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1.  5-HT2B Receptor Antagonists Inhibit Fibrosis and Protect from RV Heart Failure 
BioMed Research International  2015;2015:438403.
Objective. The serotonin (5-HT) pathway was shown to play a role in pulmonary hypertension (PH), but its functions in right ventricular failure (RVF) remain poorly understood. The aim of the current study was to investigate the effects of Terguride (5-HT2A and 2B receptor antagonist) or SB204741 (5-HT2B receptor antagonist) on right heart function and structure upon pulmonary artery banding (PAB) in mice. Methods. Seven days after PAB, mice were treated for 14 days with Terguride (0.2 mg/kg bid) or SB204741 (5 mg/kg day). Right heart function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometric methods. Total secreted collagen content was determined in mouse cardiac fibroblasts isolated from RV tissues. Results. Chronic treatment with Terguride or SB204741 reduced right ventricular fibrosis and showed improved heart function in mice after PAB. Moreover, 5-HT2B receptor antagonists diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblasts in vitro. Conclusion. 5-HT2B receptor antagonists reduce collagen deposition, thereby inhibiting right ventricular fibrosis. Chronic treatment prevented the development and progression of pressure overload-induced RVF in mice. Thus, 5-HT2B receptor antagonists represent a valuable novel therapeutic approach for RVF.
doi:10.1155/2015/438403
PMCID: PMC4312574  PMID: 25667920
2.  Effects of Dimethylarginine Dimethylaminohydrolase–1 Overexpression on the Response of the Pulmonary Vasculature to Hypoxia 
Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase–1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1tg) on HPV and chronic hypoxia–induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3′,5′-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1tg mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1tg. The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1tg mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia–induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO–cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia–induced PH.
doi:10.1165/rcmb.2012-0330OC
PMCID: PMC3824049  PMID: 23642043
asymmetric dimethylarginine; cyclic guanosine monophosphate; hypoxia-induced pulmonary hypertension; hypoxic pulmonary vasoconstriction; nitric oxide
3.  Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin 
Critical Care  2014;18(2):R73.
Introduction
Ventilator-induced lung injury (VILI) contributes to morbidity and mortality in acute respiratory distress syndrome (ARDS). Particularly pre-injured lungs are susceptible to VILI despite protective ventilation. In a previous study, the endogenous peptide adrenomedullin (AM) protected murine lungs from VILI. We hypothesized that mechanical ventilation (MV) contributes to lung injury and sepsis in pneumonia, and that AM may reduce lung injury and multiple organ failure in ventilated mice with pneumococcal pneumonia.
Methods
We analyzed in mice the impact of MV in established pneumonia on lung injury, inflammation, bacterial burden, hemodynamics and extrapulmonary organ injury, and assessed the therapeutic potential of AM by starting treatment at intubation.
Results
In pneumococcal pneumonia, MV increased lung permeability, and worsened lung mechanics and oxygenation failure. MV dramatically increased lung and blood cytokines but not lung leukocyte counts in pneumonia. MV induced systemic leukocytopenia and liver, gut and kidney injury in mice with pneumonia. Lung and blood bacterial burden was not affected by MV pneumonia and MV increased lung AM expression, whereas receptor activity modifying protein (RAMP) 1–3 expression was increased in pneumonia and reduced by MV. Infusion of AM protected against MV-induced lung injury (66% reduction of pulmonary permeability p < 0.01; prevention of pulmonary restriction) and against VILI-induced liver and gut injury in pneumonia (91% reduction of AST levels p < 0.05, 96% reduction of alanine aminotransaminase (ALT) levels p < 0.05, abrogation of histopathological changes and parenchymal apoptosis in liver and gut).
Conclusions
MV paved the way for the progression of pneumonia towards ARDS and sepsis by aggravating lung injury and systemic hyperinflammation leading to liver, kidney and gut injury. AM may be a promising therapeutic option to protect against development of lung injury, sepsis and extrapulmonary organ injury in mechanically ventilated individuals with severe pneumonia.
doi:10.1186/cc13830
PMCID: PMC4056010  PMID: 24731244
4.  Histological characterization of mast cell chymase in patients with pulmonary hypertension and chronic obstructive pulmonary disease 
Pulmonary Circulation  2014;4(1):128-136.
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.
doi:10.1086/675642
PMCID: PMC4070756  PMID: 25006428
idiopathic pulmonary arterial hypertension; chronic obstructive pulmonary disease; mast cells; chymase
5.  Nox Family NADPH Oxidases in Mechano-Transduction: Mechanisms and Consequences 
Antioxidants & Redox Signaling  2014;20(6):887-898.
Abstract
Significance: The majority of cells in a multi-cellular organism are continuously exposed to ever-changing physical forces. Mechano-transduction links these events to appropriate reactions of the cells involving stimulation of signaling cascades, reorganization of the cytoskeleton and alteration of gene expression. Recent Advances: Mechano-transduction alters the cellular redox balance and the formation of reactive oxygen species (ROS). Nicotine amide adenine dinucleotide reduced form (NADPH) oxidases of the Nox family are prominent ROS generators and thus, contribute to this stress-induced ROS formation. Critical Issues: Different types and patterns of mechano-stress lead to Nox-dependent ROS formation and Nox-mediated ROS formation contributes to cellular responses and adaptation to physical forces. Thereby, Nox enzymes can mediate vascular protection during physiological mechano-stress. Despite this, over-activation and induction of Nox enzymes and a subsequent substantial increase in ROS formation also promotes oxidative stress in pathological situations like disturbed blood flow or extensive stretch. Future Directions: Individual protein targets of Nox-mediated redox-signaling will be identified to better understand the specificity of Nox-dependent ROS signaling in mechano-transduction. Nox-inhibitors will be tested to reduce cellular activation in response to mechano-stimuli. Antioxid. Redox Signal. 20, 887–898.
doi:10.1089/ars.2013.5414
PMCID: PMC3924808  PMID: 23682993
6.  Functional and Muscular Adaptations in an Experimental Model for Isometric Strength Training in Mice 
PLoS ONE  2013;8(11):e79069.
Exercise training induces muscular adaptations that are highly specific to the type of exercise. For a systematic study of the differentiated exercise adaptations on a molecular level mouse models have been used successfully. The aim of the current study was to develop a suitable mouse model of isometric strength exercise training characterized by specific adaptations known from strength training. C57BL/6 mice performed an isometric strength training (ST) for 10 weeks 5 days/week. Additionally, either a sedentary control group (CT) or a regular endurance training group (ET) groups were used as controls. Performance capacity was determined by maximum holding time (MHT) and treadmill spirometry, respectively. Furthermore, muscle fiber types and diameter, muscular concentration of phosphofructokinase 1 (PFK), succinate dehydrogenase (SDHa), and glucose transporter type 4 (GLUT4) were determined. In a further approach, the effect of ST on glucose intolerance was tested in diabetic mice. In mice of the ST group we observed an increase of MHT in isometric strength tests, a type II fiber hypertrophy, and an increased GLUT4 protein content in the membrane fraction. In contrast, in mice of the ET group an increase of VO2max, a shift to oxidative muscle fiber type and an increase of oxidative enzyme content was measured. Furthermore strength training was effective in reducing glucose intolerance in mice fed a high fat diet. An effective murine strength training model was developed and evaluated, which revealed marked differences in adaptations known from endurance training. This approach seems also suitable to test for therapeutical effects of strength training.
doi:10.1371/journal.pone.0079069
PMCID: PMC3827300  PMID: 24236089
7.  Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD 
Disease Models & Mechanisms  2013;6(6):1378-1387.
SUMMARY
Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.
doi:10.1242/dmm.013482
PMCID: PMC3820261  PMID: 24046361
8.  NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis 
The Journal of Clinical Investigation  2013;123(11):4731-4738.
ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4–/– mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis.
doi:10.1172/JCI67603
PMCID: PMC3809780  PMID: 24216508
9.  Rhodiola: an ordinary plant or a promising future therapy for pulmonary hypertension? a brief review 
Pulmonary Circulation  2013;3(3):499-506.
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.
doi:10.1086/674303
PMCID: PMC4070792  PMID: 24618536
Rhodiola; pulmonary hypertension; pulmonary vascular remodeling; Chinese medicine; salidroside
10.  Short-term improvement in pulmonary hemodynamics is strongly predictive of long-term survival in patients with pulmonary arterial hypertension 
Pulmonary Circulation  2013;3(3):523-532.
Hemodynamic measurements provide important parameters for determining prognosis and therapy in patients with pulmonary arterial hypertension (PAH). Current guidelines do not incorporate the possible predictive value of individual changes in hemodynamic variables during the disease time course, and there is no consensus about the time point for hemodynamic reevaluation. We aimed to assess the long-term prognostic value of short-term changes in hemodynamic parameters. The study included 122 patients with PAH from the Giessen Pulmonary Hypertension Registry who underwent hemodynamic evaluation at baseline and at 16 weeks (±2.5 standard deviations [SDs]; range: 4–29 weeks) after initial assessment. At baseline, mean pulmonary vascular resistance (PVR) was 1,109 dyn s cm−5, and 82% of patients were in World Health Organization (WHO) functional class III or IV. Fifty patients died, and 2 underwent lung transplantation during long-term observation (≤10 years; mean: 4.7 years). Kaplan-Meier estimates for transplant-free survival were 93.3%, 76.1%, 57.8%, and 53.1% at 1, 3, 5, and 7 years, respectively. When assigned to prognostic groups, improvements in cardiac output of >0.22 L min−1 (hazard ratio [HR]: 2.05; ) and a decrease in PVR of >176 dyn s cm−5 (HR: 1.89; ) at 4–29 weeks were associated with long-term transplant-free survival. Changes in mean pulmonary arterial pressure did not predict long-term prognosis. Of 2 noninvasive parameters assessed in this selected patient group, change in WHO functional class, but not in 6-minute walk distance, predicted long-term prognosis. Short-term assessment of changes in hemodynamic parameters at after initial invasive evaluation is useful to determine long-term prognosis in patients with PAH.
doi:10.1086/674338
PMCID: PMC4070816  PMID: 24618538
cardiac output; Kaplan-Meier survival estimates; mean pulmonary arterial pressure; prognosis; pulmonary vascular resistance
12.  Obstructive Sleep Apnea, Oxidative Stress and Cardiovascular Disease: Lessons from Animal Studies 
Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular (CV) diseases such as arterial hypertension, heart failure, and stroke. Based on human research, sympathetic activation, inflammation, and oxidative stress are thought to play major roles in the pathophysiology of OSA-related CV diseases. Animal models of OSA have shown that endothelial dysfunction, vascular remodelling, and systemic and pulmonary arterial hypertension as well as heart failure can develop in response to chronic intermittent hypoxia (CIH). The available animal data are clearly in favour of oxidative stress playing a key role in the development of all of these CV manifestations of OSA. Presumably, the oxidative stress is due to an activation of NADPH oxidase and other free oxygen radicals producing enzymes within the CV system as evidenced by data from knockout mice and pharmacological interventions. It is hoped that animal models of OSA-related CV disease will continue to contribute to a deeper understanding of their underlying pathophysiology and will foster the way for the development of cardioprotective treatment options other than conventional CPAP therapy.
doi:10.1155/2013/234631
PMCID: PMC3603718  PMID: 23533685
13.  Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2 
Nucleic Acids Research  2013;41(4):2255-2266.
Cytochrome c oxidase (COX) is the terminal enzyme of the electron transport chain, made up of 13 subunits encoded by both mitochondrial and nuclear DNA. Subunit 4 (COX4), a key regulatory subunit, exists as two isoforms, the ubiquitous isoform 1 and the tissue-specific (predominantly lung) isoform 2 (COX4I2). COX4I2 renders lung COX about 2-fold more active compared with liver COX, which lacks COX4I2. We previously identified a highly conserved 13-bp sequence in the proximal promoter of COX4I2 that functions as an oxygen responsive element (ORE), maximally active at a 4% oxygen concentration. Here, we have identified three transcription factors that bind this conserved ORE, namely recombination signal sequence–binding protein Jκ (RBPJ), coiled-coil-helix-coiled-coil-helix domain 2 (CHCHD2) and CXXC finger protein 5 (CXXC5). We demonstrate that RBPJ and CHCHD2 function towards activating the ORE at 4% oxygen, whereas CXXC5 functions as an inhibitor. To validate results derived from cultured cells, we show using RNA interference a similar effect of these transcription factors in the gene regulation of COX4I2 in primary pulmonary arterial smooth muscle cells. Depending on the oxygen tension, a concerted action of the three transcription factors regulates the expression of COX4I2 that, as we discuss, could augment both COX activity and its ability to cope with altered cellular energy requirements.
doi:10.1093/nar/gks1454
PMCID: PMC3575822  PMID: 23303788
14.  Smoking: Is it a risk factor for pulmonary vascular diseases? 
Pulmonary Circulation  2012;2(4):395-396.
doi:10.4103/2045-8932.105027
PMCID: PMC3555409  PMID: 23372923
15.  The Soluble Guanylate Cyclase Stimulator Riociguat Ameliorates Pulmonary Hypertension Induced by Hypoxia and SU5416 in Rats 
PLoS ONE  2012;7(8):e43433.
Background
The nitric oxide (NO)–soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63–2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO–sGC–cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH.
Methods and Results
Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55±0.02, p<0.05), increased cardiac output (60.8±.8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03±0.3 mmHg min−1 ml−1 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05).
Conclusion
Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.
doi:10.1371/journal.pone.0043433
PMCID: PMC3422306  PMID: 22912874
16.  Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction 
Respiratory Research  2012;13(1):7.
Background
Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined.
Method
We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability.
Results
In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W.
Conclusion
Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.
doi:10.1186/1465-9921-13-7
PMCID: PMC3306743  PMID: 22292558
hypoxia; hypercapnia; acidosis; nitric oxide; hypoxic pulmonary vasoconstriction
21.  Therapeutic efficacy of TBC3711 in monocrotaline-induced pulmonary hypertension 
Respiratory Research  2011;12(1):87.
Background
Endothelin-1 signalling plays an important role in pathogenesis of pulmonary hypertension. Although different endothelin-A receptor antagonists are developed, a novel therapeutic option to cure the disease is still needed. This study aims to investigate the therapeutic efficacy of the selective endothelin-A receptor antagonist TBC3711 in monocrotaline-induced pulmonary hypertension in rats.
Methods
Monocrotaline-injected male Sprague-Dawley rats were randomized and treated orally from day 21 to 35 either with TBC3711 (Dose: 30 mg/kg body weight/day) or placebo. Echocardiographic measurements of different hemodynamic and right-heart hypertrophy parameters were performed. After day 35, rats were sacrificed for invasive hemodynamic and right-heart hypertrophy measurements. Additionally, histologic assessment of pulmonary vascular and right-heart remodelling was performed.
Results
The novel endothelin-A receptor antagonist TBC3711 significantly attenuated monocrotaline-induced pulmonary hypertension, as evident from improved hemodynamics and right-heart hypertrophy in comparison with placebo group. In addition, muscularization and medial wall thickness of distal pulmonary vessels were ameliorated. The histologic evaluation of the right ventricle showed a significant reduction in fibrosis and cardiomyocyte size, suggesting an improvement in right-heart remodelling.
Conclusion
The results of this study suggest that the selective endothelin-A receptor antagonist TBC3711 demonstrates therapeutic benefit in rats with established pulmonary hypertension, thus representing a useful therapeutic approach for treatment of pulmonary hypertension.
doi:10.1186/1465-9921-12-87
PMCID: PMC3141422  PMID: 21699729
22.  Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats 
Respiratory Research  2011;12(1):60.
Background
Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored.
Methods
Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies.
Results
There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling.
Conclusions
The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT- rats.
doi:10.1186/1465-9921-12-60
PMCID: PMC3104382  PMID: 21535881
23.  Glycogen Synthase Kinase 3beta Contributes to Proliferation of Arterial Smooth Muscle Cells in Pulmonary Hypertension 
PLoS ONE  2011;6(4):e18883.
Rationale
Pulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.
Methods
All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [3H]-thymidine incorporation assay.
Results
Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.
Conclusion
This study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.
doi:10.1371/journal.pone.0018883
PMCID: PMC3078925  PMID: 21533110
24.  Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling 
PLoS ONE  2011;6(4):e18136.
Phosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determine whether any of the recently identified PDEs (PDE7-PDE11) contribute to progressive pulmonary vascular remodeling in PH. All in vitro experiments were performed with lung tissue or pulmonary arterial smooth muscle cells (PASMCs) obtained from control rats or monocrotaline (MCT)-induced pulmonary hypertensive (MCT-PH) rats, and we examined the effects of the PDE10 inhibitor papaverine (Pap) and specific small interfering RNA (siRNA). In addition, papaverine was administrated to MCT-induced PH rats from day 21 to day 35 by continuous intravenous infusion to examine the in vivo effects of PDE10A inhibition. We found that PDE10A was predominantly present in the lung vasculature, and the mRNA, protein, and activity levels of PDE10A were all significantly increased in MCT PASMCs compared with control PASMCs. Papaverine and PDE10A siRNA induced an accumulation of intracellular cAMP, activated cAMP response element binding protein and attenuated PASMC proliferation. Intravenous infusion of papaverine in MCT-PH rats resulted in a 40%–50% attenuation of the effects on pulmonary hypertensive hemodynamic parameters and pulmonary vascular remodeling. The present study is the first to demonstrate a central role of PDE10A in progressive pulmonary vascular remodeling, and the results suggest a novel therapeutic approach for the treatment of PH.
doi:10.1371/journal.pone.0018136
PMCID: PMC3073929  PMID: 21494592
25.  Hypoxic pulmonary hypertension in mice with constitutively active platelet-derived growth factor receptor-β 
Pulmonary Circulation  2011;1(2):259-268.
Platelet-derived growth factor (PDGF) has been implicated in the pathobiology of vascular remodeling. The multikinase inhibitor imatinib that targets PDGF receptor (PDGFR), c-kit and Abl kinases, shows therapeutic efficacy against experimental pulmonary hypertension (PH); however, the role of PDGFR-b in experimental PH has not been examined by genetic approach. We investigated the chronic hypoxia-induced PH in mice carrying an activating point mutation of PDGFR-β (D849N) and evaluated the therapeutic efficacy of imatinib. In addition, we studied pulmonary global gene expression and confirmed the expression of identified genes by immunohistochemistry. Chronically hypoxic D849N mice developed PH and strong pulmonary vascular remodeling that was improved by imatinib (100 mg/kg/day) as evident from the significantly reduced right ventricular systolic pressure, right ventricular hypertrophy and muscularization of peripheral pulmonary arteries. Global gene expression analysis revealed that stromal cell derived factor SDF)-1α was significantly upregulated, which was confirmed by immunohistochemistry. Moreover, an enhanced immunoreactivity for SDF-1α, PDGFR-β and CXCR4, the receptor for SDF-1α was localized to the α-smooth muscle cell (SMC) actin positive pulmonary vascular cells in hypoxic mice and patients with idiopathic pulmonary arterial hypertension (IPAH). In conclusion, our findings substantiate the major role of PDGFR activation in pulmonary vascular remodeling by a genetic approach. Immunohistochemistry findings suggest a role for SDF-1α/CXCR4 axis in pulmonary vascular remodeling and point to a potential interaction between the chemokine SDF-1 and the growth factor PDGF signaling. Future studies designed to elucidate an interaction between the chemokine SDF-1 and the PDGF system may uncover novel therapeutic targets.
doi:10.4103/2045-8932.83448
PMCID: PMC3198653  PMID: 22034611
hypoxia; remodeling; PDGFR; SDF-1α; imatinib

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