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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.
PMCID: PMC4312574  PMID: 25667920
2.  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.
PMCID: PMC4070756  PMID: 25006428
idiopathic pulmonary arterial hypertension; chronic obstructive pulmonary disease; mast cells; chymase
3.  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.
PMCID: PMC4070792  PMID: 24618536
Rhodiola; pulmonary hypertension; pulmonary vascular remodeling; Chinese medicine; salidroside
4.  Expression and Activity of Phosphodiesterase Isoforms during Epithelial Mesenchymal Transition: The Role of Phosphodiesterase 4 
Molecular Biology of the Cell  2009;20(22):4751-4765.
Epithelial–mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer and is typically induced by the multifunctional cytokine transforming growth factor (TGF)-β1. The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-β1-induced EMT in the human alveolar epithelial type II cell line A549. Stimulation of A549 with TGF-β1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, and α-smooth muscle actin. Interestingly, TGF-β1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Furthermore, mRNA and protein expression demonstrated up-regulation of PDE4A and PDE4D isoforms in TGF-β1-stimulated cells. Most importantly, treatment of TGF-β1 stimulated epithelial cells with the PDE4-selective inhibitor rolipram or PDE4 small interfering RNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species, p38, and extracellular signal-regulated kinase phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin but did not result in changes of mesenchymal markers. In addition, Rho kinase signaling activated by TGF-β1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-β1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuate EMT-associated lung diseases such as pulmonary fibrosis and lung cancer.
PMCID: PMC2777105  PMID: 19759179
6.  Schistosomiasis causes remodeling of pulmonary vessels in the lung in a heterogeneous localized manner: Detailed study 
Pulmonary Circulation  2013;3(2):356-362.
Schistosomiasis is a global parasitic disease with high impact on public health in tropical areas. Schistosomiasis is a well-described cause of pulmonary arterial hypertension (PAH). The exact pathogenesis is still unclear, though inflammatory mechanisms are suspected. Another unknown is whether the changes in the pulmonary vasculature are generalized or localized. We studied 13 mice infected with cercariae for 12 weeks compared with 10 control mice. In our model, we observed that the liver was a target during infection and was enlarged more than two-fold after infection. However, right heart hypertrophy as measured by RV/(LV + S) ratio was not observed at this time point. Moreover, we noticed that 72% of the sampled lobes (92% of the lungs) harvested from these animals costained evidence of granulomatous changes, secondary to egg deposition. We systemically mapped the distribution of granulomatous lesions in right lung lobes (n = 43) of infected mice. We observed that the distribution of the granulomatous lesions was heterogeneous. Remodeled pulmonary vessels were seen in 26% of the lobes (46% of the lungs) and were observed only in close proximity to the granuloma. No remodeling was observed in the absence of granulomas. These findings support the view that pulmonary vascular remodeling is caused by the local presence of granulomas in PAH associated with schistosomiasis. The heterogeneous nature of the remodeling partly explains why many patients with schistosomiasis do not develop pulmonary hypertension.
PMCID: PMC3757830  PMID: 24015336
schistosomiasis; pulmonary hypertension; inflammation; experimental models
7.  Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation 
Cancer Immunology, Immunotherapy  2012;62(2):309-320.
Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E2 were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE2 release and proliferation in response to LPS. Synthesis of PGE2 was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.
PMCID: PMC3569588  PMID: 22923191
Lung cancer; Infection; Endotoxin; Tumor proliferation; Inflammation
8.  The Soluble Guanylate Cyclase Stimulator Riociguat Ameliorates Pulmonary Hypertension Induced by Hypoxia and SU5416 in Rats 
PLoS ONE  2012;7(8):e43433.
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).
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.
PMCID: PMC3422306  PMID: 22912874
12.  Glycogen Synthase Kinase 3beta Contributes to Proliferation of Arterial Smooth Muscle Cells in Pulmonary Hypertension 
PLoS ONE  2011;6(4):e18883.
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.
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.
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.
This study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.
PMCID: PMC3078925  PMID: 21533110
13.  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.
PMCID: PMC3073929  PMID: 21494592
14.  Inhaled tolafentrine reverses pulmonary vascular remodeling via inhibition of smooth muscle cell migration 
Respiratory Research  2005;6(1):128.
The aim of the study was to assess the chronic effects of combined phosphodiesterase 3/4 inhibitor tolafentrine, administered by inhalation, during monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.
CD rats were given a single subcutaneous injection of monocrotaline to induce PAH. Four weeks after, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system. In these animals (i) the acute pulmonary vasodilatory efficacy of inhaled tolafentrine (ii) the anti-remodeling effect of long-term inhalation of tolafentrine (iii) the effects of tolafentrine on the expression profile of 96 genes encoding cell adhesion and extracellular matrix regulation were examined. In addition, the inhibitory effect of tolafentrine on ex vivo isolated pulmonary artery SMC cell migration was also investigated.
Monocrotaline injection provoked severe PAH (right ventricular systolic pressure increased from 25.9 ± 4.0 to 68.9 ± 3.2 after 4 weeks and 74.9 ± 5.1 mmHg after 6 weeks), cardiac output depression and right heart hypertrophy. The media thickness of the pulmonary arteries and the proportion of muscularization of small precapillary resistance vessels increased dramatically, and the migratory response of ex-vivo isolated pulmonary artery smooth muscle cells (PASMC) was increased. Micro-arrays and subsequent confirmation with real time PCR demonstrated upregulation of several extracellular matrix regulation and adhesion genes, such as matrixmetalloproteases (MMP) 2, 8, 9, 10, 11, 12, 20, Icam, Itgax, Plat and serpinb2. When chronically nebulized from day 28 to 42 (12 daily aerosol maneuvers), after full establishment of severe pulmonary hypertension, tolafentrine reversed about 60% of all hemodynamic abnormalities, right heart hypertrophy and monocrotaline-induced structural lung vascular changes, including the proportion of pulmonary artery muscularization. The upregulation of extracellular matrix regulation and adhesion genes was reduced by nearly 80% by inhalation of the tolafentrine. When assessed in vitro, tolafentrine blocked the enhanced PASMC migratory response.
In conclusion, we demonstrate for the first time that inhalation of combined PDE3/4 inhibitor reverses pulmonary hypertension fully developed in response to monocrotaline in rats. This "reverse-remodeling" effect includes structural changes in the lung vascular wall and key molecular pathways of matrix regulation, concomitant with 60% normalization of hemodynamics.
PMCID: PMC1291406  PMID: 16262900
15.  Reversal of experimental pulmonary hypertension by PDGF inhibition 
Journal of Clinical Investigation  2005;115(10):2811-2821.
Progression of pulmonary hypertension is associated with increased proliferation and migration of pulmonary vascular smooth muscle cells. PDGF is a potent mitogen and involved in this process. We now report that the PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in 2 animal models of pulmonary hypertension. In rats with monocrotaline-induced pulmonary hypertension, therapy with daily administration of STI571 was started 28 days after induction of the disease. A 2-week treatment resulted in 100% survival, compared with only 50% in sham-treated rats. The changes in RV pressure, measured continuously by telemetry, and right heart hypertrophy were reversed to near-normal levels. STI571 prevented phosphorylation of the PDGF receptor and suppressed activation of downstream signaling pathways. Similar results were obtained in chronically hypoxic mice, which were treated with STI571 after full establishment of pulmonary hypertension. Moreover, expression of the PDGF receptor was found to be significantly increased in lung tissue from pulmonary arterial hypertension patients compared with healthy donor lung tissue. We conclude that STI571 reverses vascular remodeling and cor pulmonale in severe experimental pulmonary hypertension regardless of the initiating stimulus. This regimen offers a unique novel approach for antiremodeling therapy in progressed pulmonary hypertension.
PMCID: PMC1236676  PMID: 16200212
16.  Lung vasodilatory response to inhaled iloprost in experimental pulmonary hypertension: amplification by different type phosphodiesterase inhibitors 
Respiratory Research  2005;6(1):76.
Inhaled prostanoids and phosphodiesterase (PDE) inhibitors have been suggested for treatment of severe pulmonary hypertension. In catheterized rabbits with acute pulmonary hypertension induced by continuous infusion of the stable thromboxane analogue U46619, we asked whether sildenafil (PDE1/5/6 inhibitor), motapizone (PDE3 inhibitor) or 8-Methoxymethyl-IBMX (PDE1 inhibitor) synergize with inhaled iloprost. Inhalation of iloprost caused a transient pulmonary artery pressure decline, levelling off within <20 min, without significant changes in blood gases or systemic hemodynamics. Infusion of 8-Methoxymethyl-IBMX, motapizone and sildenafil caused each a dose-dependent decrease in pulmonary artery pressure, with sildenafil possessing the highest efficacy and at the same time selectivity for the pulmonary circulation. When combining a per se ineffective dose of each PDE inhibitor (200 μg/kg × min 8-Methoxymethyl-IBMX, 1 μg/kg × min sildenafil, 5 μg/kg × min motapizone) with subsequent iloprost nebulization, marked amplification of the prostanoid induced pulmonary vasodilatory response was noted and the area under the curve of PPA reduction was nearly threefold increased with all approaches, as compared to sole iloprost administration. Further amplification was achieved with the combination of inhaled iloprost with sildenafil plus motapizone, but not with sildenafil plus 8MM-IBMX. Systemic hemodynamics and gas exchange were not altered for all combinations. We conclude that co-administration of minute systemic doses of selective PDE inhibitors with inhaled iloprost markedly enhances and prolongs the pulmonary vasodilatory response to inhaled iloprost, with maintenance of pulmonary selectivity and ventilation perfusion matching. The prominent effect of sildenafil may be operative via both PDE1 and PDE5, and is further enhanced by co-application of a PDE3 inhibitor.
PMCID: PMC1180856  PMID: 16033645
17.  Evaluation of Angiogenesis Using Micro-Computed Tomography in a Xenograft Mouse Model of Lung Cancer 
Neoplasia (New York, N.Y.)  2009;11(1):48-56.
Quantitative evaluation of lung tumor angiogenesis using immunohistochemical techniques has been limited by difficulties in generating reproducible data. To analyze intrapulmonary tumor angiogenesis, we used high-resolution micro-computed tomography (micro-CT) of lung tumors of mice inoculated with mouse Lewis lung carcinoma (LLC1) or human adenocarcinoma (A549) cell lines. The lung vasculature was filled with the radiopaque silicone rubber, Microfil, through the jugular vein (in vivo application) or pulmonary artery (ex vivo application). In addition, human adenocarcinoma lung tumor-bearing mice treated site-specifically with humanized monoclonal antibody (bevacizumab) against vascular endothelial growth factor. Quantitative analysis of lung tumor microvessels imaged with micro-CT showed that more vessels (mainly small, <0.02 mm2) were filled using the in vivo (5.4%) compared with the ex vivo (2.1%) method. Furthermore, bevacizumab-treated lung tumor-bearing mice showed significantly reduced lung tumor volume and lung tumor angiogenesis compared with untreated mice as assessed by micro-CT. Interestingly, microvascularization of mainly the smaller vessels (<0.02 mm2) was reduced after bevacizumab treatment. This observation with micro-CT was nicely correlated with immunohistochemical measurement of microvessels. Therefore, micro-CT is a novel method for investigating lung tumor angiogenesis, and this might be considered as an additional complementary tool for precise quantification of angiogenesis.
PMCID: PMC2606118  PMID: 19107231

Results 1-17 (17)