Monocrotaline Pulmonary Hypertension Research Articles

The EPOR/CD131 heteroreceptoragonist has an endothelioprotective effect against the background of pulmonary hypertension caused by monocrotalin

Introduction: The abnormal increase in pulmonary pressure observed in pulmonary arterial hypertension (PAH) is a consequence of increased pulmonary vascular resistance due to progressive loss and obliteration of pulmonary arteries. The initial trigger is a combination of factors that lead to endothelial damage and impaired vascular regeneration. Aim: research the possibilities of pharmacological correction of pulmonary arterial hypertension induced by monocrotalin using the EPOR/CD131 heteroreceptor agonist with the laboratory code EP-11-3.
 Materials and Methods: The study of pharmacological activity on a model of monocrotaline induced PAH was carried out on male Sprague-Dawley rats weighing 180-220 grams. Monocrotaline (MCT) pulmonary hypertension was simulated in 30 animals using subcutaneous injection of MCT at a dose of 60 mg/kg. Seven days after the injection of MCT, the administration of the studied compounds began. The erythropoietin derivative with the laboratory code EP-11-3 and pHBSP administered subcutaneously at a dose of 25 mcg/kg once every 3 days for 21 days.
 Results: On the model of monocrotalin-induced PAH, it was shown that the erythropoietin derivative with the laboratory code EP-11-3 has a pronounced endothelioprotective effect, reducing the coefficient of endothelial dysfunction, statistically significantly increasing the expression of VEGF-R2 mRNA and reducing the expression of SDF-1 mRNA, reducing the concentrations of CT-1 and PNP, and reducing the signs of remodeling of the heart and pulmonary vessels.
 Conclusion: Erythropoietin derivative with laboratory code EP-11-3 has an endothelioprotective effect and reduces the manifestations of vascular remodeling in pulmonary hypertension caused by monocrotalin.

17β-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17β-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-β-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Combined use of arginase II inhibitors and tadalafil for the correction of monocrotaline pulmonary hypertension

Introduction: The concept of the regulatory role of endothelium in the pathogenesis of pulmonary hypertension (PH) is fundamental. Research objective: To study the protective effects of the selective arginase II inhibitors L207-0525 and L327-0346 in combination with tadalafil in a monocrotaline model of pulmonary hypertension in rats.
 Materials and methods: Monocrotaline-induced pulmonary hypertension was simulated in 10 animals by a subcutaneous injection of an alcohol-water solution of monocrotaline (MCT) in the dose of 60 mg/kg. Seven days after the injection of MCT, the administration of L207-0525 and L327-0346 in the doses of 1 mg/kg and 3 mg/kg was started. The compounds were administered intragastrically once a day for 21 days.
 Results and discussion: It was found that L207-0525 and L327-0346 in the dose of 3 mg/kg and tadalafil in the dose of 1 mg/kg prevented the development of pulmonary hypertension, which was expressed in a statistically significant decrease in the coefficient of endothelial dysfunction (CED, prevention of an increase in systolic pressure in the right ventricle, as well as Fulton, RV/BW and WT indices. The greatest activity was shown by L207-0525 and L327-0346 in the dose of 3 mg/kg in combination with tadalafil in the dose of 0.1 mg/kg.
 Conclusions: The received results suggest the dose-dependent protective activity of selective arginase II inhibitors L207-0525 and L327-0346 and the development of the additive effect of their combined use with low doses of PDE-5 inhibitor tadalafil in relation to the development of monocrotaline pulmonary hypertension

STUDY OF THE PHARMACOLOGICAL ACTIVITY OF LOWMOLECULAR ARGINASE II SELECTIVE INHIBITOR USING A MODEL OF MONOCROTALINE-INDUCED PULMONARY HYPERTENSION

The aimof the present work was to study the pharmacological activity of a low-molecular arginase II selective inhibitor (low-molecular compound with laboratory code ZB49) with the chemical formula 2-{1-[3-(3-[chloroisoxazole-5-yl) propyl] piperidin-4-yl}-6-(digibroxyboryl) norleucine dihydrochloride) using a model of monocrotaline-induced pulmonary hypertensionMaterials and methods.The work was performed on 40 adult male Wistar rats weighing 180– 220 g. The simulation of monocrotaline pulmonary hypertension was performed on 20 animals using a subcutaneous injection of an alcohol-aqueous solution of monocrotaline (MCT) at a dose of 60 mg/kg in a volume of 0.5 ml per animal. 7 days after the MCT injection, the administration of the studied pharmaceutical substance (PS) (laboratory code ZB49) was started at a dose of 5 mg/kg. ZB49 was administered intragastrically, once a day with the administration duration of 21 days.Results.Under the intragastric FS ZB49 administration at a dose of 5 mg/kg, a statistically significant decrease in the coefficient of endothelial dysfunction, systolic right ventricle pressure (SRVP), average the right ventricle pressure (ARVP), diastolic right ventricle pressure (DRVP), maximum contraction rate (dP/dt max) and minimum contraction rate (dP/dt min) was established at the background of the simulation of monocrotaline-induced pulmonary hypertension. At the same time, a statistically significant positive effect of ZB49 on the blood gas composition was established.Conclusion.The study has confirmed the possibility of using arginase inhibitors to prevent the development of endothelial dysfunction and the disorders of nitric oxide metabolism in pulmonary arterial hypertension. Among the substances of this group, arginase II selective inhibitors should be considered as the most promising.Conflict of interest: the authors declare no conflict of interest.

Endothelial Progenitor Cells Are the Bone Marrow Cell Population in Mice with Monocrotaline-Induced Pulmonary Hypertension Which Induce Pulmonary Hypertension in Healthy Mice

Rationale: We have demonstrated that whole, undifferentiated bone marrow (WBM) cells isolated from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) cause pulmonary hypertensive changes when transplanted into lethally-irradiated mice (Aliotta et al., 2014 ATS abstract). In addition, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs), sub-cellular particles shed by activated cells, can reverse features of MCT-induced PH (Aliotta et al., 2015 ATS abstract). Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis of human PH as more have been noted to be present in the circulation and remodeled pulmonary vasculature of these patients compared with healthy humans. We wished to determine if bone marrow-derived EPCs isolated from mice with MCT PH were the cells responsible for inducing PH in healthy mice and if so, whether exposure of these EPCs to MSC EVs prevented the development of PH.Methods: Sca-1+/ckit+/flk1+ cells (EPCs) and sca-1-/ckit-/flk1- cells (non-EPCs) were isolated from the bone marrow of MCT-injured and vehicle-injected mice. Cohorts of lethally-irradiated mice were transplanted with EPCs (2,000 EPCs + 300,000 helper WBM cells/recipient) or non-EPCs (200,000 non-EPCs + 200,000 helper WBM cells/recipient) from the bone marrow of MCT-injured and vehicle-injected mice. 28 days after transplantation, recipient mouse right ventricular (RV) hypertrophy was assessed by RV-to-left ventricle+septum (RV/LV+S) ratio (mg/g) and pulmonary vascular remodeling by blood vessel wall thickness-to-diameter (WT/D) ratio. In addition, WBM from MCT-injured and vehicle-injected mice was cultured with or without MSC EVs for 48 hours. EPCs (80-100 EPCs + 300,000 helper WBM cells/recipient) and non-EPCs (500,000-700,000 non- EPCs + 300,000 helper WBM cells/recipient) were separated by flow cytometry and infused into lethally-irradiated mice which were analyzed 28 days post-transplant.Results: RV/LV+S ratios of mice transplanted with EPCs from MCT-injured mice (0.256+/-0.142 mg/g) were similar to those of MCT-injured mice (0.274+/-0.211 mg/g, p=NS, n=5/cohort) but elevated compared to mice transplanted with EPCs and non-EPCs from vehicle-injected mice (0.126+/-0.111 and 0.121+/-0.134 mg/g, p<0.05, n=5/cohort) and mice transplanted with non-EPCs from MCT-injured mice (0.139+/-0.218 mg/g, n=5/cohort) . WT/D ratios of mice transplanted with EPCs from MCT-injured mice (0.145+/-0.122) were similar to those of MCT-injured mice (0.156+/-0.232, p=NS, n=5/cohort) but elevated compared to mice transplanted with EPCs and non-EPCs from vehicle-injected mice (0.076+/-0.008 and 0.072+/-0.009, p<0.05, n=5/cohort) and mice transplanted with non-EPCs from MCT-injured mice (0.081+/-0.012, n=5/cohort). Mice transplanted with EPCs from MCT-injured mice that were treated with MSC EVs prior to transplantation had significantly lower RV/LV+S and WT/D ratios compared with mice transplanted with EPCs from MCT-injured mice that were not treated with MSC EVs prior to transplantation (0.119+/-0.164 mg/g, 0.072+/-0.008 vs. 0.188+/-0.201 mg/g, 0.122+/-0.011 p<0.05, n=5/cohort).Conclusions: These findings suggest that EPCs are among the pathogenic bone marrow cells in MCT-injured mice that are responsible for inducing RV hypertrophy and pulmonary vascular remodeling, key features of PH, upon transplantation into healthy mice. MSC EVs appear to be capable of reversing this disease via interactions with EPCs. DisclosuresNo relevant conflicts of interest to declare.

Sex differences in stretch-dependent effects on tension and Ca(2+) transient of rat trabeculae in monocrotaline pulmonary hypertension.

We aim to compare the effects of stretch on isometric tension/Ca(2+) transient in the right ventricular trabeculae of control (CONT) and hypertensive (MCT, monocrotaline application) adult male and female rats. The treatment with MCT resulted in RV hypertrophy in males only. Blunted active force-length relation and substantially prolonged twitch were found in MCT-males but not MCT-females (vs same-sex CONT). Ca(2+) transient was prolonged in both MCT-treated groups but extremely so in the MCT-males. The gradual stretch resulted in a distinct "bump" on Ca(2+) transient decline in CONT and MCT-treated groups. The integral magnitude of the "bump" was unaffected by the treatment with MCT in males or females but was larger in males vs females. The rate of "bump" development was significantly slower in MCT-males. In conclusion, the sex-specific differences in the stretch-dependent regulation of [Ca(2+)] i may underlie preservation of the Frank-Starling mechanism in female rat myocardium in monocrotaline-induced pulmonary hypertension.

Imatinib attenuates monocrotaline pulmonary hypertension and has potent vasodilator activity in pulmonary and systemic vascular beds in the rat

Cardiovascular responses to the tyrosine kinase inhibitor imatinib were investigated in the rat. Intravenous injections of 0.3-30 mg/kg imatinib produced small decreases in pulmonary arterial pressure, larger dose-dependent decreases in systemic arterial pressure, and no change or small increases in cardiac output, suggesting that the systemic vasodilator response is more pronounced under baseline conditions. When pulmonary arterial pressure was increased with U-46619 or N(ω)-nitro-L-arginine methyl ester (L-NAME), intravenous injections of imatinib produced larger dose-dependent decreases in pulmonary arterial pressure. Imatinib attenuated the acute hypoxic pulmonary vasoconstrictor response. Vasodilator responses to imatinib were not inhibited by meclofenamate, glybenclamide, or rolipram, suggesting that cyclooxygenase, ATP-sensitive K(+) (KATP) channels, and cAMP were not involved in mediating the response. In a 21-day prevention study, imatinib treatment (50 mg/kg ip) attenuated the increase in pulmonary arterial pressure, right ventricular hypertrophy, and small vessel remodeling induced by monocrotaline. Imatinib reduced PDGF receptor phosphorylation and PDGF-stimulated thymidine incorporation in rat pulmonary artery smooth muscle cells. These data suggest that the beneficial effect of imatinib in pulmonary hypertension may involve inhibition of PDGF tyrosine kinase receptor-mediated effects on smooth muscle cell proliferation and on vasoconstrictor tone. These results indicate that imatinib has nonselective vasodilator activity in the pulmonary and systemic vascular beds similar to the Rho kinase inhibitor fasudil and the calcium entry antagonist isradipine. The present results are consistent with the hypothesis that imatinib may inhibit a constitutively active tyrosine kinase vasoconstrictor pathway in the pulmonary and systemic vascular beds in the rat.

Reversibility of the monocrotaline pulmonary hypertension rat model

To the Editor : Pulmonary hypertension (PH) is a disease characterised by progressive remodelling of the pulmonary vasculature eventually leading to right heart failure. Various animal models have been used to mimic the disease, involving pigs, dogs, rats and mice [1]. The most commonly used model is the monocrotaline (MCT) rat model. In this model MCT is injected subcutaneously and becomes metabolically activated, as a pyrrolizidine alkaloid, by hepatic cytochrome P450 3A [2, 3]. The active MCT pyrrole is pneumotoxic and damages the pulmonary artery endothelial cells (PAECs), which leads to a disturbed barrier function [4]. Other features of MCT-induced pulmonary vascular remodelling are arterial medial hyperplasia of axial arteries, interstitial oedema, adventitial inflammation, haemorrhage and, eventually, fibrosis [1, 2, 5, 6]. As a result, pulmonary vascular resistance (PVR) increases and the right ventricle compensates by hypertrophy and eventually fails [7, 8]. Besides the MCT PH rat model, chronic hypoxia with or without Sugen 5416 and pulmonary artery banding are used to study experimental PH [1]. The PH animal model of choice is mostly dependent on the research question that needs to be answered. Ideally, an animal model would recapitulate the progressive and irreversible pulmonary vascular remodelling, which is the hallmark of human PH [1, 4, 9]. However, none of the animal models fulfil this criterion. Concerns have been raised about the MCT rat model since many therapies were successful in MCT rats but not in humans with PH [4]. We, therefore, investigated the long-term progression and reversibility of MCT-induced PH in rats over 12 weeks, using a dose of 40 mg·kg−1 in a randomised placebo-controlled study design. Since it is known that a high dose …

The Extracellular Signal‐Regulated Kinase Is Involved in the Effects of Sildenafil on Pulmonary Vascular Remodeling

Pulmonary hypertension is a group of diseases comprising vascular constriction and obstructive changes of the pulmonary vasculature. Phosphodiesterase type 5 inhibitors, for example, sildenafil, can alleviate vascular remodeling in the monocrotaline pulmonary hypertension model in rats. We investigate the mechanisms of sildenafil on the pulmonary vascular remodeling of pulmonary hypertension induced by monocrotaline (MCT) in rats. Thirty Sprague-Dawley rats (weighing 200-220 g) were administered with MCT abdominal cavity injection or equivalent volume of normal saline (NS) (which were treated as C group n = 10) to induce pulmonary hypertension model. Fourteen days later, 20 MCT treated rats were randomly fed with sildenafil (25mg/kg/day) or placebo as S, P group (10 rats for each group), respectively. Another 6 weeks later, mean pulmonary artery pressure (mPAP), index of right ventricular hypertrophy (RV/LV+S) of all animals were measured under general anesthesia. Pulmonary tissue was collected to investigate pathological features of pulmonary arteries and to measure protein expression of ERK(1)/ERK(2) and MKP1. After 6 weeks, there were significant elevated mPAP and RV/LV+S in both P and S groups. The ratio of wall thickness to vessel diameter in pulmonary arteries with diameters <200 microm were increased in both P and S groups. But the ratio of wall thickness to vessel diameter was smaller in S group than that in P group. The phosphorylation level of ERK(1)/ERK(2) were elevated in both P and S groups, but the level of phosphorlation ERK(1)/ERK(2) were lower in S group than that in P group. Intriguingly, the expression level of MKP1 was significantly increased in both S and P groups, while it was higher in S group than that in P group. The sildenafil can decrease mPAP and inhibit the progress of pulmonary vascular remodeling in pulmonary hypertension rats. The ERK-MAP kinase signaling pathway might play a role during this process.

Calcineurin/NFAT is a common pathway in rat monocrotaline‐ and hypoxia‐induced pulmonary hypertension

The nuclear factors of activated T-cells (NFATs) are calcium sensitive transcription factors regulated by calcineurin. NFATs play critical roles in inflammation and cell proliferation, which are important processes in pulmonary hypertension (PAH) pathogenesis. We hypothesize that NFATs might be the common pathway through which PAH develops. To investigate this hypothesis, we treated monocrotaline (MCT) and hypoxia-induced PAH models with cyclosporine (CsA), a specific calcinurin/NFAT pathway inhibitor, at a dosage of 20 mg/kg/day for 24 days. As a result, right ventricular systolic pressure (RVSP) was suppressed by CsA treatment to 40.5 ± 2.8 mmHg which is otherwise increased to 72.6 ± 4.9 mmHg (p<0.01) in MCT PAH model, compared with CsA treated normal control rat of 24.6 ± 1.7 mmHg. Moreover, right ventricular hypertrophy as reflected by the right ventricle to left ventricle plus septal weight ratio (RV/LV+S) decreased from 0.65 ± 0.04 to 0.38 ± 0.03 (p<0.01) whereas it was 0.28 ± 0.004 in control CsA treated rat. RVSP in hypoxia induced PAH rat was similarly decreased from 51.6% ± 4.3 to 35.1% ± 1.7 (p<0.01) and the RV/LV+S ratio from 0.48 ± 0.01 to 0.35 ± 0.007 (p<0.01) after CsA treatment. Considering the different mechanisms between the MCT- and hypoxia-induced PAH models, we suggest that calcinurin/NFAT pathway is a pivotal common pathway in PAH development.

Smad Signaling in the Rat Model of Monocrotaline Pulmonary Hypertension

Mutations in the bone morphogenetic protein receptor type II (BMPrII) gene have been implicated in the development of familial pulmonary artery hypertension (PAH). The function of BMP signal transduction within the pulmonary vasculature and the role BMPrII mutations have in the development of PAH are incompletely understood. We used the monocrotaline (MCT) model of PAH to examine alterations in Smad signal transduction pathways in vivo. Lungs harvested from Sprague-Dawley rats treated with a single 60-mg/kg intraperitoneal (IP) injection of MCT were compared to saline-treated controls 2 weeks following treatment. Smad 4 was localized by immunohistochemistry to endothelial nuclei of the intra-acinar vessels undergoing remodeling. Smad 4, common to both BMP and transforming growth factor beta (TGFbeta) signaling, and BMP-specific Smad 1 were significantly decreased in western blot from whole lungs of treated animals, while no change was found for TGFbeta-specific Smad 2. MCT-treated rats also had increased expression of phosphorylated Smad 1 (P-Smad 1) but not phosphorylated Smad 2 (P-Smad 2). There was a decrease in the expression of the full BMPrII protein but not its short form variant in MCT-treated rat lungs. The type I receptor Alk1 had increased expression. Collectively, our data indicate that vascular remodeling in the MCT model is associated with alterations in BMP receptors and persistent endothelial Smad 1 signaling.

The Antiproliferative Effect of Sildenafil on Pulmonary Artery Smooth Muscle Cells Is Mediated via Upregulation of Mitogen-Activated Protein Kinase Phosphatase-1 and Degradation of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation

Pulmonary hypertension is a group of diseases comprising vascular constriction and by obstructive changes of the pulmonary vasculature. Phosphodiesterase type 5 inhibitors, e.g., sildenafil, can alleviate vascular remodeling in the monocrotaline pulmonary hypertension model in rats, and inhibit the proliferation of pulmonary vascular smooth muscle cells in vitro. We examined the ability of sildenafil to inhibit platelet-derived growth factor (PDGF)-induced proliferation of porcine pulmonary artery smooth muscle cells. Pulmonary artery smooth muscle cell proliferation and cell cycle analysis were assessed by MTT assay and fluorescence-activated cell sorting. Western blotting was used to examine protein expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and phosphorylation level of extracellular signal-regulated kinase (ERK1/2). PDGF increased cell proliferation and the percentage of cells in S phase. These effects were inhibited by pretreatment with sildenafil in a dose-dependent manner. Sildenafil (96 microM) also caused a 67% decrease in PDGF-stimulated ERK1/2 phosphorylation. Sildenafil inhibition of ERK1/2 was accompanied by a rapid induction of MKP-1. Inhibition of the cGMP-dependent kinase I alpha (cGK I alpha) using Rp-8-BrcGMPS (25 microM) blocked sildenafil-induced MKP-1 expression. Either vanadate (12.5 microM), a phosphatase inhibitor, or Rp-8-BrcGMPS abolished the inhibitory effect of sildenafil on PDGF-stimulated phosphorylation of ERK1/2 and restored PDGF-induced cell proliferation. This study indicates that sildenafil upregulates MKP-1 expression and promotes degradation of phosphorylation of ERK1/2, which suppresses the proliferation of pulmonary artery smooth muscle cells.

Statin therapy, alone or with rapamycin, does not reverse monocrotaline pulmonary arterial hypertension: the rapamcyin-atorvastatin-simvastatin study

Pulmonary arterial hypertension (PAH) is characterized by excessive pulmonary artery smooth muscle cell proliferation and impaired apoptosis leading to obstruction of resistance pulmonary arteries. We hypothesized that antiproliferative (rapamycin) and proapoptotic (statins) agents, already used clinically for other indications, would decrease experimental PAH, facilitating translation to human therapies. Prior studies in the rat monocrotaline-PAH model have indicated that simvastatin regresses and rapamycin prevents, but cannot reverse, PAH. Two PAH regression strategies (rapamycin monotherapy vs. rapamycin + atorvastatin) and one prevention strategy (simvastatin) were tested in a rat monocrotaline-PAH model. Adult male Sprague-Dawley rats were randomized to saline (n = 6) or monocrotaline (60 mg/kg ip, n = 36) treatment groups. Monocrotaline rats were randomized to gavage with vehicle, rapamycin (2.5 mgxkg(-1)xday(-1)), or rapamycin + atorvastatin (10 mgxkg(-1)xday(-1)) treatment groups, beginning 12 days post-monocrotaline. Echocardiographic and hemodynamic end points were assessed 2 wk later. Additional monocrotaline-PAH rats (n = 20) were randomized to vehicle or simvastatin (2 mgxkg(-1)xday(-1)) treatment groups and followed echocardiographically for 4 wk. Monocrotaline-PAH increased lung p70 S6 kinase phosphorylation, and this was reversed by rapamycin, confirming the biological activity of rapamycin. Despite the use of high doses, neither rapamcyin nor rapamycin + atorvastatin improved survival nor reduced PAH, vascular remodeling, and right ventricular hypertrophy. Although prophylactic simvastatin slowed PAH progression, by 4 wk PAH severity and mortality were not different from placebo. Apart from the new finding of p70 S6 kinase phosphorylation in monocrotaline-PAH, this is a negative therapeutic trial (none of these promising therapies improved monocrotaline-PAH). These negative results should be considered as human trials with these agents are underway (simvastatin) or proposed (rapamycin).

Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares l-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.

Overexpression of human bone morphogenetic protein receptor 2 does not ameliorate monocrotaline pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is associated with mutations of bone morphogenetic protein receptor 2 (BMPR2), and BMPR2 expression decreases with the development of experimental PAH. Decreased BMPR2 expression and impaired intracellular BMP signaling in pulmonary artery (PA) smooth muscle cells (PASMC) suppresses apoptosis and promotes proliferation, thereby contributing to the pathogenesis of PAH. We hypothesized that overexpression of BMPR2 in resistance PAs would ameliorate established monocrotaline PAH. Human BMPR2 was inserted into a serotype 5 adenovirus with a green fluorescent protein (GFP) reporter. Dose-dependent transgene expression was confirmed in PASMC using fluorescence microscopy, quantitative RT-PCR, and immunoblots. PAH was induced by injecting Sprague-Dawley rats with monocrotaline (60 mg/kg ip) or saline. On day 14, post-monocrotaline (MCT) rats received 5 x 10(9) plaque-forming units of either Ad-human BMPR2 (Ad-hBMPR2) or Ad-GFP. Transgene expression was confirmed by fluorescence microscopy, quantitative RT-PCR of whole lung samples, and laser-capture microdissected resistance PAs. Invasive hemodynamic and echocardiographic end points of pulmonary hypertension were assessed on day 24. Endogenous BMPR2 mRNA levels were greatest in resistance PAs, and expression declined with MCT PAH. Despite robust hBMPR2 expression in all lung lobes and within resistance PAs of treated rats, hBMPR2 did not lower mean PA pressure, pulmonary vascular resistance index, right ventricular hypertrophy, or remodeling of resistance PAs. Nebulized intratracheal adenoviral gene therapy with hBMPR2 reliably distributed hBMPR2 to resistance PAs but did not ameliorate PAH. Depressed BMPR2 expression may be a marker of PAH but is not central to the pathogenesis of this model of PAH.

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.

EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension.

Nitric oxide (NO) and endothelin (ET) have been implicated in the pathogenesis of pulmonary hypertension (PH). Chronic ETA antagonist therapy reduces PH in monocrotaline (MCT)-treated rats. Interactions between the L-arginine-NO pathway and the ET system have been described. We therefore studied the effect of long-term treatment with an oral ETA antagonist (LU 135252) on NO-related vasodilation in isolated lungs from control rats and rats with MCT-induced PH. Three weeks after MCT injection, PH was associated with an increase in right ventricular pressure (from 27.4 +/- 0.9 to 66.6 +/- 4.1 mm Hg) and a decrease in endothelium-independent vasodilation in response to sodium nitroprusside (10(-10) to 10(-5) mol/L; delta Emax, from 11.1 +/- 0.9 to 2.7 +/- 0.3 mm Hg). Endothelium-dependent vasodilation in response to acetylcholine (10(-9) to 10(-4) mol/L) and the calcium ionophore A23187 (10(-9) to 10(-7) mol/L) remained unaffected. Treatment with LU 135252 did not significantly affect the endothelium-dependent and -independent vasodilations in control rats. However, in MCT-treated rats, LU 135252 therapy significantly reduced right ventricular pressure (39.7 +/- 2.1 mm Hg), potentiated acetylcholine-induced vasodilatation (delta Emax, from 1.6 +/- 0.2 to 3.7 +/- 0.4 mm Hg), and improved the responses to sodium nitroprusside (delta Emax, from 2.7 +/- 0.3 to 5.6 +/- 0.6 mm Hg). LU 135252 did not significantly alter the non-receptor-mediated endothelium-dependent vasodilation to A23187 or pulmonary constitutive NO synthase activity. MCT PH is associated with a reduced smooth muscle responsiveness to NO but a maintained endothelium-dependent vasodilatory potency. Long-term ETA antagonist therapy not only restores smooth muscle responsiveness to NO but also increases endothelium-dependent dilation in response to acetylcholine. This mechanism may contribute to the therapeutic benefit of ETA antagonists in PH.

Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats

1. We studied the effects of oestradiol 17 beta on the development of pulmonary vascular changes and right ventricular (RV) hypertrophy in response to monocrotaline in male Sprague-Dawley rats. 2. Rats were treated with either placebo or oestradiol 17 beta (10 mg) in the form of slow release pellets implanted subcutaneously 48 h before monocrotaline administration. Rats were injected with either saline or a single dose of monocrotaline (60 mg kg-1, i.m.). Pulmonary vascular changes and RV hypertrophy were studied at 4 weeks following monocrotaline administration. 3. Monocrotaline induced a significant increase in the ratio of right ventricle (RV) to left ventricle-plus-septum (LV + S) weights. Monocrotaline-treated rats also showed significant myointimal proliferation in small pulmonary arteries, decrease of arterial numbers and increase in the number of abnormal alveolar macrophages. 4. Oestradiol 17 beta attenuated myointimal hyperplasia in pulmonary vessels, decreased the RV/(LV + S) ratio in monocrotaline-treated rats. Oestradiol 17 beta had no significant effect on control animals. 5. Oestradiol treatment prevented the increase in lung wet to dry weight ratio, observed 7 days post monocrotaline administration. 6. These results suggest that oestradiol 17 beta protects against the pulmonary vascular remodelling and RV hypertrophy associated with monocrotaline-induced pulmonary hypertension in the rat. Oestradiol also protects against microvascular leak observed in the early days of lesion.

Inhibition of elastolysis by SC-37698 reduces development and progression of monocrotaline pulmonary hypertension

Our previous studies showed that increased pulmonary artery elastolytic activity is associated with monocrotaline-induced pulmonary hypertension in rats, and the latter is reduced by the elastase inhibitor SC-39026. This agent, given orally, decreases monocrotaline-induced muscularization of normally nonmuscular peripheral arteries but not medial hypertrophy of muscular arteries. To establish whether constant infusion of an elastase inhibitor would reduce both vascular lesions induced by monocrotaline injection, SC-37698 (an analogue of SC-39026) was given intravenously by osmopump. To separately assess whether SC-37698 would inhibit development of the vascular changes as well as their progression, SC-37698 or vehicle was infused for the first 2 wk (2-wk study) or was delayed until 1 wk after monocrotaline injection (3-wk study). Hemodynamic data were recorded from indwelling catheters, and the lungs were evaluated morphologically. Saline-injected control rats given SC-37698 or vehicle were similar at both time points. Monocrotaline-injected rats given SC-37698 compared with those given vehicle alone had lower pulmonary artery pressures, 17.9 +/- 0.5 vs. 23.7 +/- 0.8 mmHg (P less than 0.01) in the 2-wk study and 24.0 +/- 1.8 vs. 33.5 +/- 3.1 mmHg (P less than 0.05) in the 3-wk study. This was associated with significant decreases in muscularization of peripheral arteries and reductions in medial hypertrophy of muscular arteries. In the hilar pulmonary arteries assessed at 3 wk only, SC-37698 significantly decreased monocrotaline-induced endothelial injury, subendothelial edema, migration of smooth muscle cells into subendothelium, medial hypertrophy, collagen accumulation, and abnormal distribution of elastin as interlamellar islands. Pulmonary artery elastolytic activity was reduced in SC-37698-treated compared with untreated monocrotaline-injected rats (P less than 0.05). Thus infusion of SC-37698 reduces monocrotaline-induced pulmonary hypertension when administered before or even after development of early vascular changes.

Monocrotaline-induced cardiopulmonary injury in rats: Modification by the neutrophil elastase inhibitor SC39026

Rats were killed after 6 weeks of continuous ingestion of the pneumotoxic alkaloid monocrotaline (2.2mg/kg/day), the neutrophil elastase inhibitor SC39026 (60mg/kg/day), or both. Pulmonary reactions were evaluated by light and electron microscopy. Lung endothelial function was monitored by angiotensin converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI 2) and thromboxane (TXA 2) production. Lung hydroxyproline content was measured as an index of interstitial fibrosis. Cardiac right ventricular hypertrophy was determined by the right ventricle to the left ventricle plus septum weight ratio (RV/LV + S). Rats receiving SC39026 alone did not differ significantly from untreated control animals with respect to any of the quantitative endpoints, although rarefaction of Type I pneumocytes was observed in the electron micrographs of these animals. Monocrotaline-treated rats, in contrast, developed a significant increase in RV/LV + S, and exhibited pulmonary edema, inflammation, fibrosis, and muscularization and occlusive mural thickening of the pulmonary small arteries and arterioles. These monocrotaline-induced structural changes were accompanied by decreased lung ACE and PLA activities, and increased PGI 2 and TXA 2 production, and by an increase in lung hydroxyproline content. Cotreatment with SC39026 ameliorated the monocrotaline-induced pulmonary vascular wall thickening and the cardiac right ventricular hypertrophy. These data suggest that inappropriate neutrophil elastase activity contributes to monocrotaline pulmonary vasculopathy and hypertension. On the other hand, cotreatment with SC39026 had no significant effect on the severity of the monocrotaline-induced lung inflammatory reaction, the pulmonary endothelial dysfunction, or the increase in lung hydroxyproline content.