MCT-injected Rats Research Articles

Effect of thrombomodulin on the development of monocrotaline-induced pulmonary hypertension

The purpose of the present study was to investigate whether thrombomodulin (TM) prevents the development of pulmonary hypertension (PH) in monocrotaline (MCT)-injected rats. Human recombinant TM (3 mg/kg/2 days) or saline were given to MCT-injected male Sprague-Dawley rats for 19 (n = 14) or 29 (n = 11) days. Control rats (n = 6) were run for 19 days. The mean pulmonary artery pressure (mPAP), right ventricular hypertrophy (RVH), percentages of muscularized peripheral arteries (%muscularization), and medial wall thickness of small muscular arteries (%MWT) were measured. To determine inflammatory and coagulation responses, broncho-alveolar lavage fluid (BALF) was analyzed in another set of rats (n = 29). Western blotting for endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (peNOS) in the lung tissue was performed in separate rats (n = 13). Survival was determined in 60 rats. MCT increased mPAP, RVH, %muscularization, and %MWT. TM treatment significantly reduced mPAP, %muscularization, and %MWT in peripheral arteries with an external diameter of 50-100 μm in 19 days after MCT injection, but the effect was lost after 29 days. MCT increased the levels of tumor necrosis factor alpha, monocyte chemoattractant protein-1, and thrombin-antithrombin complex in BALF. Expression of eNOS increased in MCT rats, while peNOS decreased. The relative amount of peNOS to total eNOS increased in MCT/TM rats compared to MCT/Vehicle rats. A Kaplan-Meier survival curve showed no difference with and without TM. Although the administration of TM might slightly delay the progression of MCT-induced PH, the physiological significance for treatment is limited, since the survival rate was not improved.

Pyrrolidine dithiocarbamate attenuates the development of monocrotaline-induced pulmonary arterial hypertension

We aimed to demonstrate the potential protective effects of pyrrolidine dithiocarbamate (PDTC) on monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Adult male rats were randomly assigned to 4 groups: control group, MCT-treated rats only, MCT-injected rats treated with PDTC, and PDTC-treated rats only. Blood and tissue samples were collected after the sacrifice. Levels of malondialdehyde (MDA) were measured by using the thiobarbituric acid method. Total antioxidant status (TAS) was determined using a commercially available ImAnOx kit. A histopathological evaluation was accomplished by scoring the degree of severity. Endothelial damage of the main pulmonary artery was evaluated by immunohistochemical labeling of endothelial cells using anti-rat endothelial cell antigen 1 (RECA-1) antibody. MCT-induced right ventricular hypertrophy (RVH) was reduced significantly in the MCT+PDTC-treated group. MDA levels were significantly lowered in the MCT+PDTC-treated group. TAS was significantly higher in the MCT+PDTC-treated group when compared with the rats with PAH. Histopathological examination demonstrated that PDTC treatment reduced the development of inflammation, hemorrhage and congestion, and collagen deposition. In conclusion, PDTC attenuated PAH and protected pulmonary endothelium in rats administered MCT. These findings suggest that PDTC treatment may provide a new effective therapeutic approach in the treatment of PAH.

Nicorandil Prevents Right Ventricular Remodeling by Inhibiting Apoptosis and Lowering Pressure Overload in Rats with Pulmonary Arterial Hypertension

BackgroundMost of the deaths among patients with severe pulmonary arterial hypertension (PAH) are caused by progressive right ventricular (RV) pathological remodeling, dysfunction, and failure. Nicorandil can inhibit the development of PAH by reducing pulmonary artery pressure and RV hypertrophy. However, whether nicorandil can inhibit apoptosis in RV cardiomyocytes and prevent RV remodeling has been unclear.Methodology/Principal FindingsRV remodeling was induced in rats by intraperitoneal injection of monocrotaline (MCT). RV systolic pressure (RVSP) was measured at the end of each week after MCT injection. Blood samples were drawn for brain natriuretic peptide (BNP) ELISA analysis. The hearts were excised for histopathological, ultrastructural, immunohistochemical, and Western blotting analyses. The MCT-injected rats exhibited greater mortality and less weight gain and showed significantly increased RVSP and RV hypertrophy during the second week. These worsened during the third week. MCT injection for three weeks caused pathological RV remodeling, characterized by hypertrophy, fibrosis, dysfunction, and RV mitochondrial impairment, as indicated by increased levels of apoptosis. Nicorandil improved survival, weight gain, and RV function, ameliorated RV pressure overload, and prevented maladaptive RV remodeling in PAH rats. Nicorandil also reduced the number of apoptotic cardiomyocytes, with a concomitant increase in Bcl-2/Bax ratio. 5-hydroxydecanoate (5-HD) reversed these beneficial effects of nicorandil in MCT-injected rats.Conclusions/SignificanceNicorandil inhibits PAH-induced RV remodeling in rats not only by reducing RV pressure overload but also by inhibiting apoptosis in cardiomyocytes through the activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels. The use of a mitoKATP channel opener such as nicorandil for PAH-associated RV remodeling and dysfunction may represent a new therapeutic strategy for the amelioration of RV remodeling during the early stages of PAH.

Abstract 329: Oral Administration of New Calcium Channel Antagonist Reverses Right Ventricular Dysfunction in Rats With Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, endothelial injury and vascular remodeling, right ventricular hypertrophy and increased right ventricular systolic pressure. This work investigated the effects of LASSBio-1289, a new calcium channel antagonist, in rats with PAH induced by monocrotaline (MCT). METHODS: Male Wistar rats (200 - 250 g) received a single MCT i.p. injection (60 mg/kg) for induction of PAH. The experimental groups were: control, MCT, MCT + vehicle, MCT + LASSBio-1289 (50 mg/kg p.o.). Animals were treated either with vehicle or LASSBio-1289 for 14 days after the onset of disease. The following parameters were analyzed: right ventricular systolic pressure (RVSP), RV contractility (dp/dtmax), RV relaxation (dp/dtmin)and RV weight / left ventricle + septum weight (RV/LV+S). Echocardiography was performed to determine RV area and wall thickness. The protocols used in the present study were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Results: RVSP (mmHg) increased from 25.3 ± 1.9 (control) to 49.0 ± 6.2 (P < 0.05) and 52.7 ± 6.2 (P < 0.05) in MCT and MCT + vehicle groups, respectively. When MCT-injected rats were treated with LASSBio-1289, RVSP was decreased to 36.4 ± 2.0 mm Hg (P < 0.05). RV/LV+S increased from 0.24 ± 0.02 to 0.56 ± 0.04 (P < 0.05) and 0.49 ± 0.03 in the MCT and MCT + vehicle groups (P < 0.05) and reduced to 0.30 ± 0.03 (P < 0.05) after treatment with LASSBio-1289. The dp/dtmax (mmHg/s) was 1179 ± 137, 2180 ± 238, 2482 ± 371 and 1134 ± 131 for control, MCT, MCT + vehicle and MCT + LASSBio-1289 groups. In addition to this, the dp/dtmin (mmHg/s) increased from -702 ± 83 to -1359 ± 120, -1720 ± 243 in the MCT and MCT + vehicle groups. In contrast, treatment with LASSBio-1289 reduced this parameter to -701 ± 122 (P < 0.05). RV area (mm2) was increased from 8.2 ± 0.5 to 17.8 ± 1.4 and 18.7 ± 1.0 in the PAH groups. RV area was reduced in LASSBio-1289-treated rats to 9.4 ± 0.9 (P < 0.05). PAH increased the RV wall thickness (cm) from 0.089 ± 0.002 to 0.125 ± 0.006 (P < 0.05) and was reduced to 0.098 ± 0.006 after treatment with LASSBio-1289. Conclusions: LASSBio-1289 effectively reversed right ventricular dysfunction and hypertrophy in rats with monocrotaline-induced PAH.

Bortezomib Alleviates Experimental Pulmonary Arterial Hypertension

Vascular remodeling and endothelial dysfunction are important pathogenic features of pulmonary arterial hypertension (PAH). There is a growing body of evidence that proteasome inhibitors may be beneficial in vascular diseases by inhibiting proliferation of vascular smooth muscle cells (VSMCs) and ameliorating endothelial dysfunction. Here, we evaluated whether bortezomib (BTZ) could alleviate hypoxia- and monocrotaline (MCT)-induced PAH. BTZ (at doses from 1 to 100 μg/kg, or a dose of 100 μg/kg) was administered to mice every other day for the last 2 weeks of a 5-week hypoxia (10% O(2)) period, or to rats once daily from Day 22 to Day 34 after MCT challenge, respectively. BTZ treatment substantially suppressed elevation of right ventricular (RV) systolic pressure, RV hypertrophy, and pulmonary vascular remodeling in hypoxia-exposed mice. Similarly, BTZ treatment inhibited RV hypertrophy and vascular remodeling in MCT-injected rats. Strikingly, BTZ rescued 70% of MCT-injected rats up to Day 60, along with a considerable reduction in RV systolic pressure and suppression of vascular remodeling, whereas, among MCT-injected rats not administered BTZ, there were no survivors by Day 41. BTZ significantly suppressed proliferation of pulmonary VSMCs in vivo and in vitro. Furthermore, BTZ increased not only endothelial nitric oxide (NO) synthase (eNOS), phosphorylated eNOS, and NO production in vitro, but also eNOS and p-eNOS in hypoxia-exposed mice and MCT-injected rats, respectively. In contrast to the beneficial effects, BTZ increased active caspase-3 in cardiac ventricles of MCT-injected rats. Taken together, with caution for cardiotoxicity, BTZ could be a potential therapeutic strategy in PAH, possibly acting by inhibition of VSMC proliferation and amelioration of endothelial dysfunction.

Haemodynamic and neuroendocrine effects of tezosentan in chronic experimental pulmonary hypertension

Chronic pulmonary hypertension (PH) therapy is poorly investigated in intensive care. Our aim was to evaluate haemodynamic and neuroendocrine effects of the dual endothelin-1 (ET-1) blocker tezosentan in monocrotaline (MCT)-induced PH. Male Wistar rats (180-200 g, n = 194) randomly received 60 mg kg(-1) MCT or vehicle, subcutaneously, and 2 days later, a subgroup of MCT-injected rats was gavaged with 300 mg kg(-1) day(-1) bosentan (MCT BOS, n = 46), while another (MCT, n = 125) and control rats (Ctrl, n = 23) received vehicle. At 25-30 days, 48 h after interrupting bosentan, rats randomly underwent either a dose-response evaluation (0.5-20 mg kg(-1), n = 7 each group) or a 4 h perfusion of tezosentan (20 mg kg(-1) in 10 min + 10 mg g(-1) h(-1)) or vehicle (n = 8 per group, each). Haemodynamics, including blood gas analysis, were evaluated after thoracotomy under anaesthesia. After plasma, right ventricle (RV) and lung collection, plasma ET-1, cytokines, nitrate and 6-keto-PGF1α, and lung and right ventricular gene expression and cyclooxygenase (COX) and nitric oxide synthase (NOS) activities were quantified. Monocrotaline resulted in PH, RV dilation and decreased cardiac output (CO) that were attenuated in MCT BOS. Pulmonary hypertension was attenuated by tezosentan without systemic hypotension. Tezosentan increased CO without changing ventilation-perfusion matching. Both bosentan and tezosentan reduced ET-1 and cytokine plasma levels and tissue expression, and inducible NOS and COX-2 RV activities. Bosentan increased nitrate plasma levels and non inducible NOS activities whereas tezosentan decreased circulating 6-keto-PGF1α but increased lung COX-1 activity. Tezosentan may be useful for haemodynamic handling and bosentan replacement in critically ill PH patients exerting important beneficial neuroendocrine and anti-inflammatory actions.

Spontaneous Ventricular Fibrillation in Right Ventricular Failure Secondary to Chronic Pulmonary Hypertension

Right ventricular failure (RVF) in pulmonary hypertension (PH) is associated with increased incidence of sudden death by a poorly explored mechanism. We test the hypothesis that PH promotes spontaneous ventricular fibrillation (VF) during a critical post-PH onset period characterized by a sudden increase in mortality. Rats received either a single subcutaneous dose of monocrotaline (MCT, 60 mg/kg) to induce PH-associated RVF (PH, n=24) or saline (control, n=17). Activation pattern of the RV-epicardial surface was mapped using voltage-sensitive dye in isolated Langendorff-perfused hearts along with single glass-microelectrode and ECG-recordings. MCT-injected rats developed severe PH by day 21 and progressed to RVF by approximately day 30. Rats manifested increased mortality, and ≈30% rats died suddenly and precipitously during 23-32 days after MCT. This fatal period was associated with the initiation of spontaneous VF by a focal mechanism in the RV, which was subsequently maintained by both focal and incomplete reentrant wave fronts. Microelectrode recordings from the RV-epicardium at the onset of focal activity showed early afterdepolarization-mediated triggered activity that led to VF. The onset of the RV cellular triggered beats preceded left ventricular depolarizations by 23±8 ms. The RV but not the left ventricular cardiomyocytes isolated during this fatal period manifested significant action potential duration prolongation, dispersion, and an increased susceptibility to depolarization-induced repetitive activity. No spontaneous VF was observed in any of the control hearts. RVF was associated with significantly reduced RV ejection fraction (P<0.001), RV hypertrophy (P<0.001), and RV fibrosis (P<0.01). The hemodynamic function of the LV and its structure were preserved. PH-induced RVF is associated with a distinct phase of increased mortality characterized by spontaneous VF arising from the RV by an early afterdepolarization-mediated triggered activity.

A role for coagulation factor Xa in experimental pulmonary arterial hypertension

Anticoagulation with warfarin is recommended for the treatment of patients with pulmonary arterial hypertension (PAH). However, the therapeutic benefit of anticoagulation has not yet been demonstrated experimentally or clinically. Here, rivaroxaban, an oral, direct factor Xa (FXa) inhibitor, was compared with warfarin and enoxaparin in the prevention of right ventricular (RV) dysfunction and hypertrophy in the monocrotaline (MCT) model of pulmonary hypertension. Sprague-Dawley rats (n = 10 per group) were randomized to receive rivaroxaban, warfarin, enoxaparin, or placebo before receiving a subcutaneous injection of MCT 60 mg/kg or saline. Rivaroxaban and enoxaparin were administered for 28 days starting 4 h before MCT injection; warfarin was given for 35 days initiated 7 days before MCT injection. RV haemodynamics and hypertrophy were assessed 28 days after MCT administration. Rivaroxaban dose-dependently reduced systolic and end-diastolic RV pressure increase and RV hypertrophy. Warfarin reduced RV pressure increase only. Enoxaparin had no effect on either parameter. Severe bleeding occurred in four and five rats treated with warfarin and enoxaparin, respectively, whereas no overt bleeding was observed in rats treated with rivaroxaban. Selective, direct inhibition of FXa by rivaroxaban effectively prevented RV dysfunction and hypertrophy in MCT-injected rats, indicating a role for coagulation factors in experimental pulmonary hypertension. Clinical investigation of the impact of early and continued administration of a specific FXa inhibitor such as rivaroxaban on the course of PAH should be considered.

Alterations of TRPC1 Expression and Store‐Operated Ca2+ Entry in Pulmonary Arteries of Monocrotaline‐Induced Pulmonary Hypertensive Rats

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that chronic hypoxia upregulates canonical transient receptor potential (TRPC) genes and enhances store-operated Ca2+ entry (SOCE) in PASMCs. Similar changes have not been examined in monocrotaline (MCT)-induced PH. Here PH was induced in rats by a single intraperitoneal injection of MCT. MCT-injected rats developed severe PH and right ventricular hypertrophy within three weeks. qRT-PCR and western blot showed that the expression of mRNA and protein of the store-operated TRPC1 channel were increased in pulmonary arteries (PAs) of MCT-treated rats. Activation of SOCE with cyclopiazonic acid evoked contraction of isolated PAs and Ca2+ influx in PASMCs of control rats, which were dramatically enhanced in MCT-treated animals. Moreover, endothelin-1 elicited vasoconstriction was potentiated in PAs of MCT-treated rats, and the responses were more susceptible to La3+ (30 μM) inhibition, which blocks SOCE. Our results showed that the alterations of TRPC1 expression and SOCE activity in PAs of MCT-treated rats are similar to chronic hypoxic rats, suggesting that the TRPC1-SOCE pathway might be a pivotal common mechanism in the pathogenesis of PH. (supported by NSFC 30670772, FJMU09ZD010, NSF-Fujian 2010J01173, and R01-HL075134)

Emerging Pathogenetic Mechanisms of Pulmonary Arterial Hypertension: Nitric Oxide and More

Emerging Pathogenetic Mechanisms of Pulmonary Arterial Hypertension: Nitric Oxide and More

Therapeutic efficacy of azaindole-1 in experimental pulmonary hypertension

An accumulating body of evidence incriminates Rho kinase (ROCK) in the pathogenesis of pulmonary hypertension (PH). The therapeutic efficacy of azaindole-1, a novel highly selective and orally active ROCK inhibitor, has not yet been investigated in PH. This study aimed to investigate the effects of azaindole-1 on 1) acute hypoxic pulmonary vasoconstriction (HPV), 2) proliferation of pulmonary arterial smooth muscle cells (PASMCs) and 3) animal models of PH. Azaindole-1 significantly inhibited HPV in isolated, ventilated and buffer-perfused murine lungs and proliferation of primary rat PASMCs in vitro. Azaindole-1 was administered orally from 21 to 35 days after monocrotaline (MCT) injection in rats and hypoxic exposure in mice. Azaindole-1 (10 and 30 mg per kg body weight per day in rats and mice, respectively) significantly improved haemodynamics and right ventricular hypertrophy. Moreover, the medial wall thickness and muscularisation of peripheral pulmonary arteries were ameliorated. Azaindole-1 treatment resulted in a decreased immunoreactivity for phospho-myosin phosphatase target subunit 1 and proliferating cell nuclear antigen in pulmonary vessels of MCT-injected rats, suggesting an impaired ROCK activity and reduced proliferating cells. Azaindole-1 provided therapeutic benefit in experimental PH, and this may be attributable to its potent vasorelaxant and antiproliferative effects. Azaindole-1 may offer a useful approach for treatment of PH.

Combination of sildenafil and simvastatin ameliorates monocrotaline-induced pulmonary hypertension in rats

Sildenafil, a phosphodiesterase-5 inhibitor, and simvastatin, a cholesterol lowering drug, both have therapeutic effects on PAH; however, the combination of these drugs has not been tested in the treatment of PAH. The purpose of this study was to determine whether the combination of sildenafil and simvastatin is superior to each drug alone in the prevention of MCT-induced PAH. Phosphorylated Smad levels were decreased in lung tissue in MCT-injected rats, whereas ERK protein levels were increased. This indicates a possible role for an increase in mitogenic ERK activity in addition to decreased proapoptotic Smad signaling in the MCT model of PAH. Combination sildenafil and simvastatin treatment prevented the MCT-induced increases in right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH), exerted an anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC). Our results indicate that combination therapy with sildenafil and simvastatin attenuated the development of pulmonary hypertension more than either treatment alone.

Resveratrol inhibits right ventricular hypertrophy induced by monocrotaline in rats

1. Resveratrol (RSV), a polyphenol in red wine, exhibits cardioprotective effects in vitro, such as inhibition of angiotensin II- or phenylephrine-induced cardiomyocyte hypertrophy in rat neonatal myocyte cultures and suppression of cardiac fibroblast proliferation. The aim of the present study was to investigate the protective effects of RSV against monocrotaline (MCT)-induced right ventricular (RV) hypertrophy in rats. 2. Male Sprague-Dawley rats were given a single injection of MCT (50 mg/kg, s.c.) and were then treated with either vehicle (normal saline) or RSV (10 and 30 mg/kg, i.g., twice daily) for 21 days. A separate group of control rats were not injected with MCT and were treated with normal saline for 21 days. At the end of the treatment period, all rats were subjected to echocardiography and haemodynamic measurements. In addition, after rats had been killed, the hearts were subjected to histopathological, untrastructural and immunohistochemical analyses. 3. In vehicle-treated rats, MCT injection resulted in 33% mortality, whereas mortality in RSV-treated MCT-injected rats was 0%. In vehicle-treated rats, MCT increased RV free wall thickness and RV systolic pressure and decreased pulmonary arterial acceleration time at the end of the experimental period. These dynamic changes were ameliorated by RSV in a dose-dependent manner. Histologically, MCT injection resulted in RV hypertrophy, swollen mitochrondria and cardiomyocyte apoptosis; all these morphological changes were dose-dependently improved in rats treated with RSV. 4. In conclusion, RSV inhibits the RV hypertrophy induced by MCT in rats and this effect is mediated by both a direct effect of RSV on cardiomyocytes and an indirect effect mediated via a reduction in pulmonary hypertension.

Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension

Alterations of the nitric oxide receptor, soluble guanylate cyclase (sGC) may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). In the present study, the expression of sGC in explanted lung tissue of PAH patients was studied and the effects of the sGC stimulator BAY 63-2521 on enzyme activity, and haemodynamics and vascular remodelling were investigated in two independent animal models of PAH. Strong upregulation of sGC in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was demonstrated by immunohistochemistry. Upregulation of sGC was detected, similarly to humans, in the structurally remodelled smooth muscle layer in chronic hypoxic mouse lungs and lungs from monocrotaline (MCT)-injected rats. BAY 63-2521 is a novel, orally available compound that directly stimulates sGC and sensitises it to its physiological stimulator, nitric oxide. Chronic treatment of hypoxic mice and MCT-injected rats, with fully established PAH, with BAY 63-2521 (10 mg x kg(-1) x day(-1)) partially reversed the PAH, the right heart hypertrophy and the structural remodelling of the lung vasculature. Upregulation of soluble guanylate cyclase in pulmonary arterial smooth muscle cells was noted in human idiopathic pulmonary arterial hypertension lungs and lungs from animal models of pulmonary arterial hypertension. Stimulation of soluble guanylate cyclase reversed right heart hypertrophy and structural lung vascular remodelling. Soluble guanylate cyclase may thus offer a new target for therapeutic intervention in pulmonary arterial hypertension.

Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced pulmonary artery hypertension

Monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) in rats is preceded by an inflammatory response, progressive endothelial cell membrane disruption, reduction in the expression of caveolin-1, and reciprocal activation of STAT3 (PY-STAT3). Superoxide and NF-kappaB have been implicated in PAH. To evaluate the role of caveolin-1, PY-STAT3 activation, and superoxide in PAH, MCT-injected rats were treated daily with pyrrolidine dithiocarbamate (PDTC; starting on days 1, 3, and 14 x 2 wk), an inhibitor of inflammation and NF-kappaB activation. Hemodynamic data, the expression of inhibitory (I)-kappaBalpha, caveolin-1, and Tie2 (a membrane protein), activation of PY-STAT3 and NF-kappaB, and superoxide chemiluminescence were examined. Rats developed progressive PAH at 2 wk post-MCT. There was progressive reduction in the expression of caveolin-1, Tie2, and activation of PY-STAT3 in the lungs. Reduction in I-kappaBalpha expression was present at 2 and 4 wk post-MCT. Superoxide chemiluminescence and NF-kappaB activation were observed only at 2 wk post-MCT and both decreased by 4 wk post-MCT despite progressive PAH. PDTC (starting on days 1 and 3) rescued caveolin-1 and Tie2, reversed MCT-induced PY-STAT3 activation, and attenuated PAH. In addition, PDTC restored I-kappaBalpha expression and reduced superoxide chemiluminescence at 2 wk but did not inhibit NF-kappaB activation despite attenuation of PAH. PDTC had no effect on established PAH. Increased superoxide chemiluminescence and NF-kappaB activation appear to be a transient phenomenon in the MCT model. Thus the disruption of endothelial cell membrane integrity resulting in caveolin-1 loss and reciprocal activation of PY-STAT3 plays a key role in the MCT-induced PAH.

Caveolin‐1 rescue is associated with the inhibition of STAT3 activation and attenuation of pulmonary hypertension

Monocrotaline (MCT)-induced pulmonary hypertension (PH) is associated with a loss of caveolin-1 (caveolar membrane protein) expression and reciprocal activation of STAT3 (PY-STAT3), a proproliferative facotor. In several cell systems, loss of caveolin-1 is associated with the activation of cell proliferative pathways, and reduction in the expression of p21waf1/cip1, a cyclin-dependent kinase that blocks DNA replication. To assess the role of caveolin-1 and PY-STAT3 in PH, MCT-injected rats were treated with molsidomine or pyrrolidine dithiocarbamate (PDTC). Two wks post-MCT, rats exhibited significant PH and RVH. Pulmonary arteries in these rats revealed a significant reduction in the expression of cav-1 and p21waf1/cip1, and activation of PY-STAT3. Molsidomine and PDTC significantly attenuated MCT-induced PH and RVH. This attenuation of PH was associated not only with the preservation of caveolin-1 and p21waf1/cip1, but also inhibition of PY-STAT3 activation. Furthermore, PECAM-1 and Tie2 (caveolar membrane proteins) were also rescued indicating a generalized restoration of endothelial cell membrane integrity. We conclude that the restoration of caveolin-1 led to inhibition of PY-STAT3 activation and an increase in the expression of p21waf1/cip1, resulting in the inhibition of cell proliferation and attenuation of PH. Thus, caveolin-1 and PY-STAT3 play a pivotal role in pulmonary vascular health.

Fetal gene program associated with right ventricular hypertrophy/failure-is left ventricle really spared?

Left ventricular (LV) hypertrophy is usually associated with fetal gene program, but it remains unclear whether it is the case in right ventricular (RV) hypertrophy. Recently, an alkaloid toxin, monocrotaline (MCT), has been established to induce pulmonary hypertension and RV hypertrophy. Objective: To investigate myocyte-specific gene expression associated with RV hypertrophy/failure. Methods: Rats were injected with MCT (50mg/kg) and after 6 weeks levels of mRNA expression were determined. ∗P < 0.05 vs vehicle. Results: Approximately half of MCT-injected rats died after 6 weeks. MCT markedly increased RV weight (289% ∗) with modest increases in LV weight (148% ∗). In MCT-treated RV, the mRNA level of SERCA (34.6% ∗) or alpha-MHC (12.8%∗) was decreased, while that of ANP (1100% ∗), BNP (1100% ∗), skeletal alpha-actin (462% ∗), or beta-MHC (313% ∗) was increased, i.e. fetal gene program activation. Interestingly, not only in septum but in separated LV free wall, MCT also activated significant fetal gene program except SERCA. Conclusion: MCT induced RV hypertrophy and failure, which was accompanied by typical fetal gene program in RV. Although MCT theoretically caused unloading of LV free wall, MCT-treated LV exhibited similar pattern of fetal gene activation. Thus, LV may also be affected in the case of severe pulmonary hypertension. The induction of fetal gene program in LV might be attributable to paracrine effects of neurohormonal factors produced by diseased pulmonary vasculature and/or RV.

Athymic Nude Rats Develop Severe Pulmonary Hypertension following Monocrotaline Administration

Objective: To investigate the significance of the presence or absence of the thymus in the development of pulmonary hypertension (PH) following monocrotaline (MCT) administration, the degree of MCT-induced PH (MCT-PH) in athymic nude (F344/N Jcl-rnu) rats was compared with that in their euthymic (rnu/+) littermates. Methods: Histopathological studies of the lung in terms of interstitial edema, congestion, thickening of the alveolar wall, inflammatory cell infiltration and degeneration of arteries were performed by staining with hematoxylin-eosin (HE), elastin van Gieson and Masson’s trichrome. The medial wall thickness of the small pulmonary arteries and the weight ratio of the right ventricular free wall (RV) to that of the left ventricle plus septum [RV/(LV + S) weight ratio] were used as indices of the degree of PH. Toluidine blue staining was performed to estimate the number of the mast cells in the lung interstitium. Results: Interstitial edema was significantly severer in MCT- injected euthymic rats than in MCT-injected athymic nude rats (p < 0.01); in contrast, the thickening of the alveolar wall was severer in MCT-injected athymic nude rats than in MCT-injected euthymic rats (p < 0.05). The degree of MCT-PH, as determined by the medial wall thickness of the small pulmonary arteries and RV/(LV + S) weight ratio in MCT-injected athymic nude rats, was significantly severer than in MCT-injected euthymic rats (p < 0.05 and p < 0.01, respectively). The number of mast cells was significantly greater in MCT-injected athymic nude rats than in MCT-injected euthymic rats (p < 0.01). The degree of the medial wall thickness of the small pulmonary arteries was significantly correlated with RV/(LV + S) weight ratio (p < 0.05) as well as with the number of mast cells in MCT-injected rats. Conclusions: Athymic nude rats developed severer PH than euthymic rats along with a greater number of mast cells and severer histopathological changes, such as thickening of the alveolar wall. Mast cell proliferation was considered to play a pivotal role in the development of PH in MCT-PH.

Endothelin-1 is elevated in monocrotaline pulmonary hypertension.

These studies document striking pulmonary vasoconstrictor response to nitric oxide synthase (NOS) inhibition in monocrotaline (MCT) pulmonary hypertension in rats. This constriction is caused by elevated endothelin (ET)-1 production acting on ETA receptors. Isolated, red blood cell plus buffer-perfused lungs from rats were studied 3 wk after MCT (60 mg/kg) or saline injection. MCT-injected rats developed pulmonary hypertension, right ventricular hypertrophy, and heightened pulmonary vasoconstriction to ANG II and the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). In MCT-injected lungs, the magnitude of the pulmonary pressor response to NOS inhibition correlated strongly with the extent of pulmonary hypertension. Pretreatment of isolated MCT-injected lungs with combined ETA (BQ-123) plus ETB (BQ-788) antagonists or ETA antagonist alone prevented the L-NMMA-induced constriction. Addition of ETA antagonist reversed established L-NMMA-induced constriction; ETB antagonist did not. ET-1 concentrations were elevated in MCT-injected lung perfusate compared with sham-injected lung perfusate, but ET-1 levels did not differ before and after NOS inhibition. NOS inhibition enhanced hypoxic pulmonary vasoconstriction in both sham- and MCT-injected lungs, but the enhancement was greater in MCT-injected lungs. Results suggest that in MCT pulmonary hypertension, elevated endogenous ET-1 production acting through ETA receptors causes pulmonary vasoconstriction that is normally masked by endogenous NO production.

Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats.

Endothelin-1 (ET-1), a potent vasoactive and mitogenic peptide, has been implicated in the pathogenesis of several forms of pulmonary hypertension. We hypothesized that nonspecific blockade of ET receptors would blunt the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. A single dose of the nonspecific ET blocker bosentan (100 mg/kg) given to intact rats by gavage completely blocked the pulmonary vasoconstrictor actions of Big ET-1 and partially blunted hypoxic pulmonary vasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavaged once daily with bosentan (200 mg/kg) had lower right ventricular (RV) systolic pressure (RVSP), RV-to-body weight (RV/BW) and RV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratios and less percent medial thickness of small pulmonary arteries than control MCT-injected rats. Lower dose bosentan (100 mg/kg) had no effect on these parameters after MCT or saline injection. Bosentan raised plasma ET-1 levels but had no effect on lung ET-1 levels. Bosentan (200 mg/kg) also had no effect on wet-to-dry lung weight ratios 6 days after MCT injection. When given during the last 10 days, but not the first 11 days of a 3-wk period after MCT injection, bosentan reduced RV/(LV+S) compared with MCT-injected controls. We conclude that ET-1 contributes to the pathogenesis of MCT-induced pulmonary hypertension and acts mainly during the later inflammatory rather than the acute injury phase after injection.