Pulmonary Arterial Hypertension In Mice Research Articles

Analysis of ET-1 system in mild and severe pulmonary arterial hypertension in mice

Analysis of ET-1 system in mild and severe pulmonary arterial hypertension in mice

Treatment with Anti–Gremlin 1 Antibody Ameliorates Chronic Hypoxia/SU5416–Induced Pulmonary Arterial Hypertension in Mice

The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.

Dexfenfluramine and the oestrogen-metabolizing enzyme CYP1B1 in the development of pulmonary arterial hypertension

AimsPulmonary arterial hypertension (PAH) occurs more frequently in women than men. Oestrogen and the oestrogen-metabolising enzyme cytochrome P450 1B1 (CYP1B1) play a role in the development of PAH. Anorectic drugs such as dexfenfluramine (Dfen) have been associated with the development of PAH. Dfen mediates PAH via a serotonergic mechanism and we have shown serotonin to up-regulate expression of CYP1B1 in human pulmonary artery smooth muscle cells (PASMCs). Thus here we assess the role of CYP1B1 in the development of Dfen-induced PAH.Methods and resultsDfen (5 mg kg−1 day−1 PO for 28 days) increased right ventricular pressure and pulmonary vascular remodelling in female mice only. Mice dosed with Dfen showed increased whole lung expression of CYP1B1 and Dfen-induced PAH was ablated in CYP1B1−/− mice. In line with this, Dfen up-regulated expression of CYP1B1 in PASMCs from PAH patients (PAH-PASMCs) and Dfen-mediated proliferation of PAH-PASMCs was ablated by pharmacological inhibition of CYP1B1. Dfen increased expression of tryptophan hydroxylase 1 (Tph1; the rate-limiting enzyme in the synthesis of serotonin) in PAH-PASMCs and both Dfen-induced proliferation and Dfen-induced up-regulation of CYP1B1 were ablated by inhibition of Tph1. 17β-Oestradiol increased expression of both Tph1 and CYP1B1 in PAH-PASMCs, and Dfen and 17β-oestradiol had synergistic effects on proliferation of PAH-PASMCs. Finally, ovariectomy protected against Dfen-induced PAH in female mice.ConclusionCYP1B1 is critical in the development of Dfen-induced PAH in mice in vivo and proliferation of PAH-PASMCs in vitro. CYP1B1 may provide a novel therapeutic target for PAH.

Targeting Epigenetic Changes for the Reprogramming of Vascular Walls in Pulmonary Arterial Hypertension, the Role of Histone Deacetylases and their Inhibitors

Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature as defined by an ele vated pulmonary vascular resistance (PVR) leading to a failure of the right heart and ultimately death [1]. Although the overall 5-year survival for PAH has significantly increased in the past years, none of the approved therapies have shown an ability to cure the disease. Current available treatments are limited, expensive, and often associated with significant, side effects. As such, furthering our understanding of the mechanisms underlying PAH development is important to improve patient management and outcomes. A number of evidence have shown that cellular and molecular changes occur in the pulmonary arteries in response to chronic hypoxic exposure [1,2], and primary lung diseases including chronic obstructive pulmonary disease, cystic fibrosis, etc. are associated with the presence of chronic hypoxia. In PAH, the pulmonary arteries (PAs) exhibit pathological proliferative vascular remodeling related to medial and adventitial thickening of pulmonary arterial walls. The excessive proliferation of smooth muscle cells as well as the accumulation of fibroblasts and myofibroblasts plays an important role in the medial and adventitial thickening respectively. Epigenetics describes that heritable changes in gene expression or cellular phenotype are caused by mechanisms other than changes in the underlying DNA sequence. Histone modification is one of the predominant epigenetic phenomena. It selectively modulates gene expressions that control many biological processes including cell proliferation. Histone deacetylases (HDACs) remove an acetyl group from lysine residues of target proteins to regulate a number of cellular processes. There are 18 HDAC enzymes which are identified and grouped into four classes. Class I, II, and IV share sequence similarity and are dependent on Zn2+ for enzymatic activity, whereas the class III sirtuins act through a distinct NAD+-dependent mechanism. Pulmonary vascular remodeling (PAR) is a characteristic pathological change of PAH. The development of PAR involves all layers of the vessel wall. It has been reported that adventitial fibroblasts in the pulmonary hypertensive vessel wall exhibit a hyper proliferative, inflammatory, and invasive phenotype. Hypoxia-induced PAR is characterized by the emergence of a distinct adventitial fibroblast population. This population exhibits a constitutively activated, pro-inflammatory phenotype that is capable of inducing recruitment, retension and pro-inflammatory activation of monocytes/microphages. Stenmark’s group has recently demonstrated that pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (PH-Fibs) expressed a constitutive and persistent proinflammatory phenotype. The proinflammatory phenotype of PH-Fibs was associated with epigenetic alterations as demonstrated by increased activity of HDACs. Importantly, a reversal of this proinflammatory phenotype can be achieved with the use of class I HDAC inhibitors [3]. Another hallmark feature of PAR is excessive smooth muscle cell proliferation leading to medial thickening. In our high altitude longterm (LTH) sheep model, an obvious pulmonary arterial remodeling was observed. We found that the level of global histone acetylation was significantly decreased in LTH fetus PAs, and it was accompanied by the loss of the cyclin-dependent kinase inhibitor, p21, as opposed to the control. The treatment of the pulmonary arterial smooth cells (PASMC), isolated from LTH fetal lung, with a class I HDAC inhibitor markedly decreased their proliferation rate in part due to an increased expression of p21 at both the RNA and protein level. Interestingly, a class II HDAC inhibitor also showed an inhibitory effect on cell proliferation when used with a relative higher concentration. However, a class III inhibitor exhibited no effect on cell proliferation [4]. These findings suggested that epigenetic alterations caused by chronic hypoxia led to fetal PASMC proliferation and vessel remodeling as associated with vascular proliferative disease, and that this process is regulated by p21. This is consistent with a previous study which showed that a p53 gene deficiency decreased p21 expression levels that eventually resulted in hypoxia-induced PAH and vascular remodeling in mice [5]. The above studies correlate with in vivo data showing that small molecules (MGCD0103 and MS-275) that selectively block class I HDACs reduce hypoxia-induced PAH in a manner that associates with a blunted medial thickening of PAs and a reduced proliferation of PASMC in these vessels [6]. These findings support a unique role for HDACs as important targets for PAH, and highlight the potential of isoform-specific HDAC inhibitors for the treatment of cardiovascular diseases.

Myeloperoxidase Aggravates Pulmonary Arterial Hypertension in Mice

Myeloperoxidase Aggravates Pulmonary Arterial Hypertension in Mice

Activity of the Estrogen-Metabolizing Enzyme Cytochrome P450 1B1 Influences the Development of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogen-metabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcription-polymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxic+SU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1(-/-) mice, and the potent CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS; 3 mg · kg(-1) · d(-1) IP) significantly attenuated hypoxic PAH and hypoxic+SU5416 PAH in vivo. TMS also abolished estrogen-induced proliferation in human pulmonary artery smooth muscle cells and PAH-pulmonary artery smooth muscle cells. The estrogen metabolite 16α-hydroxyestrone provoked human pulmonary artery smooth muscle cell proliferation, and this mitogenic effect was greatly pronounced in PAH-pulmonary artery smooth muscle cells. ELISA analysis revealed that 16α-hydroxyestrone concentration was elevated in PAH, consistent with CYP1B1 overexpression and activity. Finally, administration of the CYP1B1 metabolite 16α-hydroxyestrone (1.5 mg · kg(-1) · d(-1) IP) caused the development of PAH in mice. Increased CYP1B1-mediated estrogen metabolism promotes the development of PAH, likely via the formation of mitogens, including 16α-hydroxyestrone. Collectively, this study reveals a possible novel therapeutic target in clinical PAH.

Chronic Allergic Inflammation Causes Vascular Remodeling and Pulmonary Hypertension in Bmpr2 Hypomorph and Wild-Type Mice

Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations.

Development of pulmonary arterial hypertension in mice over-expressing S100A4/Mts1 is specific to females

BackgroundIdiopathic and familial forms of pulmonary arterial hypertension (PAH) occur more frequently in women than men. However, the reason for this remains unknown. Both the calcium binding protein S100A4/Mts1 (Mts1) and its endogenous receptor (receptor for advanced glycosylation end products; RAGE) have been implicated in the development of PAH. We wished to investigate if the Mts1/RAGE pathway may play a role in the gender bias associated with PAH.MethodsWe investigated the effects of gender on development of PAH in mice over-expressing Mts1 (Mts1+ mice) via measurement of pulmonary arterial remodeling, systolic right ventricular pressure (sRVP) and right ventricular hypertrophy (RVH). Gender differences in pulmonary arterial Mts1 and RAGE expression were assessed by qRT-PCR and immunohistochemistry. Western blotting and cell counts were used to investigate interactions between 17β-estradiol, Mts1 and RAGE on proliferation of human pulmonary artery smooth muscle cells (hPASMCs). Statistical analysis was by one-way analysis of variance with Dunnetts post test or two-way analysis of variance with Bonferronis post test, as appropriate.ResultsFemale Mts1+ mice developed increased sRVP and pulmonary vascular remodeling, whereas male Mts1+ mice remained unaffected. The development of plexiform-like lesions in Mts1+ mice was specific to females. These lesions stained positive for both Mts1 and RAGE in the endothelial and adventitial layers. Expression of pulmonary arterial Mts1 was greater in female than male Mts1+ mice, and was localised to the medial and adventitial layers in non plexiform-like pulmonary arteries. RAGE gene expression and immunoreactivity were similar between male and female Mts1+ mice and RAGE staining was localised to the endothelial layer in non plexiform-like pulmonary arteries adjacent to airways. In non-plexiform like pulmonary arteries not associated with airways RAGE staining was present in the medial and adventitial layers. Physiological concentrations of 17β-estradiol increased Mts1 expression in hPASMCs. 17β-estradiol-induced hPASMC proliferation was inhibited by soluble RAGE, which antagonises the membrane bound form of RAGE.ConclusionsMts1 over-expression combined with female gender is permissive to the development of experimental PAH in mice. Up-regulation of Mts1 and subsequent activation of RAGE may contribute to 17β-estradiol-induced proliferation of hPASMCs.

Pulmonary Arterial Hypertension in a Patient With Cowden Syndrome and Anorexigen Exposure

We report a case of pulmonary arterial hypertension (PAH) occurring in a patient with Cowden syndrome with a mutation in the phosphatase and tensin (PTEN) tumor suppressor gene, in the context of exposure to the appetite suppressant dexfenfluramine. Anorexigen exposure is known to be a risk factor for PAH. However, the role of PTEN in cell function and the development of pulmonary vascular remodeling and histopathologic signs of PAH in mice with a Pten depletion in smooth muscle cells suggest that the association of PAH and Cowden syndrome may be relevant. In this case report, we hypothesize that PTEN mutations may be a predisposing factor for the development of PAH, with anorexigen exposure as a potential trigger.

Obesity and Pulmonary Arterial Hypertension: Is Adiponectin the Molecular Link between these Conditions?

Pulmonary arterial hypertension (PAH) is a condition of unknown etiology whose pathological features include increased vascular resistance, perivascular inflammatory cell infiltration and pulmonary arteriolar remodeling. Although risk factors for PAH are poorly defined, recent studies indicate that obesity may be an important risk factor for this condition. The mechanisms leading to this association are largely unknown, but bioactive mediators secreted from adipose tissue have been implicated in this process. One of the most important mediators released from adipose tissue is the adipokine adiponectin. Adiponectin is highly abundant in the circulation of lean healthy individuals, and possesses well-described metabolic and antiinflammatory actions. Levels of adiponectin decrease with increasing body mass, and low levels are directly linked to the development of PAH in mice. Moreover, overexpression of adiponectin has been shown to protect mice from developing PAH in response to inflammation and hypoxia. Based on the findings from these studies, it is suggested that the effects of adiponectin are mediated, in part, through its antiinflammatory and antiproliferative properties. In this review, we discuss the emerging evidence demonstrating a role for adiponectin in lung vascular homeostasis and discuss how deficiency in this adipocyte-derived hormone might explain the recent association between obesity and PAH.

YC-1 attenuates hypoxia-induced pulmonary arterial hypertension in mice

Background Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and elevation of pulmonary arterial pressure, leading to right ventricular failure and eventual death. Currently, no curative therapy for PAH is available, and the overall prognosis is very poor. Recently, direct activators of soluble guanylyl cyclase (sGC) have been tested as a novel therapeutic modality in experimental models of pulmonary arterial hypertension (PAH). Objective In this study, we used in vitro and in vivo models to evaluate the therapeutic potential of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), a dual functioning chemical, as a direct activator of guanylyl cyclase and an inhibitor of hypoxia-inducible factor-1. Methods We analyzed the effects of YC-1 on cell proliferation and the levels of p21 and p53 in human pulmonary artery smooth muscle cells (HPASMCs) under hypoxia. We also determined the effects of YC-1 on expression of endothelin-1 (ET-1) and phosphorylation status of endothelial nitric oxide synthase (eNOS) at Ser 1179 in human pulmonary artery endothelial cells (HPAECs) under hypoxia. In mice, hypoxic PAH was induced by exposure to normobaric hypoxic conditions for 28 days. To assess preventive or therapeutic effects, randomized mice were subjected to once daily i.p. injections of YC-1 for the entire hypoxic period (5 mg/kg) or for the last seven days of a 28-day hypoxic period (5 and 10 mg/kg). On day 28, we measured the right ventricular systolic pressure (RVSP) and determined the degrees of right ventricular hypertrophy (RVH) and vascular remodeling. Results In HPASMCs, YC-1 inhibited hypoxia-induced proliferation and induction of p53 and p21 in a concentration-dependent manner. Also, YC-1 suppressed the hypoxia-induced expression of ET-1 mRNA and dephosphorylation of eNOS at Ser 1179 in HPAECs. In the preventive in vivo model, a daily dose of 5 mg/kg YC-1 significantly prevented the elevation of RVSP, development of RVH, and pulmonary vascular remodeling, which were caused by hypoxic exposure. In the therapeutic model, YC-1 at daily doses of 5 and 10 mg/kg alleviated RVH and pulmonary vascular remodeling but did not prevent the elevation of RVSP. Conclusions Our results indicate that YC-1 prevents the development of hypoxia-induced PAH in a preventive model and alleviates RVH and pulmonary vascular remodeling in a therapeutic model. Therefore, these data imply that YC-1 has therapeutic potential for use in a single or combination therapy for PAH.

Transgenic expression of human matrix metalloproteinase-1 attenuates pulmonary arterial hypertension in mice

PAH (pulmonary arterial hypertension) is a debilitating and life-threatening disease, often affecting young people. We specifically expressed human MMP-1 (matrix metalloproteinase-1) in mouse macrophages and examined its effects in attenuating the decompensating features of MCT (monocrotaline)-induced PAH. Measurement of RV (right ventricular) pressure revealed a 2.5-fold increase after treatment with MCT, which was reduced to 1.5-fold in MMP-1 transgenic mice. There was conspicuous pulmonary inflammation with chronic infiltration of mononuclear cells after the administration of MCT, which was significantly diminished in transgenic mice. Furthermore, transgenic mice showed decreased collagen deposition compared with WT (wild-type). Staining for Mac-3 (macrophage-3) and α-SMA (α-smooth muscle actin) revealed extensive infiltration of macrophages and medial hypertrophy of large pulmonary vessels with complete occlusion of small arteries respectively. These changes were markedly reduced in MMP-1 transgenic mice compared with WT. Western blotting for molecules involved in cell multiplication and proliferation depicted a significant decrease in the lung tissue of transgenic mice after the treatment with MCT. In conclusion, the present study demonstrated that transgenic expression of human MMP-1 decreased proliferation of smooth muscle cells and prevented excessive deposition of collagen in the pulmonary arterial tree. Our results indicate that up-regulation of MMP-1 could attenuate the debilitation of human PAH and provide an option for therapeutic intervention.

Disruption of PPARγ/β-catenin–mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival

Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin-dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.

Exacerbated Pulmonary Arterial Hypertension and Right Ventricular Hypertrophy in Animals With Loss of Function of Extracellular Superoxide Dismutase

Studies have demonstrated that increased oxidative stress contributes to the pathogenesis and the development of pulmonary artery hypertension (PAH). Extracellular superoxide dismutase (SOD3) is essential for removing extracellular superoxide anions, and it is highly expressed in lung tissue. However, it is not clear whether endogenous SOD3 can influence the development of PAH. Here we examined the effect of SOD3 knockout on hypoxia-induced PAH in mice and a loss-of-function SOD3 gene mutation (SOD3(E124D)) on monocrotaline (40 mg/kg)-induced PAH in rats. SOD3 knockout significantly exacerbated 2 weeks of hypoxia-induced right ventricular (RV) pressure and RV hypertrophy, whereas RV pressure in SOD3 knockout mice under normoxic conditions is similar to wild-type controls. In untreated control rats at age of 8 weeks, there was no significant difference between wild-type and SOD3(E124D) rats in RV pressure and the ratio of RV weight:left ventricular weight (0.25±0.02 in wild-type rats versus 0.25±0.01 in SOD3(E124D) rats). However, monocrotaline caused significantly greater increases of RV pressure in SOD3(E124D) rats (48.6±1.8 mm Hg in wild-type versus 57.5±3.1 mm Hg in SOD3(E124D) rats), of the ratio of RV weight:left ventricular weight (0.41±0.01 versus 0.50±0.09; P<0.05), and of the percentage of fully muscularized small arterioles in SOD3(E124D) rats (55.2±2.3% versus 69.9±2.6%; P<0.05). Together, these findings indicate that the endogenous SOD3 has no role in the development of PAH under control conditions but plays an important role in protecting the lung from the development of PAH under stress conditions.

Retracted: Mice lacking the Raf‐1 kinase inhibitor protein exhibit exaggerated hypoxia‐induced pulmonary hypertension

Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts. Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP-/-) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts. RKIP-/- mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP-/- mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP-/- mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1. The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.

Transgenic expression of human matrix metalloproteinase-9 augments monocrotaline-induced pulmonary arterial hypertension in mice

Pulmonary arterial hypertension (PAH) is characterized by intimal lesions, right ventricular hypertrophy, and adventitial thickening of pulmonary arteries with progressive pulmonary hypertension. This investigation was aimed to examine the effects of transgenic expression of human matrix metalloproteinase-9 (MMP-9) in the pathogenesis of PAH. PAH was induced using serial subcutaneous administration of monocrotaline (MCT). Right ventricular pressure was measured through the right jugular vein using a 1.4F Millar Mikro-tip catheter-transducer. Zymography, western blotting, and quantitative reverse transcription PCR (qRT-PCR) were carried out for MMP-9. Immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and Mac-3 antigen. Measurement of right ventricular pressure demonstrated 2.5-fold and 3.7-fold elevation after the administration of MCT in wild-type and MMP-9 transgenic mice, respectively. Zymography, western blotting, and qRT-PCR depicted increased activity and expression of MMP-9 after treatment with MCT, which were augmented in transgenic mice. There was marked pulmonary inflammation with extensive infiltration of mononuclear cells, which was more intense in MMP-9 transgenic mice. SMA and Mac-3 staining demonstrated hypertrophy of pulmonary arteries with occlusion of precapillary vessels and extensive infiltration of macrophages, respectively. All these changes were aggravated in MCT-treated MMP-9 transgenic mice when compared to normal littermates. Our study demonstrated that the MCT-induced PAH in mouse is a reproducible and potentially valuable animal model for the human disease. Our results further demonstrated that MMP-9 plays a significant role in the pathogenesis of PAH and effective blocking of MMP-9 could provide an option in the therapeutic intervention of human PAH.

Reduced expression of angiotensin I-converting enzyme in Caveolin-1 knockout mouse lungs

Reduced lung capillary expression of angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, and of caveolin-1, an important regulator of endothelial cell signalling, has been demonstrated in various models of pulmonary arterial hypertension (PAH). We addressed the relationship between PAH and ACE expression in caveolin-1 knockout mice ( Cav1 − / −), which have moderate PAH. Tissue ACE activity was reduced by 50% in lungs from 3-month-old Cav1 − /− mice compared to wild type (WT). A similar reduction in lung endothelial ACE expression was observed by measuring the lung uptake of 125I-labeled monoclonal anti-ACE antibody and by quantitative immunohistochemistry. These alterations in ACE are limited to capillary segments of the pulmonary circulation. Functionally, the increase in pulmonary artery pressure (PAP) in response to ACE conversion of angiotensin I to angiotensin II in isolated, perfused mouse lungs was reduced significantly in Cav1 − /− mice compared to WT. Thus, these complementary approaches demonstrate the dependence of lung microvascular endothelial cell ACE protein expression on caveolin-1 expression and underscore the vital role of caveolin-1-regulated pulmonary vascular homeostasis on endothelial ACE expression and activity. In summary, we have revealed a novel role of caveolin-1 in the regulation of ACE expression in pulmonary capillary endothelial cells. Further understanding of the mechanism by which reduced caveolin-1 expression leads altered pulmonary vascular development, PAH, and reduced ACE expression may have important clinical implications in patients with these severe lung diseases.

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension.

Voltage-gated potassium (K(v)) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K(v)7 channels has been recently proposed. We investigated the effect of the K(v)7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT(+) mice). Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg.kg(-1).day(-1)). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K(v) channel blocking drugs, including the K(v)7 channel blocker linopirdine, were measured. In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT(+) mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT(+) mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K(v) channel blockers did not. Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT(+) mice, apparently via K(v)7 channel activation. These results provide the first direct evidence that drugs activating K(v)7 channels may be of benefit in the treatment of PAH with different aetiologies.

I008 MRP4 inhibition prevents human pulmonary artery smooth muscle cells proliferation and hypoxia-induced pulmonary arterial hypertension in mice

Inhibition of the active efflux pump Multidrug Resistance Associated Protein 4 (MRP4) can increase intracellular levels of cyclic nucleotides cAMP and cGMP in smooth muscle cells, thereby limiting their proliferation. MRP4 inhibition thus appears as a promising therapeutic target in vasculoproliferative disorders such as pulmonary arterial hypertension (PAH). We investigate whether MRP4 inhibition can exert beneficial effects on pulmonary vascular remodeling in PAH. MRP4-knock out (KO) and wild-type (WT) mice were placed for 4 weeks in hypoxic (10 % O2) or normoxic conditions (21 % O2). After this period, the right ventricular systolic pressure (RSVP) was gauged. The Fulton Index (ratio of right ventricle to left ventricle plus septum weights), a marker of right ventricular hypertrophy, was calculated. Complementary in vitro studies were conducted in isolated human pulmonary artery smooth muscle cells (hPASMC). hPASMC proliferation was measured by BrdU incorporation quantification after transfection with scrambled or MRP4 small interfering RNAs (siRNA). Whereas there were no differences between WT and MRP4-KO mice in normoxic conditions (23.8±0.3 vs 23.0±0.6 mmHg, NS), chronic hypoxia resulted in pulmonary vascular remodeling and increased RVSP in WT but not in MRP4-KO mice (35.0±1.8 vs 25.8±0.7 mmHg, p<0.001). The right ventricular hypertrophy was strongly decreased in MRP4-KO compared to WT mice (0.27±0.01 vs 0.37±0.01 respectively, p<0.001). In vitro specific inhibition of MRP4 by siRNA caused an increase of the intracellular/extracellular ratio for both cAMP and cGMP (185±18 % and 162±22 %, relative to scrambled siRNA, p<0.05) and was associated with a significant decrease in proliferation compared to scrambled siRNA (219±13 % vs 432±44 %, relative to control in 0.1 % growth factors, p< 0.001). This antiproliferative effect was abolished by blocking PKA or PKG activity: 225±9 % with PKA inhibitor PKI and 194±11 % with PKG inhibitor KT5823 vs 160±7 % for MRP4 siRNA alone (p<0.05), compared to 238±16 % for siRNA scrambled. MRP4 inhibition reduces hPASMC pathological proliferation by controlling PKA and PKG pathways and prevents PAH development in vivo. MRP4 inhibition appears as a new target for therapeutic intervention in PAH.

Statin ameliorates hypoxia-induced pulmonary hypertension associated with down-regulated stromal cell-derived factor-1

Mobilization of stem cells/progenitors is regulated by the interaction between stromal cell-derived factor-1 (SDF-1) and its ligand, CXC chemokine receptor 4 (CXCR4). Statins have been suggested to ameliorate pulmonary arterial hypertension (PAH); however, the mechanisms involved, especially their effects on progenitors, are largely unknown. Therefore, we examined whether pravastatin ameliorates hypoxia-induced PAH in mice, and if so, which type of progenitors and what mechanism(s) are involved. Chronic hypoxia (10% O(2) for 5 weeks) increased the plasma levels of SDF-1 and mobilization of CXCR4(+)/vascular endothelial growth factor receptor (VEGFR)2(+)/c-kit(+) cells from bone marrow (BM) to pulmonary artery adventitia in Balb/c mice in vivo, both of which were significantly suppressed by simultaneous oral treatment with pravastatin (2 mg/kg/day). Furthermore, in vitro experiments demonstrated that hypoxia enhances differentiation of VEGFR2(+)/c-kit(+) cells into alpha-smooth muscle actin(+) cells. Importantly, pravastatin ameliorated hypoxia-induced PAH associated with a decrease in the number of BM-derived progenitors accumulating in the pulmonary artery adventitia. The expression of intercellular adhesion molecule-1 (ICAM-1) and its ligand, CD18 (beta2-integrin), were enhanced by hypoxia and were again suppressed by pravastatin. These results suggest that pravastatin ameliorates hypoxia-induced PAH through suppression of SDF-1/CXCR4 and ICAM-1/CD18 pathways with a resultant reduction in the mobilization and homing of BM-derived progenitor cells.