Pulmonary Artery Fibroblasts Research Articles

Histone deacetylase inhibitors synergize with sildenafil to suppress purine metabolism and proliferation in pulmonary hypertension

RationaleSildenafil, a well-known vasodilator known to interfere with purinergic signaling through effects on cGMP, is a mainstay in the treatment of pulmonary hypertension (PH). However, little is known regarding its effects on the metabolic reprogramming of vascular cells, which is a hallmark of PH. Purine metabolism, especially intracellular de novo purine biosynthesis is essential for vascular cell proliferation. Since adventitial fibroblasts are critical contributors to proliferative vascular remodeling in PH, in this study we aimed to investigate if sildenafil, beyond its well-known vasodilator role in smooth muscle cells, impacts intracellular purine metabolism and proliferation of fibroblasts derived from human PH patients. MethodsIntegrated omics approaches (plasma and cell metabolomics) and pharmacological inhibitor approaches were employed in plasma samples and cultured pulmonary artery fibroblasts from PH patients. Measurements and main resultsPlasma metabolome analysis of 27 PH patients before and after treatment with sildenafil, demonstrated a partial, but specific effect of sildenafil on purine metabolites, especially adenosine, adenine, and xanthine. However, circulating markers of cell stress, including lactate, succinate, and hypoxanthine were only decreased in a small subset of sildenafil-treated patients. To better understand potential effects of sildenafil on pathological changes in purine metabolism (especially purine synthesis) in PH, we performed studies on pulmonary fibroblasts from PAH patients (PH-Fibs) and corresponding controls (CO-Fibs), since these cells have previously been shown to demonstrate stable and marked PH associated phenotypic and metabolic changes. We found that PH-Fibs exhibited significantly increased purine synthesis. Treatment of PH-Fibs with sildenafil was insufficient to normalize cellular metabolic phenotype and only modestly attenuated the proliferation. However, we observed that treatments which have been shown to normalize glycolysis and mitochondrial abnormalities including a PKM2 activator (TEPP-46), and the histone deacetylase inhibitors (HDACi), SAHA and Apicidin, had significant inhibitory effects on purine synthesis. Importantly, combined treatment with HDACi and sildenafil exhibited synergistic inhibitory effects on proliferation and metabolic reprogramming in PH-Fibs. ConclusionsWhile sildenafil alone partially rescues metabolic alterations associated with PH, treatment with HDACi, in combination with sildenafil, represent a promising and potentially more effective strategy for targeting vasoconstriction, metabolic derangement and pathological vascular remodeling in PH.

Pulmonary Vascular Remodeling in Pulmonary Hypertension.

Pulmonary vascular remodeling is the critical structural alteration and pathological feature in pulmonary hypertension (PH) and involves changes in the intima, media and adventitia. Pulmonary vascular remodeling consists of the proliferation and phenotypic transformation of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs) of the middle membranous pulmonary artery, as well as complex interactions involving external layer pulmonary artery fibroblasts (PAFs) and extracellular matrix (ECM). Inflammatory mechanisms, apoptosis and other factors in the vascular wall are influenced by different mechanisms that likely act in concert to drive disease progression. This article reviews these pathological changes and highlights some pathogenetic mechanisms involved in the remodeling process.

Biomechanical and Mechanobiological Drivers of the Transition From PostCapillary Pulmonary Hypertension to Combined Pre-/PostCapillary Pulmonary Hypertension.

Combined pre-/postcapillary pulmonary hypertension (Cpc-PH), a complication of left heart failure, is associated with higher mortality rates than isolated postcapillary pulmonary hypertension alone. Currently, knowledge gaps persist on the mechanisms responsible for the progression of isolated postcapillary pulmonary hypertension (Ipc-PH) to Cpc-PH. Here, we review the biomechanical and mechanobiological impact of left heart failure on pulmonary circulation, including mechanotransduction of these pathological forces, which lead to altered biological signaling and detrimental remodeling, driving the progression to Cpc-PH. We focus on pathologically increased cyclic stretch and decreased wall shear stress; mechanotransduction by endothelial cells, smooth muscle cells, and pulmonary arterial fibroblasts; and signaling-stimulated remodeling of the pulmonary veins, capillaries, and arteries that propel the transition from Ipc-PH to Cpc-PH. Identifying biomechanical and mechanobiological mechanisms of Cpc-PH progression may highlight potential pharmacologic avenues to prevent right heart failure and subsequent mortality.

Abstract 15671: Targeting Chromatin Structure: The Combined Inhibition of Histone Deacetylases and Bromodomain Proteins in Normalizing Persistently Activated Pulmonary Hypertensive Fibroblasts

Rationale: The multifactorial nature of pulmonary hypertension (PH) suggests epigenetic changes as potential determinants of vascular remodeling through regulating gene expression (both up-regulated and down-regulated genes) in vascular cells. Changes in the chromatin state (especially histone acetylation) of specific genes can lead to their repression or activation. We and others have demonstrated histone deacetylases inhibitors (HDACi) and histone acetylation readers inhibitors (BRDi) can prevent and ameliorate PH changes by normalizing pathological gene expression. However, the non-histone protein targets and the narrow therapeutic window of currently available HDACi limits their clinical utility. In this study, we evaluated the effect of combination treatments using a new HDACi (OKI-005, US patent) with high potency and selectivity for HDACs (targeting decreased genes) together with BRDi (targeting increased genes), in low doses. Methods and Results: Cultured human pulmonary artery fibroblasts derived from patients with IPAH (PH-Fibs) or from control donors (CO-Fibs) were used as in vitro model system. PH-Fibs exhibit increased pro-inflammatory genes (e.g. CXCL12), proliferation markers (e.g. Mki67) and anti-apoptosis genes (e.g. BCL2) as well as decreased anti-inflammatory genes (e.g. HMOX1), anti-proliferation genes (e.g. P21) and pro-apoptotic genes (e.g. PERP). In PH-Fibs, the increased expressed CXCL2 gene exhibited more opened chromatin structure and BRD4 binding (ChIP-assay), and BRDi (JQ1) can decrease its expression in a dose dependent trend. At the low doses, JQ1 (100 nM) alone is sufficient to normalize most of increased genes, while the new HDACi (OKI-005) was more effective in normalizing decreased genes in PH- Fibs. The combination of low dose of JQ1 (100 nM) and OKI-005 (64 nM) was sufficient to normalize both increased genes and decreased genes thus inhibiting the persistently activated PH-phenotype without deleterious effects on CO-Fibs. Conclusion: Combination of HDACi and BRDi in low doses is effective in normalizing the persistent activation of PH-Fibs without deleterious effects on control cells, offering promise for epigenetic-targeted therapies in PH.

Epigenetic reactivation of transcriptional programs orchestrating fetal lung development in human pulmonary hypertension.

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.

Non-Coding RNA Networks in Pulmonary Hypertension.

Non-coding RNAs (ncRNAs) are involved in various cellular processes. There are several ncRNA classes, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). The detailed roles of these molecules in pulmonary hypertension (PH) remain unclear. We systematically collected and reviewed reports describing the functions of ncRNAs (miRNAs, lncRNAs, and circRNAs) in PH through database retrieval and manual literature reading. The characteristics of identified articles, especially the experimental methods, were carefully reviewed. Furthermore, regulatory networks were constructed using ncRNAs and their interacting RNAs or genes. These data were extracted from studies on pulmonary arterial smooth muscle cells, pulmonary artery endothelial cells, and pulmonary artery fibroblasts. We included 14 lncRNAs, 1 circRNA, 74 miRNAs, and 110 mRNAs in the constructed networks. Using these networks, herein, we describe the current knowledge on the role of ncRNAs in PH. Moreover, these networks actively provide an improved understanding of the roles of ncRNAs in PH. The results of this study are crucial for the clinical application of ncRNAs.

Mechanisms Contributing to the Dysregulation of miRNA-124 in Pulmonary Hypertension.

Chronic pulmonary hypertension (PH) is a fatal disease characterized by the persistent activation of pulmonary vascular cells that exhibit aberrant expression of genes including miRNAs. We and others reported that decreased levels of mature microRNA-124 (miR-124) plays an important role in modulating the activated phenotype of pulmonary vascular cells and HDAC inhibitors (HDACi) can restore the levels of mature miR-124 and reverse the persistently activated phenotype of PH vascular cells. In this study, we sought to determine the mechanisms contributing to reduced levels of miRNAs, as well as how HDACi restores the levels of reduced miRNA in PH vascular cells. We found that pulmonary artery fibroblasts isolated from IPAH patients (PH-Fibs) exhibit reduced levels of mature miR-124 and several other miRNAs including let-7i, miR-224, and miR-210, and that these reduced levels can be restored by HDACi. Using miR-124 expression in human PH-Fibs as a model, we determined that reduced miR-124 gene transcription, not decreased expression of miRNA processing genes, is responsible for reduced levels of mature miR-124 in human PH-Fibs. Using both DNase I Sensitivity and chromatin immunoprecipitation assays, we found that the miR-124-1 gene exhibits a more condensed chromatin structure in human PH-Fibs, compared to corresponding controls. HDACi relaxed miR-124-1 chromatin structure, evidenced by increased levels of the open chromatin mark H3K27Ac, but decreased levels of closed chromatin mark H3K27Me3. Most importantly, the delivery of histone acetyltransferase (HAT) via CRISPR-dCas9-HAT and guiding RNAs to the promoter of the miR-124-1 gene increased miR-124-1 gene transcription. Thus, our data indicate epigenetic events play important role in controlling miR-124 and likely other miRNA levels and epigenetic regulators such as HDACs appear to be promising therapeutic targets for chronic PH.

Connexin 43 plays a role in proliferation and migration of pulmonary arterial fibroblasts in response to hypoxia.

Pulmonary hypertension (PH) is a disease associated with vasoconstriction and remodelling of the pulmonary vasculature. Pulmonary artery fibroblasts (PAFs) play an important role in hypoxic-induced remodelling. Connexin 43 (Cx43) is involved in cellular communication and regulation of the pulmonary vasculature. Using both in vitro and in vivo models of PH, the aims of this study were to (i) investigate the role of Cx43 in hypoxic-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) and (ii) determine whether Cx43 expression is dysregulated in the rat sugen5416/hypoxic model of PH. The role of Cx43 in hypoxic-induced proliferation and migration was investigated using Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic exposure, in the presence of serum, resulted in an increase in proliferation of rPAFs but not rPASMCs. Hypoxic exposure caused migration of rPAFs but not rPASMCs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen5416/hypoxic rat lung; this increased expression was not observed in sugen5416/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. In conclusion, Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via the MAPK signalling pathway.

Apoptosis signal-regulating kinase 1 inhibition in in vivo and in vitro models of pulmonary hypertension.

Pulmonary arterial hypertension, group 1 of the pulmonary hypertension disease family, involves pulmonary vascular remodelling, right ventricular dysfunction and cardiac failure. Oxidative stress, through activation of mitogen-activated protein kinases is implicated in these changes. Inhibition of apoptosis signal-regulating kinase 1, an apical mitogen-activated protein kinase, prevented pulmonary arterial hypertension developing in rodent models. Here, we investigate apoptosis signal-regulating kinase 1 in pulmonary arterial hypertension by examining the impact that its inhibition has on the molecular and cellular signalling in established disease. Apoptosis signal-regulating kinase 1 inhibition was investigated in in vivo pulmonary arterial hypertension and in vitro pulmonary hypertension models. In the in vivo model, male Sprague Dawley rats received a single subcutaneous injection of Sugen SU5416 (20 mg/kg) prior to two weeks of hypobaric hypoxia (380 mmHg) followed by three weeks normoxia (Sugen/hypoxic), then animals were either maintained for three weeks on control chow or one containing apoptosis signal-regulating kinase 1 inhibitor (100 mg/kg/day). Cardiovascular measurements were carried out. In the in vitro model, primary cultures of rat pulmonary artery fibroblasts and rat pulmonary artery smooth muscle cells were maintained in hypoxia (5% O2) and investigated for proliferation, migration and molecular signalling in the presence or absence of apoptosis signal-regulating kinase 1 inhibitor. Sugen/hypoxic animals displayed significant pulmonary arterial hypertension compared to normoxic controls at eight weeks. Apoptosis signal-regulating kinase 1 inhibitor decreased right ventricular systolic pressure to control levels and reduced muscularised vessels in lung tissue. Apoptosis signal-regulating kinase 1 inhibition was found to prevent hypoxia-induced proliferation, migration and cytokine release in rat pulmonary artery fibroblasts and also prevented rat pulmonary artery fibroblast-induced rat pulmonary artery smooth muscle cell migration and proliferation. Apoptosis signal-regulating kinase 1 inhibition reversed pulmonary arterial hypertension in the Sugen/hypoxic rat model. These effects may be a result of intrinsic changes in the signalling of adventitial fibroblast.

Hypoxia induces pulmonary arterial fibroblast proliferation, migration, differentiation and vascular remodeling via the PI3K/Akt/p70S6K signaling pathway.

The present study was designed to examine whether hypoxia induces the proliferation, migration and differentiation of pulmonary arterial fibroblasts (PAFs) via the PI3K/Akt/p70S6K signaling pathway. PAFs were subjected to normoxia (21% O2) or hypoxia (1% O2). The proliferation, migration, differentiation and cellular p110α, p-Akt, and p-p70S6K expression levels of the PAFs were examined in vitro. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and right ventricular weight/body weight ratio (RV/BW) were examined. The expression levels of p110α, p-Akt, p70S6K, fibronectin and α-SMA in the rat pulmonary vessels were also examined. Hypoxia significantly elevated the proliferation, migration and differentiation of rat PAFs. It also strongly elevated the expression of p110α, p-Akt and p-p70S6K in PAFs in vitro. NVP-BEZ235 was revealed to significantly reduce the hypoxia-induced proliferation, migration and differentiation. In vivo experiments demonstrated that hypoxia significantly induced the elevation of RVSP, RVHI, RV/BW, medial thickening, adventitious thickening, and fibronectin and collagen deposition around pulmonary artery walls. The expression of p110α, p-Akt and p70S6K was evident in the pulmonary arteries of the hypoxic rats. NVP-BEZ235 significantly reduced the hypoxia-induced hypoxic pulmonary vascular remodeling, as well as fibronectin and collagen deposition in the pulmonary arteries. Therefore, hypoxia was demonstrated to induce the proliferation, migration and differentiation of PAFs and the hypoxic pulmonary vascular remodeling of rats via the PI3K/Akt/p70S6K signaling pathway.

Regulatory effects of endogenous sulfur dioxide on collagen accumulation in pulmonary artery fibroblasts of rats and its mechanisms

Objective To investigate the regulatory effects of endogenous sulfur dioxide (SO2) on collagen accumulation in pulmonary arterial fibroblasts of rats and its mechanisms. Methods Primary rat pulmonary artery fibroblasts were used in the experiment and were divided into 3 groups: the control group, the L-aspartate-beta -hydroxamate(HDX) group and the HDX+ SO2 group.SO2 content of pulmonary artery fibroblasts supernatant was detected by adopting high performance liquid chromatography (HPLC). Collagen type Ⅰ and collagen type Ⅲ in pulmonary artery fibroblasts were determined by using immunofluorescence.Phosphorylation of Smad2/3, protein expression of matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 were detected by using Western blot.One-way ANOVA was used for multiple group comparisons followed by Bonferroni test for each group.P<0.05 was considered as significant difference. Results Compared with control group, endogenous SO2 content in HDX group was significantly decreased [(14.30±0.48) μmol/L vs.(20.14±0.49) μmol/L, P<0.01], the level of Smad2/3 increased (1.03±0.31 vs.0.48±0.20, P<0.01), protein expressions of MMP-13 and TIMP-1 in pulmonary artery fibroblasts were decreased (MMP-13: 0.28±0.06 vs.0.75±0.11, P<0.01; TIMP-1: 0.40±0.05 vs.0.66±0.20, P<0.01), and the ratio of MMP-13/TIMP-1 was decreased (0.71±0.12 vs.1.23±0.45, P<0.01). However, contents of collagen Ⅰ and collagen Ⅲ were significantly increased.Compared with HDX group, the level of Smad2/3 phosphorylation in HDX+ SO2 group decreased (0.57±0.16 vs.1.03±0.31, P<0.01), protein expression of MMP-13 and TIMP-1 upregulated (MMP-13: 0.63±0.06 vs.0.28±0.06, P<0.01; 0.59±0.11 vs.0.40±0.05, P=0.015), the ratio of MMP-13/TIMP-1 (1.10±0.22 vs.0.71±0.12, P=0.033) increased, but contents of collagen type Ⅰ and type Ⅲ were reduced obviously. Conclusions SO2 promotes the degradation of collagen and collagen accumulation in pulmonary artery fibroblasts of rats probably by inhibiting Smad2/3 signal pathway, increasing protein expression of MMP-13 and TIMP-1, and upregulating the ratio of MMP-13/TIMP-1. Key words: Sulfur dioxide; Collagen; Fibroblasts

Sulfur Dioxide Protects Against Collagen Accumulation in Pulmonary Artery in Association With Downregulation of the Transforming Growth Factor β1/Smad Pathway in Pulmonary Hypertensive Rats.

BackgroundWe aimed to explore the role of endogenous sulfur dioxide (SO 2) in pulmonary vascular collagen remodeling induced by monocrotaline and its mechanisms.Methods and ResultsA rat model of monocrotaline‐induced pulmonary vascular collagen remodeling was developed and administered with l‐aspartate‐β‐hydroxamate or SO 2 donor. The morphology of small pulmonary arteries and collagen metabolism were examined. Cultured pulmonary arterial fibroblasts stimulated by transforming growth factor β1 (TGF‐β1) were used to explore the mechanism. The results showed that in monocrotaline‐treated rats, mean pulmonary artery pressure increased markedly, small pulmonary arterial remodeling developed, and collagen deposition in lung tissue and pulmonary arteries increased significantly in association with elevated SO 2 content, aspartate aminotransferase (AAT) activity, and expression of AAT1 compared with control rats. Interestingly, l‐aspartate‐β‐hydroxamate, an inhibitor of SO 2 generation, further aggravated pulmonary vascular collagen remodeling in monocrotaline‐treated rats, and inhibition of SO 2 in pulmonary artery smooth muscle cells activated collagen accumulation in pulmonary arterial fibroblasts. SO 2 donor, however, alleviated pulmonary vascular collagen remodeling with inhibited collagen synthesis, augmented collagen degradation, and decreased TGF‐β1 expression of pulmonary arteries. Mechanistically, overexpression of AAT1, a key enzyme of SO 2 production, prevented the activation of the TGF‐β/type I TGF‐β receptor/Smad2/3 signaling pathway and abnormal collagen synthesis in pulmonary arterial fibroblasts. In contrast, knockdown of AAT1 exacerbated Smad2/3 phosphorylation and deposition of collagen types I and III in TGF‐β1–treated pulmonary arterial fibroblasts.ConclusionsEndogenous SO 2 plays a protective role in pulmonary artery collagen accumulation induced by monocrotaline via inhibition of the TGF‐β/type I TGF‐β receptor/Smad2/3 pathway.

44 ASK-1 inhibition prevents hypoxia-induced pulmonary artery fibroblast proliferation and migration in anin vitrocellular model of pulmonary hypertension

Rationale Pulmonary arterial hypertension (PAH) involves progressive remodelling of the pulmonary arteries, increasing pulmonary vascular resistance leading to right ventricular dysfunction. The remodelling is due to increased pulmonary adventitial fibroblast proliferation, migration, and release of cytokines. These fibroblast responses involve members of the Mitogen Activated Protein Kinases (MAPK) signalling pathway. Apoptosis Signal-regulating Kinase-1 (ASK1) is an apical MAPK, which undergoes auto-phosphorylation and activation in response to oxidative stress, activating downstream p38 MAPK and c-Jun N-terminal kinase (JNK). The work presented here investigates the possible cellular mechanisms and downstream targets of ASK1 in the pulmonary artery fibroblast (PAF) in a model of PAH. Methodology PAF were obtained from adult Sprague Dawley rats. Hypoxia (5% O2) was used as an in vitro model of PAH. Proliferation was determined using [3H] thymidine incorporation. Migration was determined by scratch assay. Targets of ASK1 were determined by Western Blotting. ASK1 inhibition was achieved by the addition of a selective, small molecule inhibitor (GS-444217). Results Hypoxia increased PAF proliferation and migration; both were significantly reduced by the addition of 1 µM GS-444217. Hypoxia increased phosphorylation of both ASK1 and p38 MAPK, and increased Hypoxia-Inducible Factor 1 alpha (HIF1a) protein abundance; 1 µM GS-444217 attenuated these effects. ASK1 inhibition reduced hypoxia-induced expression of key mediators of vascular remodelling, tissue Inhibitor of Metaloproteinase-1 (TIMP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and endothelin-1 (ET-1). Conclusions ASK1 inhibitor (GS-444217) reduced hypoxia-induced proliferation and migration of PAF, and reduced disease-associated signalling pathways. ASK1 inhibition may be a novel target for preventing maladaptive vascular remodelling.

40 Pulmonary artery fibroblasts in sleep disordered breathing conditions: increased proliferation and discovery of humoral factors eliciting p38 MAPK kinase proliferation

Introduction Obstructive Sleep Apnoea (OSA) and Obesity Hypoventilation Syndrome (OHS) are sleep breathing disorders (SBD); respectively characterised by nocturnal intermittent hypoxaemia, and sustained hypoxaemia. Studies of pulmonary hypertension have found pulmonary arterial fibroblasts to proliferate excessively in hypoxic conditions due to activation of the p38MAPK pathway. Aims i) Determine if an elevated proliferative response occurs in various degrees of intermittent hypoxia. ii) To explore the presence of humoral factors in SDB patient serum that stimulate p38 MAPK activation. Methods Rat pulmonary arterial fibroblasts (RPAFs) were exposed to contrasting levels of hypoxia/normoxia in bovine serum. RPAF cell division was quantified via [3H] Thymidine proliferation assays. Western Blot analysis, for total and phospho-p38 MAP kinase, was undertaken in RPAFs exposed to 24 h of normoxia and patient serum. Results 8 h of hourly intermittent or sustained acute hypoxia followed by 16 h of normoxia triggered elevated RPAF proliferation, similar to 24 h of acute hypoxia. In contrast, no increase in RPAF proliferation was seen with 18 h of normoxia followed by 6 h of hypoxia. Exposure of RPAFs to OHS patient serum elicited a signficant increase in pp38 MAPK vs control and OSA serum; this was reduced in serum taken post treatment. Conclusion Conditions mimicking SBD elicit hyper-proliferative responses in RPAFs. This study has questioned the existence of a vulnerable point within the cell cycle to which hypoxia will elicit fibroblast proliferation. The differences in RPAF p38 activation by OSA/OHS patient serum, including evidence of an early treatment effect, indicate pathogenic pathways.

The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension.

The p38 mitogen-activated protein kinase (MAPK) system is increasingly recognized as an important inflammatory pathway in systemic vascular disease but its role in pulmonary vascular disease is unclear. Previous in vitro studies suggest p38 MAPKα is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodeling (PVremod). In this study the role of the p38 MAPK pathway was investigated in both in vitro and in vivo models of pulmonary hypertension and human disease. Pharmacological inhibition of p38 MAPKα in both chronic hypoxic and monocrotaline rodent models of pulmonary hypertension prevented and reversed the pulmonary hypertensive phenotype. Furthermore, with the use of a novel and clinically available p38 MAPKα antagonist, reversal of pulmonary hypertension was obtained in both experimental models. Increased expression of phosphorylated p38 MAPK and p38 MAPKα was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in the PVremod A reduction of IL-6 levels in serum and lung tissue was found in the drug-treated animals, suggesting a potential mechanism for this reversal in PVremod. This study suggests that the p38 MAPK and the α-isoform plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6. Selective inhibition of this pathway may provide a novel therapeutic approach that targets both remodeling and inflammatory pathways in pulmonary vascular disease.

15‐LO/15‐HETE Mediated Vascular Adventitia Fibrosis via p38 MAPK‐Dependent TGF‐β

15-Lipoxygenase/15-hydroxyeicosatetraenoic acid (15-LO/15-HETE) is known to modulate pulmonary vascular medial hypertrophy and intimal endothelial cells migration and angiogenesis after hypoxia. However, it is unclear whether 15-HETE affects the adventitia of the pulmonary arterial wall. We performed immunohistochemistry, adventitia fibrosis, pulmonary artery fibroblasts phenotype and extracellular matrix (ECM) deposition to determine the role of 15-HETE in hypoxia-induced pulmonary vascular adventitia remodeling. Our studies showed that O2 deprivation induced adventitia hypertrophy of pulmonary arteries with ECM accumulation in both humans with pulmonary arterial hypertension and hypoxic rats. Hypoxia induced 15-LO expression in adventitia. With the inhibitor, NDGA depressed the hypoxia induced ECM deposition and 15-LO production in hypoxic rats. Hypoxia up-regulated the expression of α-SMA, type-Ia collagen and fibronectin in cultured fibroblasts, which seemed to be due to the increased 15-LO/15-HETE. Exogenous 15-HETE mediated the ECM and phenotypic alterations of the fibroblasts as well. The 15-LO/15-HETE induced adventitia fibrosis and fibroblasts phenotypic alterations depended on signaling of the transforming growth factor-β1 (TGF-β1)/Smad2/3 pathway. P38 mitogen-activated protein kinase (p38 MAPKs) was likely to mediate 15-LO induced TGF-β1 and Smad2/3 activation after hypoxia. The results suggest that adventitia fibrosis is an important event in the hypoxia induced pulmonary arterial remodeling, which relies on 15-LO/15-HETE induced p38 MAPK-dependent TGF-β1/Smad2/3 intracellular signaling systems.

S36 P38 MAPK: An Important Pathway in the Pathobiology of Pulmonary Hypertension and Pulmonary Vascular Remodelling

The p38 MAPK pathway is increasingly recognised as important in inflammation leading to systemic vascular disease but its role in pulmonary vascular disease is unclear. Our group has previously identified...

Hypoxia-induced changes in expression levels of miR-210, -183 and -744 in human lung fibroblasts

Aims: Micro-RNA (miRNA) profiles of human pulmonary arterial fibroblasts (hPAFb) were measured in order to reveal hypoxia-induced changes in miRNA profiles. Pathway analysis based on miRNA target prediction was compared with pathway analysis based on mRNA microarrays. Methods: Fibroblasts from pulmonary arteries of a healthy human lung were cultured and kept under normoxic and hypoxic (FiO2=0.1) conditions for 24h. Total RNA was isolated using a phenol-/guanidine-based method. Success of the hypoxic treatment was confirmed by measuring the expression of the PGK1 gene by quantitative real-time PCR (qRT-PCR). The miRNA was labelled by T4-Ligase-mediated linking of Cy-Dye-conjugated oligoribonucleotides. Labelled miRNAs were hybridized to miChip microarrays (EMBL, miRBase V11.0). Validation of miRNA expression levels was done with qRT-PCR. miRNA target prediction and KEGG pathway analysis were performed with newly developed target-weighting-algorithms. Results were compared with KEGG pathway analysis based on Agilent Whole Genome microarrays. Results: miR-210, -744 and -296–5p (up) and miR-183 (down) were found regulated in response to hypoxia in microarrays successfully validated by qRT-PCR. Pathway analysis based on the miRNA target scoring algorithm resulted in 31 candidate pathways potentially perturbed by hypoxia-dependet regulation of miRNAs (based on 5% FDR). Most significant significantly enriched pathways are involved in cytoskeletal devopment (Focal Adhesion, ECM-Receptor interaction, Regulation of actin cytoskeleton, Adherens junction, Axon guidance), signalling (Wnt-signalling, mTOR-signalling, TGFβ-signalling) and cell division (Pathways in cancer). 60% of the predicted pathways were confirmed by mRNA expression profiles. Conclusions: Since the HIF-1α- and -2α-induced mir-210 is a major hypoxia-regulated miRNA, its regulation confirms the validity of our results. It is involved in cell cycle arrest, stem cell survival, mitochondrial metabolism, DNA-repair, angiogenesis and cell differentiation. However, none of the other regulated miRNAs has yet been linked to hypoxia in hPAFb. miR-183 is connected to cellular stress and also differenzially expressed in several cancers. miR-744 is a post-transcriptional regulator of TGFβ-1. The pathway prediction based on weighting algorithms seems to be a viable approach as its results are comparable to results based on real mRNA expression data. The reliability of results will likely improve with better target gene annotations as they will be available in future.

Activated Adventitial Pulmonary Artery Fibroblasts Drive Macrophages Toward a Distinct M2-like Phenotype through STAT3 Signaling in Pulmonary Hypertension (120.36)

Abstract We have previously documented that in the adventitia of distal pulmonary arteries (dPA) in calves with pulmonary hypertension (PH) macrophages that express CD163 (marker of alternative activation, AAM) accumulate in proximity to adventitial fibroblasts (adFibs) in areas of remodeling. AAMs have been implicated in promoting remodeling in PH in response to STAT6 signaling. Our recently published data suggest that in the dPA, adFibs express an epigenetically stable pro-inflammatory phenotype capable of activating macrophages. We hypothesized that adFibs activate AAMs through STAT3 but not STAT6. adFibs were isolated and cultured from the dPA of hypertensive (PH-fibs) and control (CO-fibs) calves and used to generate conditioned media (CM). Bovine, murine and rat bone marrow derived macrophages (BMDMs) were incubated in CM for 16hrs followed by qRT-PCR analysis. In response to PH-fibs CM, bovine BMDMs expressed Cd163, Cd206, and IL4ra consistent with AAMs. Furthermore, in response to PH-Fib but not CO-Fib CM both murine and rat BMDMs expressed Cd206, Cd204, Irf4, Klf, jmjd3; STAT3-dependent genes Socs3, Cebp/b, Pim1, Nfatc2, Il4ra, Arg1, but not STAT6-dependent genes Chi3I3 and Retnla1; phospho-STAT3 but not phospho-STAT6; and mRNA for Arg1 (previously thought to be STAT6 dependent) in murine Stat6-/- BMDMs. These results define the activated dPA adFibs as a key locus of paracrine signaling that triggers STAT3 in macrophages, which then express a distinct AAM phenotype.

Low-Dose Fluvastatin Reverses the Hypoxic Pulmonary Adventitial Fibroblast Phenotype in Experimental Pulmonary Hypertension

Hypoxic pulmonary hypertension is a worldwide public health problem. Statins attenuate hypoxic pulmonary hypertension in animal models, but the mechanism of action and applicability of these results to human treatment are not established. In hypoxic models, pulmonary artery fibroblast proliferation contributes substantially to pulmonary vascular remodeling. We previously showed that acute hypoxic pulmonary adventitial fibroblast proliferation can be selectively inhibited by statins and p38 mitogen-activated protein (MAP) kinase inhibitors. Here we used complementary chronic hypoxic and acute hypoxic coculture models to obtain necessary preclinical information regarding the utility of fluvastatin in the treatment of chronic hypoxic pulmonary hypertension. The effects of fluvastatin, cholesterol pathway intermediates, and related inhibitors on hypoxic adventitial fibroblast proliferation, p38 MAP kinase phosphorylation, and pulmonary artery smooth muscle cell proliferation were determined, using complementary chronic hypoxic rat and acute hypoxic bovine cell models. Fluvastatin reversed the proliferative phenotypic switch in adventitial fibroblasts from chronic hypoxic animals. This effect was circulation-specific, and implicated a Rac1-p38 MAP kinase signaling pathway. Coculture and conditioned media experiments also implicated this statin-sensitive signaling pathway in the release of pulmonary artery smooth muscle cell mitogens by hypoxic pulmonary adventitial fibroblasts. Treprostinil, sildenafil, and bosentan exerted no effect on the hypoxic fibroblast phenotype. Phenotypic changes (increased proliferation and mitogen release) in pulmonary artery fibroblasts during chronic hypoxia are dependent on a Rac1-p38 MAP kinase signaling pathway. The inhibition of these phenotypic changes with fluvastatin may be therapeutically relevant in high-altitude residents and in patients with hypoxic lung disease.