxia treated with ADM were markedly decreased compared with the untreated hypoxic group. The results suggest that ADM ameliorates the development of hypoxic pulmonary vascular structural remodeling. Intramolecular regulation of ADM might play an important role in the regulation of hypoxic pulmonary hypertension by ADM.30,31 Recent studies showed that inhalation of ADM in IPAH patients reduced pulmonary artery pressure but did not change systemic arterial pressure and heart rate.32 This indicated that ADM could possibly become a new drug for the treatment of pulmonary hypertension. Endothelin-1 (ET-1) was discovered in 1988 and is a vasoconstrictive substance. ET stimulates and proliferation of cultured pulmonary artery smooth muscle cells in vitro. Its function is mediated by ETA and ETB receptors. Our research team and others found that ET precursors and ET-1, as well as the expressions of mRNA for ETA and ETB receptors, are significantly increased in the pulmonary arteries and lung tissues of pulmonary hypertensive rats.33 The ET receptor antagonist bosentan improves the hemodyanamics and functions in pulmonary hypertensive patients and is currently a recommended treatment for pulmonary hypertension in many countries.34 Angiotension (AT) is changed to AT II in the presence of angiotension trasferase, which is a strong vasoconstrictive substance. It also stimulates the proliferation of the smooth muscle cells of pulmonary vessels. It is already proven that angiotension trasferase antagonists, not only decrease pulmonary blood pressure, but also improve structural remodeling of pulmonary vessels.35 AT II mediates the high pulmonary blood flow-induced pulmonary vascular structural remodeling. 5-Hydroxytryptamine (serotonin, 5-HT) can constrict blood vessels and also stimulate the proliferation of smooth muscle cells. A significant raise of 5-HT is seen in pulmonary hypertensive patients. In 1999, Eddahibi and colleagues36 found that there was a significant raise in the expression of the 5-HT transporter in response to hypoxia in pulmonary smooth muscle cells. The deletion of the 5-HT transporter gene significantly protects from pulmonary hypertension in hypoxic rats. In IPAH patients, the 5-HT transporter shows polymorphism, which makes pulmonary artery smooth, cells sensitive to 5-HT's proliferation function.37 In addition, platelet-derived growth factor, vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, TGF, and the platelet-activating factor, urotensin, etc. may be involved in the development of pulmonary hypertension. Potassium channel The voltage dependent potassium channel (Kv, a subtype of potassium channel) is the major potassium channel which is responsible for the contraction of pulmonary artery smooth muscle cells. The inhibition of Kv results in a reduction in outflow of potassium from cells, which causes of depolarization of cell membrane and then the opening of calcium channels increase the calcium level in the cytoplasm, which resultes in vasoconstriction. The disturbance in the Kv channel plays an important role in the pathogenesis of IPAH. Selective Kv1.5 expression is reduced in IPAH patients, accompanied by Kv functional impairment, which leads to membrane depolarization and vasoconstriction.38 Dexfenfluraine plays a role in the treatment of pulmonary hypertension. This drug inhibits the activity of the Kv2.1 channel, which indicates potassium channel participates in the incidence of pulmonary hypertension.39 Potassium channels represent a new treatment target with potential therapeutic value for pulmonary hypertension patients. The regulation in the expression or activity of potassium channels can affect both pulmonary vascular tension and structure. GENETIC ISSUES IN PULMONARY HYPERTENSION Dresdale and colleagues40 discovered in 1954 that IPAH has genetic tendency. Later, a series of researches found that 6% of IPAH patients had a familial incidence and its clinical and pathological characteristics are similar to Sporadic IPAH patients. Based on the research of the families of IPAH patients, it was found that IPAH was an autosomal dominant inherited disease. But only 10%— 20% of the related mutation carriers show the features of pulmonary hypertension. The explication of features of pulmonary hypertension is more often seen in females than in males. Future generations of IPAH patients will gradually advance to a serious condition or there may be a skipped generation. In 1997, Morse et al41 found a susceptible gene located at 2q 31-33.41 On this basis, in 2000 Deng42 and Lane43 independently determined that the gene mutations in the bone morphogenetic protein receptor II (BMPR2) are one of the major causes in the development of IPAH. Currently, it is believed that a mutation in the BMPR2 gene causes a functional defect in BMPR2 receptors which is important in the development of IPAH. Germline mutations in the gene coding for the BMPR2 are present in more than 70% of familial pulmonary arterial hypentension (IPAH) and up to 26% of IPAH.44 Bone morphogenetic protein (BMP) belongs to the TGF-beta super family, and is synthesized and secreted by smooth muscle cells and endothelial cells. It mainly regulates those cells which exhibit major function in the embryonic development and stativity of tissues and inhibits proliferation of vascular smooth muscle cells and induces apoptosis. Similar to TGF-β, the regulatory signal transduction pathways of BMP involve a BMP type I receptor (BMPR1a and BMPR1b) and a BMP type II receptor (BMPR2). The type II receptor is the activator of the type I receptor, the two receptors form a complex unit. They activate Smad signaling protein and LIM-kinase to regulate gene transcription and maintain blood vessels.45 There are 46 types of BMPR2 gene mutations, 60% of which may lead to early termination of transcription. A point mutation and an anomaly in the structural domain of BMPR2 kinase may inhibit its receptor functions, rendering it unable to form a heterologous dimer complex or to lose kinase activity and block the signaling pathway. This can lead to excessive cell proliferation and apoptotic inhibition causing vascular structural remodeling and pulmonary hypertension. CONCLUSIONS Though the pathogenesis of most forms of pulmonary hypertension is unknown, there have been many recent developments, especially pertaining to the molecular genetics and cell biology of pulmonary hypertension. Since the range of medical conditions and environmental exposures associated with pulmonary hypertension is wide, it is difficult to envision a unifying pathogenic mechanism. Although there probably are genetic determinants, environmental exposures and acquired disorders that predispose patients to pulmonary hypertension, it is clear that none of the factors described in this review are sufficient alone to activate the pathways essential to the development of this vascular disease.

Platelet-derived growth factor is one of the major growth factors found in human and mammalian serum and tissues. Abnormal activation of platelet-derived growth factor signaling pathway through platelet-derived growth factor receptors may contribute to the development and progression of pulmonary vascular remodeling and obliterative vascular lesions in patients with pulmonary arterial hypertension. In this study, we examined the expression of platelet-derived growth factor receptor isoforms in pulmonary arterial smooth muscle and pulmonary arterial endothelial cells and investigated whether platelet-derived growth factor secreted from pulmonary arterial smooth muscle cell or pulmonary arterial endothelial cell promotes pulmonary arterial smooth muscle cell proliferation. Our results showed that the protein expression of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β in pulmonary arterial smooth muscle cell was upregulated in patients with idiopathic pulmonary arterial hypertension compared to normal subjects. Platelet-derived growth factor activated platelet-derived growth factor receptor α and platelet-derived growth factor receptor β in pulmonary arterial smooth muscle cell, as determined by phosphorylation of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β. The platelet-derived growth factor-mediated activation of platelet-derived growth factor receptor α/platelet-derived growth factor receptor β was enhanced in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal cells. Expression level of platelet-derived growth factor-AA and platelet-derived growth factor-BB was greater in the conditioned media collected from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell than from normal pulmonary arterial endothelial cell. Furthermore, incubation of idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell with conditioned culture media from normal pulmonary arterial endothelial cell induced more platelet-derived growth factor receptor α activation than in normal pulmonary arterial smooth muscle cell. Accordingly, the conditioned media from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell resulted in more pulmonary arterial smooth muscle cell proliferation than the media from normal pulmonary arterial endothelial cell. These data indicate that (a) the expression and activity of platelet-derived growth factor receptor are increased in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal pulmonary arterial smooth muscle cell, and (b) pulmonary arterial endothelial cell from idiopathic pulmonary arterial hypertension patients secretes higher level of platelet-derived growth factor than pulmonary arterial endothelial cell from normal subjects. The enhanced secretion (and production) of platelet-derived growth factor from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell and upregulated platelet-derived growth factor receptor expression (and function) in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell may contribute to enhancing platelet-derived growth factor/platelet-derived growth factor receptor-associated pulmonary vascular remodeling in pulmonary arterial hypertension.

See related article, pages 1323–1330 Serotonin (5-HT, 5-hydroxytryptamine) has long been recognized as one of the most potent naturally occurring pulmonary vasoconstrictors.1 It was first implicated in the pathogenesis of pulmonary arterial hypertension (PAH) after an outbreak of the disease in Switzerland in the 1960’s among patients taking aminorex fumarate, an appetite suppressant that inhibits serotonin uptake by platelets.2 Since that time further outbreaks of PAH have been identified in Europe and the USA associated with the use of fenfluramine-derivate anorexigens,3–5 eventually leading to their withdrawal from the world market in 1997. Although this was, at least in retrospect, a predictable tragedy, it has ironically opened avenues of research into the biology of serotonin signaling in PAH. As fenfluramine-derivatives are substrates for the serotonin transporter (5-HTT, SERT) proteins,6 this suggests that abnormal SERT expression or functional activity could play a role in the pathogenesis of PAH. There is now a body of evidence supporting this hypothesis that provides hope for the development of effective therapeutic strategies targeting specific components of this signaling pathway in patients with these diseases. Most of the serotonin produced in the body is secreted by enterochromaffin cells of the intestine into the portal circulation where it is partially metabolized by the liver. However, levels of free circulating serotonin are maintained in the low nanomolar range through energy-dependent SERT-mediated transport into platelets. This led some researchers to hypothesize that fenfluramines might cause PAH by increasing free plasma levels of serotonin. However, this hypothesis is inconsistent with the observation that chronic treatment with fenfluramine-derivatives if anything reduces plasma levels of serotonin.6 This suggests that other SERT-related effects promote PAH in susceptible patients. This is supported by the observation that patients with idiopathic PAH have increased frequency of the so called L-type polymorphism …

Pulmonary arterial hypertension (PAH) is a syndrome in which pulmonary arterial obstruction increases pulmonary vascular resistance, which leads to right ventricular (RV) failure and a 15% annual mortality rate. The present review highlights recent advances in the basic science of PAH. New concepts clarify the nature of PAH and provide molecular blueprints that explain how PAH is initiated and maintained. Five basic science concepts provide a framework to understand and treat PAH: (1) Endothelial dysfunction creates an imbalance that favors vasoconstriction, thrombosis, and mitogenesis. Restoration of this balance by inhibition of endothelin and thromboxane or augmentation of nitric oxide (NO) and prostacyclin is the paradigm on which most current therapy is based. (2) PAH has a genetic component. Mutations (bone morphogenetic protein receptor-2 [BMPR2]) and single-nucleotide polymorphisms (SNPs; ion channels and transporter genes) predispose to PAH. (3) Excess proliferation, impaired apoptosis, and glycolytic metabolism in pulmonary artery smooth muscle, fibroblasts, and endothelial cells suggest analogies to cancer. Many experimental therapies reduce PAH by decreasing the proliferation/apoptosis ratio; these include inhibitors of pyruvate dehydrogenase kinase (PDK), serotonin transporters (SERT), survivin, 3-hydroxy-3-methylglutaryl coenzyme A reductase, transcription factors (hypoxia-inducible factor [HIF]-1α and nuclear factor of activated T lymphocytes [NFAT]), and tyrosine kinases. Augmentation of voltage-gated K+ channels (Kv1.5) and BMPR2 signaling also addresses this imbalance. Tyrosine kinase inhibitors used to treat cancer are currently in phase 1 PAH trials. (4) Refractory vasoconstriction may occur due to rho kinase activation. Fewer than 20% of PAH patients respond to conventional vasodilators; however, refractory vasoconstriction may respond to rho kinase inhibitors. (5) The RV can be targeted therapeutically. Although increased afterload initiates RV failure, which is the major cause of death/dysfunction in PAH, the RV may be amenable to cardiac-targeted therapies. The RV in PAH has features of ischemic, hibernating myocardium. Guided by these new …

pulmonary arterial hypertension (PAH) is a rare human disease displaying a very poor prognosis of survival. The chronic increase in pulmonary vascular resistance associated with PAH eventually leads to right ventricular hypertrophy and failure, and ultimately death. It is well accepted that the

BackgroundWhile chemerin has been shown to increase proliferation and migration of systemic vascular smooth muscle cells (SMCs) contributing therefore to the development of hypertension, this remains to be clarified for the pulmonary circulation.MethodsExpression of chemerin and its three receptors (CMKRL1, CCRL2, GPR1) was examined by immunohistochemistry and RTq-PCR in lungs, pulmonary artery, and thoracic aorta from Wistar rats. Primary cultured rat pulmonary artery and thoracic aorta SMCs treated with recombinant chemerin (tested from 5.10–9 to 10–7 mol/L) were assessed for proliferation and migration (both with 10–7 mol/L endothelin-1), as well as for staurosporine-induced apoptosis.ResultsIn pulmonary artery and thoracic aorta, CMKLR1 expression was detected in both endothelial cells and SMCs. In primary cultured pulmonary artery SMCs, chemerin and its three receptors were expressed, and CMKLR1 expression was higher than those of CCRL2 and GPR1. Chemerin added to endothelin-1 increased pulmonary artery SMC proliferation, while chemerin or endothelin-1 alone did not. This effect was less pronounced in thoracic aorta SMCs. Chemerin induced pulmonary artery and thoracic aorta SMC migration, which was exacerbated by endothelin-1 and more pronounced in thoracic aorta SMCs. Chemerin concentration-dependently reduced staurosporine-induced apoptosis in both pulmonary artery and thoracic aorta SMCs. In pulmonary artery SMCs, endothelin-1 treatment increased the expression of CMKLR1, CCRL2, and GPR1, while these expressions were not altered in thoracic aorta SMCs.ConclusionChemerin/CMKRL1 signaling, in conjunction with a key mediator in the pathogenesis of pulmonary hypertensive diseases, endothelin-1, stimulated proliferation and migration, and increased resistance to apoptosis in rat primary cultured pulmonary artery SMCs. Our results suggest that this signaling could play a role in pulmonary artery remodeling observed in pulmonary hypertension.

Elevated circulating hepcidin levels have been reported in patients with pulmonary artery hypertension (PAH). Hepcidin has been shown to promote proliferation of human pulmonary artery smooth muscle cells (PASMCs) in vitro, suggesting a potential role in PAH pathogenesis. However, the role of human pulmonary artery endothelial cells (PAECs) as either a source of hepcidin, or the effect of hepcidin on PAEC function is not as well described. The objective of this study was to define the role of the hepcidin-ferroportin axis on the phenotype of PAEC and to study potential PAEC-PASMC interactions relevant to the pathogenesis of pulmonary vascular remodeling and PAH. PAECs treated with hepcidin, or interleukin-6 were investigated for both ferroportin and hepcidin release and regulation using immunofluorescence, mRNA levels and cellular release assays. Effects of hepcidin on PASMC and PAEC mitochondrial function was investigated using immunofluorescence and seahorse assay. Migration and proliferation of PASMCs treated with conditioned media from hPAEC treated with hepcidin was investigated using the xCELLigence system and other tools. We demonstrate in this study that PAECs express ferroportin; hepcidin treatment of PAECs resulted in mitochondrial iron accumulation and intracellular hepcidin biosynthesis and release. Conditioned media from hepcidin treated PAECs caused PASMCs to down-regulate ferroportin expression whilst promoting migration and proliferation. Inhibition of hepcidin in PAEC conditioned media limited these responses. PASMC cellular and mitochondrial iron retention are associated with migratory and proliferative responses. This study confirms that the hepcidin ferroportin axis is present and operational in PAECs. Modulation of this axis shows distinct differences in responses seen between PAECS and PASMCs. Stimulation of this axis in PAECs with hepcidin may well institute proliferative and migratory responses in PASMCs of relevance to pathogenesis of PAH offering potential novel therapeutic targets.

Idiopathic pulmonary arterial hypertension (IPAH) is associated with proliferation of smooth muscle cells (SMCs) in small pulmonary arteries. Inhibition of proliferation of pulmonary artery smooth muscle cells (PASMCs) may be an effective treatment of patients with idiopathic pulmonary arterial hypertension. Recent studies have shown that carvedilol, an alpha- and beta-blocker with antioxidant and calcium channel blocking properties, inhibits the proliferation of cultured normal human pulmonary artery smooth muscle cells. In this study, we tested the hypothesis that carvedilol has antiproliferative effects on pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension. Pulmonary artery smooth muscle cells from six idiopathic pulmonary arterial hypertension patients who had undergone lung transplantation were cultured. To determine cell proliferation, H-thymidine incorporation was measured. Platelet-derived growth factor-induced proliferation of IPAH-PASMCs was significantly greater than that of normal control pulmonary artery smooth muscle cells. Carvedilol (0.1 microM to 10 microM) inhibited the proliferation of idiopathic pulmonary arterial hypertension-pulmonary artery smooth muscle cells in a concentration-dependent manner. Prazosin (an alpha-blocker) and N-acetyl L cysteine (an antioxidant agent) (0.1 microM to 10 microM) did not inhibit their proliferation, but the high concentration of propranolol (a beta-blocker) and nifedipine (a calcium channel blocker) (10 microM) inhibited the proliferation. The combination of propranolol and nifedipine inhibited the proliferation but only at a high concentration (10 microM) combination. Cell cycle analysis revealed that carvedilol (10 microM) significantly decreased the number of cells in S and G2/M phases. These results indicate that carvedilol inhibits the exaggerated proliferation of pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension partially via its beta-blocking [corrected] and calcium channel blocking effects in vitro.

Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline

BACKGROUND Pulmonary Arterial Hypertension (PAH) is an incurable disease and remains a significant cause of morbidity and mortality in children and adults. In PAH, pulmonary arteries narrow in diameter which increases blood flow resistance in the lungs. Eventually the increase in pulmonary blood pressure causes irreversible damage to the heart. There are a variety of reasons why the pulmonary arteries become obstructed, known and unknown; yet, the molecular mechanisms driving this disease remain to be elucidated. Approximately 1000 new cases of PAH are diagnosed each year in the U.S. alone. The average survival rate for patients diagnosed with PAH is 7 years. Therefore, any insight into the molecular mechanisms that promote PAH progression may represent new strategies to treat patients with PAH. OBJECTIVE Up to 20% of PAH patients with no family history of PAH carry a bone morphogenetic protein receptor 2 (BMPR2) mutation, whereas in patients with a family history of PAH up to 82% carry a BMPR2 mutation. Therefore, it appears extremely likely that BMPR2 loss of function promotes the dysfunctional angio‐proliferative phenotype of PAH. In fact, it has recently been shown that in endothelial cells (EC) deficient in BMPR2 exposed to exogenous bone morphogenetic protein 2 (BMP2), a ligand for BMPR2, are migratory compared to control cells. To determine if the loss of BMPR2 promotes the PAEC dysfunctional angiogenesis observed in patients with PAH, we used cells from control and PAH patients to determine the role of pulmonary artery smooth muscle cell (PASMC) secreted BMP2 in promoting the migration of pulmonary artery endothelial cells (PAEC) and angiogenesis. METHODS PASMC and PAEC from control and PAH patients were examined for the expression of BMPR2 and BMP2. Cell migration, cell invasion, and angiogenetic assays were performed using PASMC and PAEC from control and PAH patients to assess differences in migration, invasion, and angiogenesis. RESULTS PAEC from PAH patients have a significant reduction in the expression of BMPR2, consistent with published reports. PASMC from PAH secrete BMP2, while BMP2 is undetectable in control cells. Co‐culturing PAH PASMC with PAH PAEC promotes PAEC migration. This migration is further enhanced in the presence of exogenous BMP2. PAH PASMC co‐cultured with PAH PAEC promote the development of PAEC‐derived angiogenetic masses. This dysfunctional angiogenetic response is further amplified in the presence of exogenous BMP2. In contrast, control PAEC exposed to control PASMC respond normally and do not form disorganized masses of vessels. Control PAEC exposed to exogenous BMP2 further promotes a normal angiogenetic response. CONCLUSIONS PASMC from PAH patients secrete BMP2. BMP2 exposure promotes the migration of PAH PAEC and a dysfunctional angiogenetic response. In contrast, BMP2 promotes a normal angiogenetic response when added to co‐cultures of control PASMC and PAEC. These data suggest that in PAH, angiogenesis becomes dysfunctional in part due to PAEC exposure to BMP2; therefore, inhibitors of BMP2 may represent a therapeutic strategy to pursue in the treatment of PAH.

Exosomes Derived from Human Pulmonary Artery Endothelial Cells Shift the Balance between Proliferation and Apoptosis of Smooth Muscle Cells

The tumor-suppressive role of p53, a transcription factor that regulates the expression of many genes, has been linked to cell cycle arrest, apoptosis, and senescence. The noncanonical function or the pathogenic role of p53 has more recently been implicated in pulmonary vascular disease. We previously reported that rapid nuclear accumulation of hypoxia-inducible factor (HIF)-1α in pulmonary arterial smooth muscle cells (PASMCs) upregulates transient receptor potential channels and enhances Ca2+ entry to increase cytosolic Ca2+ concentration ([Ca2+]cyt). Also, we observed differences in HIF-1α/2α expression in PASMCs and pulmonary arterial endothelial cells (PAECs). Here we report that p53 is increased in PAECs, but decreased in PASMCs, isolated from mice with hypoxia-induced pulmonary hypertension (PH) and rats with monocrotaline (MCT)-induced PH (MCT-PH). The increased p53 in PAECs from rats with MCT-PH is associated with an increased ratio of Bax/Bcl-2, while the decreased p53 in PASMCs is associated with an increased HIF-1α. Furthermore, p53 is downregulated in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension compared with PASMCs from normal subjects. Overexpression of p53 in normal PASMCs inhibits store-operated Ca2+ entry (SOCE) induced by passive depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum, while downregulation of p53 enhances SOCE. These data indicate that differentially regulated expression of p53 and HIF-1α/2α in PASMCs and PAECs and the cross talk between p53 and HIF-1α/2α in PASMCs and PAECs may play an important role in the development of PH via, at least in part, induction of PAEC apoptosis and PASMC proliferation.

Treatment of Pulmonary Arterial Hypertension: A Step Forward

The pathogenesis of pulmonary hypertension has not been elucidated. We investigated the role of a circular ribonucleic acid, circDiaph3, in the proliferation and migration of pulmonary artery smooth muscle cells during pulmonary hypertension. CircDiaph3 overexpression in blood samples of patients with pulmonary hypertension was analyzed by real-time quantitative polymerase chain reaction. Subsequently, a rat model of pulmonary arterial hypertension was established under hypoxic conditions. Pulmonary artery smooth muscle cells were harvested from the rat model for subsequent experiments with small interfering ribonucleic acid-mediated knockdown of circDiaph3. In cell model, we found that PI3K, AKT, mTOR and insulin-like growth factor 1 signaling pathway (IGF1R) and smooth muscle cell marker genes (α-SMA, Vcam1) were significantly downregulated. The overexpression of Igf1r in pulmonary artery smooth muscle cells rescued the downregulated smooth muscle cell genes, IGF1R signaling pathway proteins, increased smooth muscle cell proliferation, and reduced apoptosis. CircDiaph3 regulates the PI3K/AKT/mTOR signaling pathway via IGF1R to inhibit apoptosis and promote proliferation of smooth muscle cells. Additionally, adenovirus-mediated in vivo inhibition of circDiaph3 was carried out in rats with pulmonary arterial hypertension, followed by harvesting of their pulmonary artery smooth muscle cells for subsequent experiments. Excessive proliferation of smooth muscle cells in the pulmonary artery has narrowed the pulmonary artery lumen, thereby causing pulmonary hypertension, and our results suggest that circDiaph3 has important value in the treatment of pulmonary hypertension.

PULMONARY ARTERIAL HYPERTENSION (PAH) is a progressive disease manifested by maladaptation of the pulmonary vasculature. The development of PAH can be influenced by genetic predisposition and/or by diverse endogenous or environmental stimuli. Regardless of the initial pathogenic factors, pulmonary vascular remodeling, sustained pulmonary vasoconstriction, in situ thrombosis, and increased pulmonary vascular wall stiffness are the major contributors to elevated pulmonary vascular resistance (PVR). The increase in PVR can lead to right ventricular failure and death in patients with PAH. While treatments for this disease are improving, it continues to be a life-threatening condition. MicroRNAs (miRNAs) have been implicated in the development and progression of PAH. MiRNAs are small, noncoding RNAs that regulate gene and protein expression by promoting degradation or suppressing translation of target mRNAs. Several studies have demonstrated aberrant expres