FHL-1 is not involved in pressure overload-induced maladaptive right ventricular remodeling and dysfunction.

AIMS: The cytoskeletal signaling protein four and-a-half LIM domains 1 (FHL-1) has recently been identified as a novel key player in pulmonary hypertension as well as in left heart diseases. In this regard, FHL-1 has been implicated in dysregulated hypertrophic signaling in pulmonary arterial smooth muscle cells leading to pulmonary hypertension. In mice, FHL-1-deficiency (FHL-1-/-) led to an attenuated hypertrophic signaling associated with a blunted hypertrophic response of the pressure-overloaded left ventricle (LV). However, the role of FHL-1 in right heart hypertrophy has not yet been addressed. METHODS AND RESULTS: We investigated FHL-1 expression in C57Bl/6 mice subjected to chronic biomechanical stress and found it to be enhanced in the right ventricle (RV). Next, we subjected FHL-1-/- and corresponding wild-type mice to pressure overload of the RV by pulmonary arterial banding for various time points. However, in contrast to the previously published study in LV-pressure overload, which was confirmed here, RV hypertrophy and hypertrophic signaling was not diminished in FHL-1-/- mice. In detail, right ventricular pressure overload led to hypertrophy, dilatation and fibrosis of the RV from both FHL-1-/- and wild-type mice. RV remodeling was associated with impaired RV function as evidenced by reduced tricuspid annular plane systolic excursion. Additionally, PAB induced upregulation of natriuretic peptides and slight downregulation of phospholamban and ryanodine receptor 2 in the RV. However, there was no difference between genotypes in the degree of expression change. CONCLUSION: FHL-1 pathway is not involved in the control of adverse remodeling in the pressure overloaded RV.

Chymase: a multifunctional player in pulmonary hypertension associated with lung fibrosis.

Limited literature sources implicate mast-cell mediator chymase in the pathologies of pulmonary hypertension and pulmonary fibrosis. However, there is no evidence on the contribution of chymase to the development of pulmonary hypertension associated with lung fibrosis, which is an important medical condition linked with increased mortality of patients who already suffer from a life-threatening interstitial lung disease.The aim of this study was to investigate the role of chymase in this particular pulmonary hypertension form, by using a bleomycin-induced pulmonary hypertension model.Chymase inhibition resulted in attenuation of pulmonary hypertension and pulmonary fibrosis, as evident from improved haemodynamics, decreased right ventricular remodelling/hypertrophy, pulmonary vascular remodelling and lung fibrosis. These beneficial effects were associated with a strong tendency of reduction in mast cell number and activity, and significantly diminished chymase expression levels. Mechanistically, chymase inhibition led to attenuation of transforming growth factor ß1 and matrix-metalloproteinase-2 contents in the lungs. Furthermore, chymase inhibition prevented big endothelin-1-induced vasoconstriction of the pulmonary arteries.Therefore, chymase plays a role in the pathogenesis of pulmonary hypertension associated with pulmonary fibrosis and may represent a promising therapeutic target. In addition, this study may provide valuable insights on the contribution of chymase in the pulmonary hypertension context, in general, regardless of the pulmonary hypertension form.

PAR-2 inhibition reverses experimental pulmonary hypertension.

RATIONALE: A hallmark of the vascular remodeling process underlying pulmonary hypertension (PH) is the aberrant proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). Accumulating evidence suggests that mast cell mediators play a role in the pathogenesis of PH. OBJECTIVE: In the present study we investigated the importance of protease-activated receptor (PAR)-2 and its ligand mast cell tryptase in the development of PH. METHODS AND RESULTS: Our results revealed strong increase in PAR-2 and tryptase expression in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline (MCT)-treated rats. Elevated tryptase levels were also detected in plasma samples from IPAH patients. Hypoxia and platelet-derived growth factor (PDGF)-BB upregulated PAR-2 expression in PASMC. This effect was reversed by HIF (hypoxia inducible factor)-1α depletion, PDGF-BB neutralizing antibody, or the PDGF-BB receptor antagonist Imatinib. Attenuation of PAR-2 expression was also observed in smooth muscle cells of pulmonary vessels of mice exposed to hypoxia and rats challenged with MCT in response to Imatinib treatment. Tryptase induced PASMC proliferation and migration as well as enhanced synthesis of fibronectin and matrix metalloproteinase-2 in a PAR-2- and ERK1/2-dependent manner, suggesting that PAR-2-dependent signaling contributes to vascular remodeling by various mechanisms. Furthermore, PAR-2(-/-) mice were protected against hypoxia-induced PH, and PAR-2 antagonist application reversed established PH in the hypoxia mouse model. CONCLUSIONS: Our study identified a novel role of PAR-2 in vascular remodeling in the lung. Interference with this pathway may offer novel therapeutic options for the treatment of PH.

Cofilin, a hypoxia-regulated protein in murine lungs identified by 2DE: role of the cytoskeletal protein cofilin in pulmonary hypertension.

Chronic alveolar hypoxia induces vascular remodeling processes in the lung resulting in pulmonary hypertension (PH). However, the mechanisms underlying pulmonary remodeling processes are not fully resolved yet. To investigate functional changes occurring during hypoxia exposure we applied 2DE to compare protein expression in lungs from mice subjected to 3 h of alveolar hypoxia and those kept under normoxic conditions. Already after this short-time period several proteins were significantly regulated. Subsequent analysis by MALDI-MS identified cofilin as one of the most prominently upregulated proteins. The regulation was confirmed by western blotting and its cellular localization was determined by immunohisto- and immunocytochemistry. Interestingly, enhanced cofilin serine 3 phosphorylation was observed after short-term and after chronic hypoxia-induced PH in mice, in pulmonary arterial smooth muscle cells (PASMC) from monocrotaline-induced PH in rats, in lungs of idiopathic pulmonary arterial hypertension patients and in hypoxic or platelet-derived growth factor BB-treated human PASMC. Furthermore, elevated cofilin phosphorylation was attenuated by curative treatment of monocrotaline-induced PH in rats and hypoxia-induced PH in mice with the PDGF-BB receptor antagonist imatinib. In conclusion, short-term hypoxic exposure induced prominent changes in lung protein regulation. These very early changes allowed us to identify potential triggers of PH. Thus, respective 2DE analysis can lead to the identification of new target proteins for the possible treatment of PH.

Role of Src tyrosine kinases in experimental pulmonary hypertension.

OBJECTIVE: Pulmonary arterial hypertension is a progressive pulmonary vascular disorder with high morbidity and mortality. Compelling evidence suggests that receptor tyrosine kinases, such as platelet-derived growth factor (PDGF) are closely involved in the pathogenesis of pulmonary arterial hypertension. We investigated the effects of 2 novel PDGF inhibitors, nilotinib/AMN107 (Abl kinases/PDGF receptor inhibitor) and dasatinib/BMS-354825 (Abl kinases/PDGF receptor/Src inhibitor), on the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and on the hemodynamics and pulmonary vascular remodeling in experimental pulmonary hypertension, and determined the expression and regulation of Src family kinases. METHODS AND RESULTS: Human PASMCs were stimulated by PDGF alone or multiple growth factors to induce proliferation and migration in vitro. Dasatinib (0.03 µmol/L), nilotinib (0.3 µmol/L), and imatinib (1 µmol/L) potently inhibited PDGF-induced signal transducer and activator of transcription 3 and Akt phosphorylation. All 3 inhibitors decreased PDGF-induced proliferation, cell cycle gene regulation, and migration. In contrast, only dasatinib inhibited multiple growth factor-induced PASMC proliferation, and this was associated with the inhibition of Src phosphorylation. Combination of specific Src inhibitors (phosphoprotein phosphatase 1, phosphoprotein phosphatase 2) with either imatinib or nilotinib reduced multiple growth factor-induced proliferation to a similar extent as dasatinib. Importantly, Src phosphorylation increased in pulmonary arterial hypertension PASMCs compared with control PASMCs. Finally, in vivo dasatinib (15 mg/kg per body weight) treatment caused a complete reversal of pulmonary vascular remodeling and achieved similar effectiveness as imatinib (100 mg/kg per body weight) in both monocrotaline- and hypoxia-induced pulmonary hypertension models. CONCLUSIONS: We suggest that dual inhibition of PDGF receptor and Src kinases potently inhibits mitogenic and motogenic responses to growth factors in PASMCs and pulmonary vascular remodeling in vivo so that dual inhibition may represent an alternative therapeutic approach for pulmonary arterial hypertension.

Role of epidermal growth factor inhibition in experimental pulmonary hypertension.

RATIONALE: Epidermal growth factor (EGF) and its receptors play a role in cell proliferation and survival and are implicated in the pathobiology of pulmonary arterial hypertension (PAH). OBJECTIVES: To study the role of EGF inhibition on experimental pulmonary hypertension. METHODS: We investigated (1) the effects of three clinically approved EGF receptor (EGFR) antagonists in vitro on rat pulmonary arterial smooth muscle cell proliferation and in vivo on experimental pulmonary hypertension (PH) induced by monocrotaline injection in rats and by chronic hypoxia in mice, and (2) the expression of EGFR in the lung tissues from experimental and clinical PH. MEASUREMENTS AND MAIN RESULTS: The EGFR inhibitors gefitinib, erlotinib, and lapatinib inhibited the EGF-induced proliferation of pulmonary arterial smooth muscle cells. In rats with established PH, gefitinib and erlotinib significantly reduced right ventricular systolic pressure and right ventricular hypertrophy. In addition, the medial wall thickness and muscularization of pulmonary arteries were improved. In contrast, lapatinib did not provide therapeutic benefit. These EGFR antagonists at their highest tolerable dose did not yield significant improvement in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice with chronic hypoxic PH. Moreover, no significant alteration in the EGFR expression was detected in the lung tissues from patients with idiopathic PAH. CONCLUSIONS: The partial therapeutic efficacy of the EGFR antagonists in animal models of pulmonary hypertension and the absence of significant alteration in EGFR expression in the lungs from patients with idiopathic PAH suggest that EGFRs do not represent a promising target for the treatment of pulmonary hypertension.

The soluble guanylate cyclase activator HMR1766 reverses hypoxia-induced experimental pulmonary hypertension in mice.

Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose-response curve was shifted leftward when the effects of HMR1766 were investigated in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed PH, right heart hypertrophy, and pulmonary vascular remodeling. Treatment with HMR1766 (10 mg x kg(-1) x day(-1)), after full establishment of PH from day 21 to day 35, significantly reduced PH, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH.

Pressure overload leads to an increased accumulation and activity of mast cells in the right ventricle.

Right ventricular (RV) remodeling represents a complex set of functional and structural adaptations in response to chronic pressure or volume overload due to various inborn defects or acquired diseases and is an important determinant of patient outcome. However, the underlying molecular mechanisms remain elusive. We investigated the time course of structural and functional changes in the RV in the murine model of pressure overload-induced RV hypertrophy in C57Bl/6J mice. Using magnetic resonance imaging, we assessed the changes of RV structure and function at different time points for a period of 21 days. Pressure overload led to significant dilatation, cellular and chamber hypertrophy, myocardial fibrosis, and functional impairment of the RV Progressive remodeling of the RV after pulmonary artery banding (PAB) in mice was associated with upregulation of myocardial gene markers of hypertrophy and fibrosis. Furthermore, remodeling of the RV was associated with accumulation and activation of mast cells in the RV tissue of PAB mice. Our data suggest possible involvement of mast cells in the RV remodeling process in response to pressure overload. Mast cells may thus represent an interesting target for the development of new therapeutic approaches directed specifically at the RV.

miR-223-IGF-IR signalling in hypoxia- and load-induced right-ventricular failure: a novel therapeutic approach.

AIMS: Pulmonary hypertension is a progressive disease with poor prognosis, characterized by pathological inward remodelling and loss of patency of the lung vasculature. The right ventricle is co-affected by pulmonary hypertension, which triggers events such as hypoxia and/or increased mechanical load. Initially the right ventricle responds with 'adaptive' hypertrophy, which is often rapidly followed by 'maladaptive' changes leading to right heart decompensation and failure, which is the ultimate cause of death. METHODS AND RESULTS: We report here that miR-223 is expressed in the murine lung and right ventricle at higher levels than in the left ventricle. Moreover, lung and right-ventricular miR-223 levels were markedly down-regulated by hypoxia. Correspondingly, increasing right-ventricular load by pulmonary artery banding, induced right-ventricular ischaemia, and the down-regulation of miR-223. Lung and right ventricle miR-223 down-regulation were linked with increased expression of the miR-223 target; insulin-like growth factor-I receptor (IGF-IR) and IGF-I downstream signalling. Similarly, miR-223 was decreased and IGF-IR increased in human pulmonary hypertension. Notably in young mice, miR-223 overexpression, the genetic inactivation or pharmacological inhibition of IGF-IR, all attenuated right-ventricular hypertrophy and improved right heart function under conditions of hypoxia or increased afterload. CONCLUSION: These findings highlight the early role of pulmonary and right-ventricular miR-223 and the IGF-IR in the right heart failure programme initiated by pulmonary hypoxia and increased mechanical load and may lead to the development of novel therapeutic strategies that target the development of PH and right heart failure.

Histological characterization of mast cell chymase in patients with pulmonary hypertension and chronic obstructive pulmonary disease.

Our previous findings demonstrated an increase in pulmonary mast cells (MCs) in idiopathic pulmonary arterial hypertension (IPAH). Also, literature suggests a potential role for MCs in chronic obstructive pulmonary disease (COPD). However, a comprehensive investigation of lungs from patients is still needed. We systematically investigated the presence/expression of MCs/MC chymase in the lungs of IPAH and COPD patients by (immuno)histochemistry and subsequent quantification. We found that total and perivascular chymase-positive MCs were significantly higher in IPAH patients than in donors. In addition, chymase-positive MCs were located in proximity to regions with prominent expression of big-endothelin-1 in the pulmonary vessels of IPAH patients. Total and perivascular MCs around resistant vessels were augmented and a significant majority of them were degranulated (activated) in COPD patients. While the total chymase-positive MC count tended to increase in COPD patients, the perivascular number was significantly enhanced in all vessel sizes analyzed. Surprisingly, MC and chymase-positive MC numbers positively correlated with better lung function in COPD. Our findings suggest that activated MCs, possibly by releasing chymase, may contribute to pulmonary vascular remodeling in IPAH. Pulmonary MCs/chymase may have compartment-specific (vascular vs. airway) functions in COPD. Future studies should elucidate the mechanisms of MC accumulation and the role of MC chymase in pathologies of these severe lung diseases.

Rhodiola: an ordinary plant or a promising future therapy for pulmonary hypertension? a brief review.

Abstract Pulmonary hypertension (PH) is a chronic, complex, and progressive disease that eventuates in fatality. Research efforts over the past decades have resulted in therapeutic options that improve quality of life and prolong survival of patients, but they do not offer a cure. We propose a philosophical model that a disturbed balance of yin and yang results in pulmonary vascular remodeling, the hallmark of PH pathology. The model may be useful in exploring the wisdom of traditional Chinese medicine and incorporating it into mainstream PH research. In this context, the medicinal plant Rhodiola can be of profound interest owing to its variety of health-friendly attributes. Rhodiola has been shown to be beneficial in high-altitude-related symptoms and acute exacerbation of PH; moreover, improvement of PH has been demonstrated experimentally in chronically hypoxic rats. The beneficial effects of Rhodiola in PH may be attributable to its potential targeting of the signaling pathways, such as endothelin-1, nitric oxide, vascular endothelial growth factor, angiotensin-converting enzyme, nuclear factor κ-B, tumor necrosis factor α, and interleukin-6. Alterations in these mediators are implicated in PH pathogenesis, the characteristics of which include chronic pulmonary vasoconstriction, vasoproliferation, and vascular inflammation. Salidroside, one of the compounds extracted from Rhodiola, has been found to provide therapeutic benefits in experimental PH. As the data are limited and the field is in its infancy, further studies including in-depth analysis of the therapeutic effects on various animal models of PH are desirable. We believe that future PH research should place an adequate and special emphasis on exploring and promoting the potential of traditional Chinese medicine, and to this end, the medicinal plant Rhodiola offers a promising field on which to embark.

Mast cell chymase: an indispensable instrument in the pathological symphony of idiopathic pulmonary fibrosis?

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal lung disease with no known etiology and treatment options. The hallmarks of the histopathology, which is characteristic of usual interstitial pneumonia (UIP) pattern, include interstitial fibrosis, honeycomb changes and fibroblast foci that develop owing to fibroblast proliferation and excessive matrix deposition. Although the complete pathomechanism is not yet understood, several molecular culprits, including transforming growth factor (TGF)-ß, Angiotensin (Ang) II, endothelin (ET)-1, matrix metalloproteinases (MMPs) and cytokines have been identified. IPF is increasingly believed to be an epithelial-driven disease; however, the literature does support an implication of altered immune response and inflammatory processes in the onset or progression of the disease. Mast cells (MCs) are multifunctional tissue resident cells involved in the inflammatory and immune response. An increasing body of evidence suggests a role of MCs and their mediator chymase in the pathology of IPF. With regard to the underlying mechanisms, it is conceivable that MC chymase may function via activation or processing of factors such as proteases, cytokines and growth factors. In this review, we will discuss how MC chymase is linked to and can potentially contribute to the development of IPF. Moreover, the findings from animal model studies will be discussed to highlight the chymase inhibitors as a promising strategy for the treatment of pulmonary fibrosis.

The role of dimethylarginine dimethylaminohydrolase in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, dysregulated response to alveolar injury that culminates in compromised lung function from excess extracellular matrix production. Associated with high morbidity and mortality, IPF is generally refractory to current pharmacological therapies. We examined fibrotic lungs from mice and from patients with IPF and detected increased expression of dimethylarginine dimethylaminohydrolases (DDAHs)--key enzymes that metabolize asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase, to form l-citrulline and dimethylamine. DDAHs are up-regulated in primary alveolar epithelial type II cells from these mice and patients where they are colocalized with inducible nitric oxide synthase. In cultured alveolar epithelial type II cells from bleomycin-induced fibrotic mouse lungs, inhibition of DDAH suppressed proliferation and induced apoptosis in an ADMA-dependent manner. In addition, DDAH inhibition reduced collagen production by fibroblasts in an ADMA-independent but transforming growth factor/SMAD-dependent manner. In mice with bleomycin-induced pulmonary fibrosis, the DDAH inhibitor L-291 reduced collagen deposition and normalized lung function. In bleomycin-induced fibrosis, inducible nitric oxide synthase inhibition decreased fibrosis, but an even stronger reduction was observed after inhibition of DDAH. Thus, DDAH inhibition reduces fibroblast-induced collagen deposition in an ADMA-independent manner and reduces abnormal epithelial proliferation in an ADMA-dependent manner, offering a possible therapeutic avenue for attenuation of pulmonary fibrosis.