MCT-induced Pulmonary Arterial Hypertension Rats Research Articles

Signaling pathways underlying skeletal muscle wasting in experimental pulmonary arterial hypertension

BackgroundSkeletal muscle wasting contributes to the poor functional status and quality of life of patients with pulmonary arterial hypertension (PAH). The present study aims to characterize the molecular mechanism underlying skeletal muscle wasting in experimental PAH induced by monocrotaline (MCT). MethodsMale Wistar rats were randomly injected with saline solution (CONT; n=10) or MCT (MCT; 60mg/kg, s.c.; n=15). After 4weeks of MCT or vehicle administration, animals were anesthetized and submitted to right ventricular (RV) hemodynamic evaluation. Blood and gastrocnemius samples were collected and stored for analysis. ResultsMCT group developed PAH (70% increase in RV peak systolic pressure) RV dysfunction (increased end-diastolic pressure and Tau), and body and muscle wasting (reduction of 20%, 16% and 30% on body weight, gastrocnemius mass and fiber cross sectional area, respectively). Muscle atrophy was associated with a decrease in type I MHC. Circulating (C reactive protein, myostatin and IL-1beta) and local catabolic markers (MAFbx/atrogin-1, protease activity) were increased in MCT animals, while Akt/mTOR pathway was preserved. Mitochondria isolated from gastrocnemius of MCT animals showed decreased activity of ATP synthase, lower levels of Tfam, accumulation of oxidatively modified proteins together with reduced levels of paraplegin. ConclusionsOur data suggests an anabolic/catabolic imbalance in gastrocnemius from MCT-induced PAH rats. Accumulation of dysfunctional mitochondria due to the inefficiency of protein quality control systems to eliminate damaged proteins could also contribute to muscle atrophy in PAH.

Farnesoid-X-receptor expression in monocrotaline-induced pulmonary arterial hypertension and right heart failure

ObjectiveThe farnesoid-X-receptor (FXR) is a metabolic nuclear receptor superfamily member that is highly expressed in enterohepatic tissue and is also expressed in the cardiovascular system. Multiple nuclear receptors, including FXR, play a pivotal role in cardiovascular disease (CVD). Pulmonary arterial hypertension (PAH) is an untreatable cardiovascular system disease that leads to right heart failure (RHF). However, the potential physiological/pathological roles of FXR in PAH and RHF are unknown. We therefore compared FXR expression in the cardiovascular system in PAH, RHF and a control. Methods and resultsHemodynamic parameters and morphology were assessed in blank solution-exposed control, monocrotaline (MCT)-exposed PAH (4 weeks) and RHF (7 weeks) Sprague–Dawley rats. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blot (WB), immunohistochemistry (IHC) analysis and immunofluorescence (IF) analysis were performed to assess FXR levels in the lung and heart tissues of MCT-induced PAH and RHF rats. In normal rats, low FXR levels were detected in the heart, and nearly no FXR was expressed in rat lungs. However, FXR expression was significantly elevated in PAH and RHF rat lungs but reduced in PAH and RHF rat right ventricular (RV) tissues. FXR expression was reduced only in RHF rat left ventricular (LV) tissues. ConclusionsThe differential expression of FXR in MCT-induced PAH lungs and heart tissues in parallel with PAH pathophysiological processes suggests that FXR contributes to PAH.

Hydrogen ameliorates pulmonary hypertension in rats by anti-inflammatory and antioxidant effects

ObjectiveThe pathogenesis of pulmonary arterial hypertension (PAH) involves reactive oxygen species and inflammation. Beneficial effects of molecular hydrogen, which exerts both anti-inflammatory and antioxidative effects, have been reported for various pathologic conditions. We therefore hypothesized that molecular hydrogen would improve monocrotaline (MCT)-induced PAH in rats. MethodsNineteen male Sprague-Dawley rats (body weight: 200-300 g) were divided into groups, receiving: (1) MCT + hydrogen-saturated water (group H); (2) MCT + dehydrogenized water (group M); or (3) saline + dehydrogenized water (group C). Sixteen days after substance administration, we evaluated hemodynamics, harvested the lungs and heart, and performed morphometric analysis of the pulmonary vasculature. Macrophage infiltration, antiproliferating cell nuclear antigen–positive cells, 8-hydroxy-deoxyguanosine (8-OHdG)–positive cells, and expressions of phosphorylated signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T-cells (NFAT) were evaluated immunohistochemically. Stromal cell–derived factor-1 and monocyte chemoattractant protein-1 expressions were evaluated by quantitative reverse-transcription polymerase chain reaction. ResultsPulmonary arterial hypertension was significantly exacerbated in group M compared to group C, but was significantly improved in group H. Vascular density was significantly reduced in group M, but not in group H. Adventitial macrophages, antiproliferating cell nuclear antigen - and 8-OHdG–positive cells, and stromal cell–derived factor-1 and monocyte chemoattractant protein-1 expressions were significantly increased in group M, but improved in group H. Expressions of phosphorylated STAT3 and NFAT were up-regulated in group M, but improved in group H. ConclusionsMolecular hydrogen ameliorates MCT-induced PAH in rats by suppressing macrophage accumulation, reducing oxidative stress and modulating the STAT3/NFAT axis.

Abstract 582: A Novel Adipocytokine, Omentin, Prevents Monocrotaline-induced Pulmonary Arterial Hypertension In Rats

Background: Omentin is a novel adipocytokine mainly expressed in visceral rather than subcutaneous adipose tissue. Several epidemiological reports demonstrate the negative relationship between serum omentin level and occurrence of obesity, type 2 diabetes and hypertension. Increase of inflammatory responses, contractile reactivity and structural remodeling of vascular wall contributes to hypertension development. Our in vitro and ex vivo studies previously demonstrated that omentin prevented those hypertension-related pathological processes. In addition, our in vivo study demonstrated that intravenously injected omentin acutely inhibited agonists-induced increases of blood pressure in rats. However, the chronic effects of omentin on hypertension development are not determined. In the present study, we tested the hypothesis that chronic omentin treatment may prevent monocrotaline (MCT)-induced pulmonary arterial (PA) hypertension (PAH). Methods and Results: MCT-induced PAH was induced by a single intraperitoneal injection of MCT (60 mg/kg) to rats. Omentin (18 μg/kg/day) was intraperitoneally treated for 14 days. Chronic omentin inhibited MCT-induced increases in PA pressure (n=8-10, p<0.05). Omentin inhibited MCT-induced increases in right ventricular hypertrophy (n=6-8, p<0.01) as well as lung to body weight ratio (n=8, p<0.01). Histologically, omentin inhibited MCT-induced PA hyperplasia (n=8, p<0.01). Omentin prevented the impairment of both endothelium-dependent and-independent relaxations mediated by acetylcholine and sodium nitroprusside, respectively (n=7-13, p<0.01). Conclusion: We for the first time demonstrate that chronic omentin treatment inhibits MCT-induced PAH in rats via preventing endothelial dysfunction and/or vascular structural remodeling.

A novel adipocytokine, omentin, inhibits monocrotaline-induced pulmonary arterial hypertension in rats

Omentin is a novel adipocytokine mainly expressed in visceral rather than subcutaneous adipose tissue. Several epidemiological studies demonstrated the negative relationship between blood omentin level and occurrence of obesity, type 2 diabetes and hypertension. Increases of inflammatory responses, contractile reactivity and structural remodeling of vascular wall contribute to hypertension development. Our in vitro studies previously demonstrated that omentin inhibited those hypertension-related pathological processes. In addition, our in vivo study demonstrated that intravenously injected omentin acutely inhibited agonists-induced increases of blood pressure in rats. However, the chronic effects of omentin on hypertension development are not determined. In the present study, we tested the hypothesis that chronic omentin treatment may inhibit pulmonary arterial (PA) hypertension (PAH). PAH was induced by a single intraperitoneal injection of monocrotaline (MCT: 60mg/kg) to rats. Omentin (18μg/kg/day) was intraperitoneally treated for 14days. Chronic omentin treatment inhibited MCT-induced increases in PA pressure. Omentin inhibited MCT-induced right ventricular hypertrophy as well as increase of lung to body weight ratio. Histologically, omentin inhibited MCT-induced PA hyperplasia. Further, omentin inhibited the impairment of both endothelium-dependent and -independent relaxations mediated by acetylcholine and sodium nitroprusside, respectively. In conclusion, we for the first time demonstrate that chronic omentin treatment inhibits MCT-induced PAH in rats via inhibiting vascular structural remodeling and abnormal contractile reactivity.

HMGB1 promotes the development of pulmonary arterial hypertension in rats.

RationalePulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.ObjectivesTo elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.MethodsMale Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.ResultsHMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.ConclusionsOur results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.

Benefit of combined therapy with nicorandil and colchicine in preventing monocrotaline-induced rat pulmonary arterial hypertension

This study tested the hypothesis that combined therapy with nicorandil and colchicine is superior to either alone in attenuating monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH). Adult male Sprague–Dawley rats (n=50) were equally randomized into group 1 (sham control), group 2 [MCT (60mg/kg i.p.)], group 3 [MCT–Nicorandil (5.0mg/kg/day)], group 4 [MCT-Colchicine (1.0mg/kg/day)], and group 5 (MCT–Nicorandil–Colchicine). Drugs were given on day 5. All animals were sacrificed on day 90 after MCT administration. Right ventricular systolic blood pressure (RVSBP) and RV weight were increased in group 2 compared to group 1, reduced in groups 3 and 4 compared to group 2, and further reduced in group 5, whereas arterial-oxygen saturation showed an opposite pattern (all p<0.001). Pulmonary damage severity (thickened alveolar septum and pulmonary arteriolar wall, decreased alveolar-sac numbers), number of CD3+ cells, and protein expressions of inflammatory (MMP-9, NF-κB, VCAM-1, angiotensin II-receptor), apoptotic (Bax, caspase 3, cleaved PARP), and fibrotic (TGF-β, Smad3) biomarkers showed an identical pattern compared to that of RVSBP, whereas pulmonary expressions of anti-apoptotic (Bcl-2) and anti-fibrotic (BMP-2, Smad1/5) biomarkers displayed a reverse pattern (all p<0.01). The protein expressions of RV damage markers (BNP, caspase 3) were increased, whereas expression of biomarker for RV functional preservation (Cx43) was reduced in group 2 compared with group 1, elevated in groups 3 and 4 compared to group 2, and further increased in group 5 (all p<0.01). Combined therapy with nicorandil and colchicine is superior to either alone in attenuating MCT-induced PAH in rats.

GW24-e1370 Effect of atorvastatin on endothelium-independent relaxation of small Pulmonary arteries in monocrotaline-induced PAH rats

ObjectivesTo examine the preventive effect of atorvastatin on sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EDiR) in small pulmonary artery (SPA) rings in monocrotaline (MCT)-induced pulmonary hypertension rats.Methods72 Male SD rats were...

GW24-e1368 Effect of atorvastatin on endothelium-dependent relaxation of small pulmonary arteries in monocrotaline-induced PAH rats

ObjectivesTo examine the preventive effect of atorvastatin on acetylcholine (Ach)-induced endothelium-dependentrelaxation (EDdR) in small pulmonary artery (SPA) rings in monocrotaline (MCT)-inducedpulmonary hypertension rats.Methods72 MaleSD rats were randomly assigned into four...

Benefit of Combined Therapy with Nicorandil and Colchicine in Reversing Monocrotaline-Induced Rat Pulmonary Arterial Hypertension

Purpose This study tested the hypothesis that combined therapy with and colchicine is superior to either alone in attenuating monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH). Methods and Materials Adult male Sprague-Dawley rats (n=50) were equally randomized into group 1 (sham control), group 2 [MCT (60 mg/kg i.p.)], group 3 [MCT-Nicorandil; (5.0 mg/kg/day)], group 4 [MCT-Colchicine (1.0 mg/kg/day)], and group 5; (MCT-Nicorandil-Colchicine). Drugs were given on day 5. All animals were hemodynamically assessed on day 90 after MCT administration and then were euthanized with the hearts and lungs harvested for morphometric studies, and for protein and mRNA analyses. Results Right ventricular systolic blood pressure (RVSBP) and RV weight were increased in group 2 compared to group 1, reduced in groups 3 and 4 compared to group 2, and further reduced in group 5, whereas arterial-oxygen saturation showed an opposite pattern (all p Conclusions Combined therapy with nicorandil and colchicine is superior to either alone in attenuating MCT-induced PAH in rats.

A novel Ca²+ channel antagonist reverses cardiac hypertrophy and pulmonary arteriolar remodeling in experimental pulmonary hypertension

This work investigates the actions of LASSBio-1289, (E)-N-methyl-N′-(thiophen-3-methylene)benzo[d][1,3]dioxole-5-carbohydrazide, on monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. Two weeks following the MCT injection, LASSBio-1289 (50 or 75mg/kg, p.o.) or vehicle was administrated once daily for 14 days. LASSBio-1289 (75mg/kg) treatment caused a significant decrease in right ventricular systolic pressure (31.89±0.82mmHg) compared to the MCT-vehicle group (52.74±6.19mmHg; P<0.05). Oral treatment with LASSBio-1289 (50 or 75mg/kg) effectively decreased pulmonary artery diameter and right ventricle (RV) area, assessed by echocardiography. LASSBio-1289 (75mg/kg) reduced RV area (10.00±0.58mm2) compared to the MCT-vehicle group (20.50±1.44mm2; P<0.05). LASSBio-1289 (75mg/kg) also partially recovered the pulmonary artery acceleration time in MCT-treated rats. Oral treatment with LASSBio-1289 (50mg/kg) decreased the pulmonary arteriolar wall thickness (68.57±2.21%) compared to the MCT-vehicle group (81.07±1.92%; P<0.05). In experiments with isolated pulmonary arteries, the concentration of LASSBio-1289 necessary to produce 50% relaxation in the phenylephrine- or KCl-induced contraction was 27.31±6.94 and 2.72±0.99μM, respectively, P<0.05. In the presence of LASSBio-1289 (50μM), the maximal contraction induced by 10mM CaCl2 was reduced to 36.00±8.28% of the maximal contraction of the control curve (P<0.05). LASSBio-1289 was effective in attenuating MCT-induced PAH in rats, and its beneficial effects were likely mediated by the inhibition of extracellular Ca2+ influx through L-type voltage-gated Ca2+ channels in the pulmonary artery.

Protective effects of methylsulfonylmethane on hemodynamics and oxidative stress in monocrotaline-induced pulmonary hypertensive rats.

Methylsulfonylmethane (MSM) is naturally occurring organic sulfur that is known as a potent antioxidant/anti-inflammatory compound. The aim of this study was to investigate the effect of MSM on hemodynamics functions and oxidative stress in rats with monocrotaline- (MCT-) induced pulmonary arterial hypertension (PAH). Wistar rats were randomly assigned to 38-days treatment. MSM was administered to rats at 100, 200, and 400 mg/kg/day doses 10 days before a single dose of 60 mg/kg, IP, MCT. Hemodynamics of ventricles were determined by Powerlab AD instrument. Blood samples were obtained to evaluate changes in the antioxidative system including activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and the level of reduced glutathione (GSH) and malondialdehyde (MDA). Improvements in cardiopulmonary hemodynamics were observed in the MSM-treated pulmonary arterial hypertensive rats, with a significant reduction in right ventricular systolic pressure (RSVP) and an increase in the mean arterial pressure (MAP). The values of CAT, SOD, GSH-px activities, and GSH were significantly lower in MCT-induced PAH (P < 0.01), but they were recovered to control levels of MSM-treated groups. Our present results suggest that long-term administration of the MSM attenuates MCT-induced PAH in rats through modulation of oxidative stress and antioxidant defense.

Enhanced protection against pulmonary hypertension with sildenafil and endothelial progenitor cell in rats

BackgroundSildenafil and bone marrow-derived endothelial progenitor cells (BMDEPCs) have been shown to ameliorate monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in the rat. We test whether combined sildenafil and BMDEPC treatment exerts additional protection against MCT-induced PAH in rats. MethodsMale Sprague–Dawley rats were randomized to receive saline injection only (group 1), MCT (70mg/kg) only (group 2), MCT plus autologous BMDEPC (2.0×106cells) transplantation (group 3), MCT with sildenafil (30mg/kg/day) (group 4), and MCT with combined BMDEPCs–sildenafil (30mg/kg/day) (group 5). Intravenous BMDEPC and oral sildenafil were given on day 3 after MCT administration. Hemodynamics were analyzed using Labchart software, whereas cellular and molecular parameters were measured using flow cytometry, real-time PCR, TUNEL assay, Western blot, and immunohistochemical staining. ResultsBy day 35 following MCT treatment, lower expression of connexin43, protein kinase C-ε, Bcl-2, and endothelial nitric oxide synthase and higher expression of tumor necrosis factor-α and caspase 3 were found in right ventricle (RV) and lung in group 2 compared with other groups (all p<0.05). The number of alveolar sacs and lung arterioles were also lower in group 2 than in other groups (all p<0.05). Furthermore, RV systolic pressure (RVSP), RV weight, and RV-to-final body weight ratio were substantially increased in group 2 than in other groups, and notably higher in groups 3 and 4 than in groups 1 and 5 (all p<0.0001). ConclusionsCombined therapy with autologous BMDEPC and sildenafil is superior to either BMDPEC or sildenafil alone for preventing MCT-induced PAH.

Glycogen Synthase Kinase 3beta Contributes to Proliferation of Arterial Smooth Muscle Cells in Pulmonary Hypertension

RationalePulmonary 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.MethodsAll 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.ResultsIncreased 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.ConclusionThis study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.

Terguride ameliorates monocrotaline-induced pulmonary hypertension in rats

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterised by vasoconstriction and remodelling of the pulmonary vasculature. The serotonin (5-hydroxytryptamine (5-HT)) pathway has been shown to play a major role in the pathogenesis of PAH, but pharmacological modulation of this pathway for treatment of PAH is, to date, at a pre-clinical level. Terguride is a 5-HT receptor (5-HTR) antagonist that is well tolerated and clinically approved for ovulation disorders. Immunohistochemistry against 5-HTR(₂A/B) on human lungs revealed their localisation to the vascular smooth muscle layer and quantitative RT-PCR showed 5-HTR(₂B) upregulation in pulmonary artery smooth muscle cells (PASMC) isolated from PAH patients. Proliferation and migration of cultured primary human PASMC were dose-dependently blocked by terguride. Therapeutic 5-HT signalling inhibition was 1) demonstrated in isolated, ventilated and perfused rat lungs and 2) by chronic terguride treatment of rats with monocrotaline (MCT)-induced pulmonary hypertension in a preventive or curative approach. Terguride inhibited proliferation of PASMCs and abolished 5-HT-induced pulmonary vasoconstriction. Chronic terguride treatment prevented dose-dependently the development and progression of MCT-induced PAH in rats. Thus, terguride represents a valuable novel therapeutic approach in PAH.

Systemic Evaluation of Platelet and Leukocyte Activation and Interaction in a Rat Model of Pulmonary Arterial Hypertension

Objectives: Thrombosis and inflammation are associated with the pathogenesis of pulmonary arterial hypertension (PAH). However, there are no solid data supporting the involvement of platelet and leukocyte activation and interaction in PAH. The present study thus investigated the activation and interaction of circulating platelets and leukocytes in a rat model of monocrotaline (MCT)-induced pulmonary hypertension. Methods: Mean pulmonary arterial pressure (mPAP) was monitored in rats (n = 24) before and 2, 3 and 7 weeks after MCT (60 mg/kg)injection. In parallel, activation of circulating platelets and leukocytes and platelet-leukocyte aggregates were measured by whole-blood flow cytometry. Results: Two weeks after MCT injection, mPAP had increased significantly, i.e. from 11.25 ± 0.92 mm Hg at baseline to 15.71 ± 1.66 mm Hg (p < 0.05), and it had increased even further at week 7 (26.83 ± 3.29 mm Hg; p < 0.01). Fibrinogen binding of circulating platelets had increased from the basal level of 1.45 ± 0.61 to 4.08 ± 1.59% 3 weeks after MCT injection (p < 0.01). Platelet responsiveness to ADP was also significantly enhanced. CD11b expression of circulating neutrophils was elevated; i.e. mean fluorescence intensity increased from 1.67 ± 0.38 before MCT injection to 2.37 ± 0.31 3 weeks after MCT injection (p < 0.01), and N-formyl-methionyl-leucyl-phenylalanine (1 × 10^–7M) stimulation induced more marked elevation of neutrophil CD11b expression in MCT-treated animals. Circulating platelet-neutrophil aggregates were already increased 2 weeks after MCT treatment (14.93 ± 4.22%; p < 0.01) compared to baseline (6.01 ± 2.91%) and remained elevated at 3 weeks (15.19 ± 4.78%; p < 0.01). Conclusions: MCT-induced PAH in rats is associated with increased platelet and leukocyte activation and platelet-leukocyte interaction in vivo, which may play an important role in the pathogenesis of PAH.

Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline

Mutations in the gene for the transforming growth factor (TGF)-beta superfamily receptor, bone morphogenetic protein receptor II, underlie heritable forms of pulmonary arterial hypertension (PAH). Aberrant signaling via TGF-beta receptor I/activin receptor-like kinase 5 may be important for both the development and progression of PAH. We investigated the therapeutic potential of a well-characterized and potent activin receptor-like kinase 5 inhibitor, SB525334 [6-(2-tert-butyl-5-{6-methyl-pyridin-2-yl}-1H-imidazol-4-yl)-quinoxaline] for the treatment of PAH. In this study, we demonstrate that pulmonary artery smooth muscle cells from patients with familial forms of idiopathic PAH exhibit heightened sensitivity to TGF-beta1 in vitro, which can be attenuated after the administration of SB525334. We further demonstrate that SB525334 significantly reverses pulmonary arterial pressure and inhibits right ventricular hypertrophy in a rat model of PAH. Immunohistochemical studies confirmed a significant reduction in pulmonary arteriole muscularization induced by monocrotaline (used experimentally to induce PAH) after treatment of rats with SB525334. Collectively, these data are consistent with a role for the activin receptor-like kinase 5 in the progression of idiopathic PAH and imply that strategies to inhibit activin receptor-like kinase 5 signaling may have therapeutic benefit.

Angiotensin-converting enzyme inhibitor preserves p21 and endothelial nitric oxide synthase expression in monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is associated with structural changes in the pulmonary vasculature characterized by the proliferation of cellular components of the vessels. ACE inhibitor (ACEI) may have beneficial effects in treating PAH, but its precise mechanism of action in the remodeling process is unclear. p21 is a cyclin-dependent kinase inhibitor that may have a protective role in this process by inhibiting cellular proliferation. Endothelial nitric oxide synthase (eNOS) has also been shown to be protective by its vasodilatory effect. Therefore, we investigated whether expression of p21 and eNOS was modulated by ACEI treatment in a rat model. Monocrotaline (MCT) was administered to 2 groups of Sprague-Dawley rats fed a high-cholesterol diet, ie, one group received MCT concomitantly with enalapril treatment (MCT(+)/ACEI(+) rats), and the other group did not receive enalapril (MCT(+)/ACEI(-) rats). After 5 weeks, MRI showed right ventricular hypertrophy in MCT(+)/ACEI(-) rats. MCT(+)/ACEI(+) rats showed a preserved right ventricular morphology. Isolated pulmonary perfusion studies showed that ACEI significantly upregulated NO production, as measured by nitrite levels. Addition of N-methyl-D-glucamine dithiocarbamate-Fe solution, an NO-trapping agent, reversed the basal vasodilatory effect of ACEI in the pulmonary vasculature. Immunoblot analysis showed decreased p21 and eNOS expression in the lung in MCT(+)/ACEI(-) rats, whereas their expression was preserved with enalapril treatment. ACEI suppresses the development of MCT-induced PAH in rats. The mechanism of action might involve the preservation of p21 and eNOS expression. Both p21 and endothelium-derived NO appear to have protective roles in the development of PAH.

Angiotensin 1-7 Reduces Right Ventricular Hypertrophy and Fibrosis in a Rat Model of monocrotaline Induced Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH), characterized by high pressure in the pulmonary arteries, is fatal due to the development of right ventricular (RV) failure. Angiotensin-1-7 (Ang-(1-7)) is a metabolite of Angiotensin I/ II that has beneficial effects on pulmonary vascular remodelling in animal models of PAH. However its effect on RV myocardial remodelling in PAH is unknown.Objective: To study the effects of Ang-(1-7) on RV hypertrophy and fibrosis in an experimental PAH model.Methods: 220 gram Sprague-Dawley rats were randomized to: sham-controls (n=2), monocrotaline (MCT) induced PAH (n=3) and PAH + Ang-1-7 administered via subcutaneous injection for 5-weeks following injection of MCT (n=4). Animals were sacrificed at 5-weeks and the heart harvested for investigation of RV wall thickness and collagen content.Results: RV wall thickness was increased in the MCT-group (70.3  2.3 pixels vs. sham-controls 50.42  2.1 pixels p=0.0001) and decreased towards control values with the addition of Ang-(1-7) (52.7 1.9, p=0.0001 vs. MCT). RV myocardial collagen increased in the MCT-group (6.70.8 % vs. shams 2.70.2 %, p=0.0001) and decreased with the addition of Ang-(1-7) (5.60.2 %, p=0.04 vs. MCT).Conclusions: Ang-(1-7) reduces development of RV hypertrophy and fibrosis in MCT-induced PAH in rats, despite similar pulmonary arterial pressures. Further study is needed to investigate whether Ang-(1-7) improves RV performance and clinical outcomes in experimental and clinical settings.