Abstract 263: Differential Effects of 17ß-Estradiol and 16a-Hydroxyestrone in Oxidative Stress and Proliferative Responses in Human Pulmonary Artery Smooth Muscle Cells - Implications in Pulmonary Arterial Hypertension

Gender differences in pulmonary arterial hypertension (PAH) may be, in part, due to increased formation of the deleterious estrogen metabolite, 16α-hydroxyestrone (16αOHE1). Oxidative stress and Noxs have been implicated in the pathogenesis of PAH. We hypothesised that 17β-estradiol (E2) and 16αOHE1, specifically in human pulmonary artery smooth muscle cells (PASMCs), leads to Nox-induced oxidative stress, which promotes PASMC damage. Cultured human PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM). ROS production was assessed by chemiluminescence (O2-) and Amplex Red (H2O2); antioxidants, regulators of Nrf2, and PCNA (marker of growth) expression by immunoblotting; and Nrf2 activity by ELISA. E2 increased superoxide (219%) and H2O2 (52%) in PASMCs (p<0.05 vs vehicle). E2 induced ROS was blocked by PHTPP (ERβ antagonist), tempol (SOD mimetic), ML171 (Nox1 inhibitor) and GKT137831 (Nox1/4 inhibitor). Thioredoxin (71%), NQ01 (78%) and peroxiredoxin1 (69%) protein levels were decreased by E2, even though Nrf2 activity was increased (38%), p<0.05 vs vehicle. 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in superoxide (355%) and a decrease in H2O2 (65%) production, p<0.05. 16αOHE1-induced ROS was blocked by MPP (ERα antagonist), G15 (GPR30 antagonist), tempol and ML171. 16αOHE1 increased BACH1 (129%; p<0.05), a competitor of Nrf2, which was decreased (92%). E2 stimulation resulted in decreased PCNA expression (30%), while 16αOHE1 increased PCNA levels (150%); p<0.05. E2 and 16αOHE1 induced a rapid and sustained ROS generation in PASMCs derived from PAH subjects. E2 and 16αOHE1 did not increase superoxide production in VSMCs from resistance arteries of healthy subjects. In conclusion, E2 induces ROS production through ERβ-Nox-dependent mechanisms, while 16αOHE1 increases superoxide through an ERα/GPR30-Nox-dependent manner. The effects of E2 and 16αOHE1 on oxidative stress is present in PASMCs but not in VSMCs from peripheral arteries. and seems to be related to a dysregulation of the Nrf2 pathway. These processes may impact on molecular processes contributing to vascular remodeling in PAH.

<b>Introduction:</b> Pulmonary arterial hypertension (PAH) is a fatal disease characterized by remodelling of pulmonary arteries, smooth muscle cell hyperplasia and hypertrophy. TGF-β, regulating cell proliferation, migration, and cell death, is elevated in PAH, and has been implicated in its pathogenesis based on clinical and experimental data. TGF-β binding to its receptor activates downstream signalling cascades, such as SMAD proteins. Recent data suggest that SMAD3 is downregulated in PAH. We thus hypothesize that loss of SMAD3 contributes to two major features of PAH: proliferation and hypertrophy (via disinhibition of the myocardin-related transcription factor (MRTF), a myogenic gene inducer). Here, we investigated the regulation of SMAD3 and its interaction with MRTF in human pulmonary arterial smooth muscle cells (PASMC) <i>in vitro</i> and <i>in vivo</i>, and its potential role in PAH. <b>Results:</b> TGF-β treatment for 72 h caused a significant downregulation of SMAD3 mRNA and protein levels in PASMC. Loss of SMAD3 was also evident in pulmonary arteries from rats with monocrotaline-induced PAH. Silencing of SMAD3 in PASMCs increased the proliferative response upon stimulation with fetal calf serum as determined by western blotting for proliferating cell nuclear antigen protein, Ki-67 positive cells, and bromodeoxyuridine assay. Co-immunoprecipitation revealed a reduced interaction between SMAD3 and MRTF in TGF-β treated PASMCs compared to control cells. <b>Conclusion:</b> The present data suggest that SMAD3 downregulation, which occurs in PASMC both <i>in vitro</i> and <i>in vivo</i>, contributes to increased proliferation and - through MRTF liberation - to hypertrophy of PASMC, key features of lung vascular remodeling in PAH.

See related article, pages 1109–1114 Idiopathic pulmonary artery hypertension (IPH) is a rare illness with a poor prognosis. Whereas chronic intravenous prostacyclin relieves some of the symptoms of progressive dyspnea and prolongs survival, most patients ultimately require a lung transplant.1 Newer therapies such as nonintravenously administered prostacyclin derivatives,2,3,4 endothelin receptor blockers,5,6 and, to some extent, phosphodiesterase inhibitors,7 hold some promise as alternatives for intravenous prostacyclin, but current expectation is that, like prostacyclin, they will, at best, retard disease progression, serving as a bridge to transplant rather than as an alternative. The pathological features of IPH are loss of small distal precapillary pulmonary arteries, obliterative changes (plexogenic lesions) in more proximal pulmonary arteries associated with migration and proliferation of smooth muscle cells, and increased extracellular matrix deposition. There is also dysregulation of endothelial cells associated with increased proliferation.8 The mechanism underlying the evolution of these changes is unknown, so there was great interest when 2 groups independently identified a mutation in bone morphogenetic protein receptor 11 (BMP-RII) in 60% of families with IPH.9,10 A BMP-RII mutation also occurs in 20% of sporadic cases of IPH,11 but the biological connection between the mutation and the pathobiology of IPH has been relatively elusive. Recent studies using pulmonary artery smooth muscle cells from patients with IPH, including those with and without a BMP-RII mutation, showed similar abnormal proliferation in response to agents such as transforming growth factor-β (TGF-β) or BMP-2.12 In other studies, pulmonary artery smooth muscle cells were transfected with constructs encoding different mutant forms of BMP-RII expressing aberrant kinase or cytoplasmic domains, and impaired signaling was observed related to alterations in the induction of Smads and p38.13 Specifically, suppression of Smad1/5 and activation of p38 were related to smooth muscle cell proliferation. It …

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+)cyt] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

Rationale: Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH.Objectives: To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH.Methods: PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH.Measurements and Main Results: Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs.Conclusions: Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2.

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.

Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1-/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT1B receptor signaling and Nox1, confirmed in PASMCs from Nox1-/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT1B receptors contribute to experimental pulmonary hypertension by inducing lung ROS production. Our results suggest that 5-HT1B receptor-dependent cellular Src-related kinase-Nox1-pathways contribute to vascular remodeling in PAH.

Pulmonary arterial hypertension (PAH) is a serious disease and a major public health problem with approximately 1000 new patients diagnosed every year in the United States.1,2 PAH is a progressive disease involving impaired pulmonary vascular structure and function and is ultimately lethal as a result of right ventricular failure. Recent insights into the pathogenesis of PAH have led to more promising therapeutic approaches and improved outcomes; however, the mortality rates associated with PAH remain unacceptably high.1,3,4 PAH affects all age groups and both genders; however, there is a striking preponderance of female patients with PAH, which remains unexplained.5 PAH can be idiopathic, familial, or associated with other cardiovascular disorders, but the underlying pathology and pathophysiology are shared by all forms of the disease. PAH is characterized by progressive fibroproliferative remodeling of the pulmonary arterioles, various degrees of pulmonary vasoconstriction and inflammation, thrombosis, and right ventricular hypertrophy and failure. The mainstay of current pharmacologic therapies is pulmonary vasodilation, although only a small percentage of patients have demonstrable pulmonary vasoconstriction when they undergo cardiac catheterization.6 Despite the lack of a demonstrable vasoconstrictor component, many patients with PAH improve with long-term vasodilator therapy, and it is believed that pulmonary vascular remodeling could also be ameliorated as a result of vasodilator therapies. Vasoconstrictors such as angiotensin II and endothelin-1 (ET-1) are potent stimulators of vascular smooth muscle (VSM) growth and proliferation. Although many vasodilator mediators also have antiproliferative effects on VSM, there is no definitive evidence that pulmonary vascular remodeling in humans with PAH is reversible. In addition, current vasodilator therapies are not universally successful in altering PAH progression and increasing survival. Therefore, novel approaches that directly target pulmonary vessel wall pathology are needed to reverse the established pulmonary vascular pathology in patients with PAH. Pathophysiological Mechanisms of Pulmonary Arterial Hypertension Studies in human PAH and experimental models of the disease have suggested the involvement of several molecular and signaling pathways in the development and progression of PAH.7-20 Mutations in bone morphogenetic protein receptor type II, a transforming growth factor beta receptor, have been associated with familial primary PAH.8 Decreased release or activity of endothelium-derived nitric oxide (NO) in the pulmonary circulation and loss of NO-cGMP relaxation through degradation of cGMP through phosphodiesterase 1 are major factors in the pathogenesis of PAH (Fig. 1).7,12 Several other pathways have been implicated in PAH pathogenesis as demonstrated by loss of function and interventional studies; these include cyclo-oxygenase-2 and prostacyclin (PGI2),17,19 ET-1,15 and platelet-derived growth factor signaling,13 the Rho-kinase,14 and Notch3 signaling pathways20 as well as heme oxygenase-1/carbon monoxide.18,21 Nevertheless, PAH appears to be a multifactorial disorder, and more than one gene or signaling pathway is likely to be involved.FIGURE 1: Pathophysiological mechanisms and molecular targets in a monochrotaline-treated and hypoxia-induced model of pulmonary arterial hypertension (PAH). The PAH-associated endothelial cell dysfunction and decreased pulmonary artery relaxation and the vascular smooth muscle dysfunction and increased pulmonary artery constriction and remodeling are reduced by treatment with the phosphodiesterase inhibitor sildenafil or the endothelin-1 receptor antagonist bosentan. 2-Methoxy-estradiol enhances the stimulatory effects of sildenafil on pulmonary artery relaxation and the inhibitory effects of bosentan on vasoconstriction and remodeling.As demonstrated in human studies as well as studies in experimental animals, PAH is a pan-vasculopathy characterized by endothelial dysfunction, medial hypertrophy and VSM hyperplasia and adventitial fibrosis. Using the monochrotaline (MCT) and hypoxic rat models of PAH, we and others have shown reduced pulmonary artery contraction to vasoconstrictors, decreased endothelium-dependent NO-cGMP-mediated pulmonary artery relaxation, and decreased pulmonary artery responsiveness to endogenous and exogenous nitrovasodilator.7,22-25 The changes in pulmonary artery function are associated with extensive pulmonary artery thickening and remodeling and increased pulmonary VSM cell growth and proliferation (Fig. 1).26-29 These observations have made the MCT and hypoxic rat models the most commonly used animal models to investigate the pathophysiology of PAH and to test the effects of potential therapies of the disease. In addition to the significant pulmonary artery remodeling, perivascular inflammation and transdifferentiation of circulating and resident progenitor cells are thought to contribute to the pathogenesis of PAH through mechanisms that are incompletely understood.30-33 Common Therapies of Pulmonary Arterial Hypertension The characterization of the molecular mechanisms underlying PAH has been critical to identifying novel targets for therapeutic intervention. However, as novel targets are identified, multiple obstacles to clinical application have to be overcome because bioavailability, selectivity, and potential toxicity of novel therapies need to be carefully evaluated in clinical trials. As a result, despite the identification of multiple potential new targets for intervention, only three classes of therapies are currently in use for PAH. These include phosphodiesterase-5 inhibitors, PGI2 analogs, and endothelin receptor A antagonists. These therapies target molecular pathways that are known to be dysregulated in the setting of PAH. Phosphodiesterase inhibitors such as sildenafil prevent the breakdown of cGMP and consequently enhance the NO-cGMP pulmonary arterial relaxation pathway. PGI2 analogs such as iloprost stimulate the PGI2 receptors and enhance the PGI2-cAMP relaxation pathway. Nonspecific ET-1 receptor antagonists such as bosentan and specific endothelin receptor A antagonists like sitaxentan and ambrisentan block ET-1-induced signaling and its effects on both VSM contraction and cell growth. To enhance their effectiveness, these therapeutic approaches are often used in combination, although the optimal combination therapy is still under investigation.34,35 Whether used separately or combined, the goal of these approaches is to restore the balance between the NO-cGMP and PGI2-cAMP vasodilator and VSM antiproliferative pathways and the ET-1-induced vasoconstrictor and VSM proliferative pathway in the pulmonary vasculature. Ca2+ channel blockers may have some benefits in certain patients with PAH.36,37 Also, ample experimental evidence supports that more specific antiproliferative, proapoptotic, immunomodulatory, and cell-based therapies could be effective in PAH;13,38-44 however, translation to clinical application is lagging behind these new discoveries. Effects of Sex Hormones in Pulmonary Arterial Hypertension The preponderance of PAH in females is unexplained yet very intriguing particularly because it is opposite of the known preponderance of systemic cardiovascular disease in males. It has become increasingly appreciated that the cardiovascular effects of sex steroids and their metabolites are far more complex than initially thought. Despite attempts to define the role of sex steroid hormones in pulmonary vascular disease, significant knowledge gaps exist in this area.44 Most studies on the role of sex steroids in pulmonary vascular homeostasis focus on the vasodilator properties of estrogens, and possibly androgens, especially in the settings of experimental hypoxia.45 Specifically, 17-hydroxy-estradiol has been reported to have multiple effects on the endothelial production of NO and PGI2, which in turn lead to endothelium-dependent vasodilation. In addition, 17-hydroxy-estradiol has endothelium-independent vasodilatory effects by activating voltage-activated potassium channels in VSM cells. Similarly, progesterone and testosterone have been reported to reduce vascular tone by blocking both voltage-gated and receptor-operated Ca2+ channels in VSM cells.45 Recently, 2-methoxy-estradiol (2-ME) has been recognized as a biologically active metabolite of estradiol with estrogen receptor-independent antiproliferative properties,46 and its therapeutic potential in cardiovascular and pulmonary vascular diseases warrants further investigation. In this issue of the Journal of Cardiovascular Pharmacology, Tofovic and coworkers present experimental evidence supporting that 2-ME treatment ameliorates MCT-induced PAH not only in adult females, but also in adult male rats. 2-ME was as efficacious as the phosphodiesterase inhibitor sildenafil and the ET-1 receptor antagonist bosentan in ameliorating MCT-induced PAH in both male and female rats. The authors also demonstrate that the combination of 2-ME with either sildenafil or bosentan confers additional protection and improves survival in MCT-treated rats (Fig. 1). Importantly, these pharmacologic interventions were initiated 12 days after administration of MCT and the establishment of PAH and pulmonary vascular remodeling thus supporting effective reversal of established disease. The authors propose that the mechanism of protection involves antiproliferative and anti-inflammatory effects of 2-ME in the pulmonary vasculature and the lung, respectively. Future Directions The study by Tofovic and colleagues opens an important area for investigation with regard to the sex differences in the incidence of PAH and the role of sex hormones in the pathogenesis and management of PAH. An important question is whether the increase in the incidence of PAH in females is related to estrogen. This will be difficult to reconcile with the apparent protective effects of 2-ME in the MCT-treated model of PAH. One possibility is that the effects of estradiol on the pulmonary vasculature are different from those of estrogen metabolites. In this respect, it is important to compare the effects of 2-ME on pulmonary vessels with those of estrogen, specific estrogen receptor modulators, and other estrogen metabolites. It will also be important to study the differential effects of estrogens as compared with androgens on the pulmonary vasculature. The specific mechanisms underlying the effects of estrogen and its metabolites on pulmonary vascular structure and function also need to be further examined. Estrogen-induced endothelium-dependent vasodilator effects on NO, PGI2, and endothelium-derived hyperpolarizing factor have been described in blood vessels of the systemic circulation.47 Additional inhibitory effects of estrogen on VSM cell contraction and growth have been suggested.47 Studies have suggested inhibitory effects of estrogen on the Ca2+-dependent mechanisms of VSM contraction.48 Other studies have suggested an inhibitory effect of estrogen on the expression/activity of protein kinase C and Ca2+-sensitization pathways of VSM contraction (Fig. 1).49 In this respect, studies have suggested a role for Rho-kinase as Ca2+-sensitization pathway of VSM contraction as well as in VSM cell growth and proliferation.50 Given the beneficial effects of Rho-kinase inhibitors in experimental PAH,14 it would be important to test whether estrogen or its metabolites function by inhibiting Rho-kinase. Estrogen and its metabolites may also affect the expression/activity of matrix metalloproteinases and the composition of the extracellular matrix,51 and such effects could also improve pulmonary arterial remodeling in PAH. Finally, whereas studying the effects of 2-ME in MCT-treated rat model highlights potential pulmonary vascular protective effects in PAH, it would be important to study the effects of estrogen and its metabolites in other animal models such as the hypoxia-induced model of PAH. Careful evaluation of the findings in experimental animal models could spearhead studies in human and clinical trials to determine the effects of sex hormones on the course of PAH.

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.

Pulmonary arterial hypertension (PAH) is a devastating and life-threatening clinical syndrome characterized by elevated pulmonary artery pressures leading to progressive symptoms, including shortness of breath, fatigue, and a decline in functional ability. The hemodynamic definition of PAH is a mean pulmonary artery pressure of >25 mm Hg at rest or >30 mm Hg during exercise, with a normal pulmonary arterial wedge pressure ≤15 mm Hg.1 Recent data from a French registry suggests that the prevalence of PAH is about 15 cases per 1 million,1 and the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL) demonstrates that the mean age at diagnosis is 48 years and that mostly women (80%) are affected.2 The pathophysiology of PAH includes endothelial dysfunction, vascular remodeling with progressive obstruction and obliteration of pulmonary arteries, and ultimately, high right atrial pressure, right ventricular hypertrophy, right ventricular failure, and death.1 Endothelial dysfunction is believed to be an early event in PAH, and is characterized by overproduction of vasoconstrictor/mitogenic compounds, such as endothelin and thromboxane A2, and by insufficient production of vasodilators, such as prostacyclin and nitric oxide (NO). These observations led to the development of 3 different classes of therapies that are currently in clinics, either alone or in combination: prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase (PDE) inhibitors. Although current therapies can improve symptoms and reduce severity of the hemodynamic disorders, gradual deterioration of pulmonary and cardiac functions often necessitates lung transplantation. The prognosis of PAH is reportedly poor, with ≈15% mortality within 1 year of modern therapy.1 Therefore, a renewed interest has been focused on the mechanisms of PAH pathogenesis to identify a novel therapeutic target. Article see p 1194 It is now well established that disruption or dysfunction of the endothelium promotes vascular lesion formation. This …

Neointimal lesion and medial wall thickness of pulmonary arteries (PAs) are common pathological findings in pulmonary arterial hypertension (PAH). Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling contribute to intimal and medial vascular remodeling in PAH. Nintedanib is a tyrosine kinase inhibitor whose targets include PDGF and FGF receptors. Although the beneficial effects of nintedanib were demonstrated for human idiopathic pulmonary fibrosis, its efficacy for PAH is still unclear. Thus, we hypothesized that nintedanib is a novel treatment for PAH to inhibit the progression of vascular remodeling in PAs. We evaluated the inhibitory effects of nintedanib both in endothelial mesenchymal transition (EndMT)-induced human pulmonary microvascular endothelial cells (HPMVECs) and human pulmonary arterial smooth muscle cells (HPASMCs) stimulated by growth factors. We also tested the effect of chronic nintedanib administration on a PAH rat model induced by Sugen5416 (a VEGF receptor inhibitor) combined with chronic hypoxia. Nintedanib was administered from weeks 3 to 5 after Sugen5416 injection, and we evaluated pulmonary hemodynamics and PAs pathology. Nintedanib attenuated the expression of mesenchymal markers in EndMT-induced HPMVECs and HPASMCs proliferation. Phosphorylation of PDGF and FGF receptors was augmented in both intimal and medial lesions of PAs. Nintedanib blocked these phosphorylation, improved hemodynamics and reduced vascular remodeling involving neointimal lesions and medial wall thickening in PAs. Additionally, expressions Twist1, transcription factors associated with EndMT, in lung tissue was significantly reduced by nintedanib. These results suggest that nintedanib may be a novel treatment for PAH with anti-vascular remodeling effects.

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.

The high mortality associated with PAH is in part, due to the poorly understood pathogenesis. Endoplasmic reticulum (ER)-stress is a potential commonality among many molecular triggers of PAH, including mutations, viruses, inflammation and hypoxia. The ER forms a functional unit with the mitochondria (the ER-mito unit), allowing for exchange of Ca2+. Recently, we showed that ER-mito unit disruption was critical in PAH pathogenesis. In pulmonary artery smooth muscle cells (PASMCs), ER-stress activated the transcription factor ATF6, increased levels of the ER protein Nogo, disrupted the ER-mito unit, and resulted in the apoptosis-resistance that is central to pulmonary vascular remodeling in PAH. Chemical chaperones including the FDA-approved 4-phenylbutyrate (PBA) attenuate ER-stress and have been studied in metabolic diseases and cancer. We hypothesized that attenuation of ER stress-induced ATF6 activation with PBA will prevent the disruption of the ER-mito unit and prevent/reverse PAH. Hypoxic mice or monocrotaline-injected rats were treated with PBA in their drinking water (~500mg/kg/day) in prevention (days 0-28) and reversal (days 14-28) protocols. Mechanistic studies were performed in lungs and PASMCs exposed to hypoxia. PBA decreases ATF6 activation (nuclear localization, luciferase, target gene (Nogo/GRP78) expression) and normalizes mitochondrial function (mitochondrial-Ca2+, membrane potential, reactive oxygen species). Both in vivo and in vitro, PBA suppresses proliferation (Ki67) and induces apoptosis (TUNEL). Treated mice had less pulmonary artery remodeling, improved hemodynamics, decreased right ventricular hypertrophy and better functional capacity compared to vehicle-treated controls (see table; data presented as mean±SEM). Treated rats responded similarly. Chemical chaperones improve PAH by inhibiting ATF6 activation, a pathway compatible with several PAH-triggers that induce ER-stress.

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

CGRP attenuates pulmonary vascular remodeling by inhibiting the cGAS-STING-NFκB pathway in pulmonary arterial hypertension