Aspirin Attenuates Pulmonary Arterial Hypertension in Rats by Reducing Plasma 5-Hydroxytryptamine Levels

Pulmonary arterial hypertension (PAH) is characterized by increasing pulmonary pressure, right ventricular failure, and death. The typical pathological changes include medial hypertrophy, intimal fibrosis and in situ thrombosis. Serotonin (5-HT) and other factors contribute to the development of pathologic lesions. Aspirin (ASA), a platelet aggregation inhibitor, inhibits 5-HT release from platelets. The aim of this study was to determine the efficacy of ASA in preventing or attenuating PAH. Sprague–Dawley rats injected with monocrotaline (MCT) developed severe PAH within 31 days. One hundred forty rats were randomized to receive either vehicle or ASA (0.5, 1, 2, or 4 mg/kg/day). The pre-ASA group was treated with ASA (1 mg/kg/day) for 30 days before the MCT injection. Thirty-one days after the injection (day 61 for the pre-ASA group), pulmonary arterial pressure (PAP), right ventricular hypertrophy and pulmonary arteriole thickness were measured. Plasma 5-HT was measured by high-performance liquid chromatography. Aspirin suppressed PAH and increased the survival rate compared with the control group (84 vs. 60%, P < 0.05). Aspirin treatment also reduced right ventricular hypertrophy and pulmonary arteriole proliferation in ASA-treated PAH model. In addition, plasma 5-HT was decreased in our ASA-treated PAH model. The degree of 5-HT reduction was associated with systolic PAP, right ventricular hypertrophy and wall thickness of pulmonary arterioles in rats. These results showed that ASA treatment effectively attenuated MCT-induced pulmonary hypertension, right ventricular hypertrophy, and occlusion of the pulmonary arteries. The effects of ASA was associated with a reduction of 5-HT.     

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.     

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: 60 mg/kg) to rats. Omentin (18 μg/kg/day) was intraperitoneally treated for 14 days. 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.     

脂肪干细胞移植对野百合碱诱发的肺动脉高压大鼠肺动脉压的量效和时效作用

目的探索脂肪干细胞（ADSC）移植治疗野百合碱（MCT）诱导的肺动脉高压（PAH）大鼠的适宜细胞数和干预时间。 方法（1）MCT的建模时效和量效：雄性SD大鼠48只分为正常对照组,20 mg/kg、30 mg/kg、40 mg/kg MCT组分别予腹腔注射生理盐水、MCT 20 mg/kg、30 mg/kg、40 mg/kg,4和8周后,右心室插管法检测平均肺动脉压（mPAP）,称重法计算右心室肥厚指数（RVHI）。（2）ADSC的治疗量效作用：雄性SD大鼠分别予腹腔注射MCT（30只）和生理盐水（30只）,1周后通过颈静脉注射分别移植0.5×10⁶、1.0×10⁶、3.0×10⁶、5.0×10⁶ADSC,其他组予等量生理盐水。移植3周后检测mPAP和RVHI。（3）ADSC的治疗时效作用：雄性SD大鼠30只,分别注射40 mg/kg MCT（24只）和生理盐水（6只）。MCT腹腔注射1 d,1、2周后分别移植1.0×10⁶个ADSC。MCT注射4周后检测mPAP和RVHI。多组间比较采用单因素或双因素方差分析,两两比较采用LSD检验。 结果（1）腹腔注射4周后,30 mg/ kg或40 mg/kg MCT组mPAP和RVHI均升高[mPAP值（24.89±3.31）mmHg,（27.19±2.11）mmHg比（15.80±0.42）mmHg,差异有统计学意义（P均< 0.05）;RVHI值0.42±0.06,0.47±0.04比0.25±0.02,差异有统计学意义（P均< 0.05）]。8周后,20 mg/kg或30 mg/ kg MCT组mPAP和RVHI均恢复正常,而40 mg/kg MCT组大鼠全部死亡。（2）40 mg/ kg MCT诱导的PAH大鼠mPAP和RVHI均升高。移植1.0×10⁶个ADSC可降低PAH大鼠的mPAP[（17.24±0.66）mmHg比（27.19±1.73）mmHg,P < 0.05]。移植0.5×10⁶、3.0×10⁶、5.0× 10⁶个ADSC不能降低PAH大鼠的mPAP和RVHI。（3）MCT腹腔注射1周和2周后,移植1.0×10⁶个ADSC可降低PAH大鼠的mPAP。 结论40 mg/kg MCT造模4周可建立稳定的PAH大鼠模型;造模1或2周后移植1.0×10⁶个ADSC能有效降低PAH大鼠的mPAP。     

Capsaicin pre- and post-treatment on rat monocrotaline pneumotoxicity

Monocrotaline (MCT) produces respiratory dysfunction, pulmonary hypertension (PH), and right ventricular hypertrophy (RVH) in rats. Tachykinins, such as substance P (SP) and neurokinin A (NKA), may mediate these effects. The purpose of this study was to investigate the length of tachykinin depletion (via capsaicin treatment) is needed to prevent (or attenuate) PH and/or RVH. Six groups of rats were injected subcutaneously with saline (3 ml/kg); capsaicin followed by saline or MCT (60 mg/kg); or MCT followed 7, 11, or 14 days later by capsaicin. Capsaicin (cumulative dose, 500 mg/kg) was given over a period of 4-5 days. Respiratory function, pulmonary vascular parameters, lung tachykinin levels, and tracheal neutral endopeptidase (NEP) activity were measured 21 days after MCT or saline injection. Capsaicin significantly decreased lung levels of SP but not NKA. Both capsaicin pretreatment and posttreatment blocked the following MCT-induced alterations: increases in lung SP and airway constriction; decreases in tracheal NEP activity and dynamic respiratory compliance. Administration of capsaicin before or 7 days after MCT blocked MCT-induced PH and RVH. The above data suggest that the early tachykinin-mediated airway dysfunction requires only transient elevated tachykinins, while progression of late tachykinin-mediated effects (PH and RVH) requires elevated tachykinins for more than one week.     

Involvement of BMPR2 in the protective effect of fluoxetine against monocrotaline-induced endothelial apoptosis in rats

Mutations in bone morphogenetic protein (BMP) receptor II (BMPR2) are associated with the apoptosis of the pulmonary artery endothelial cells and the loss of the pulmonary small vessels. The present study was designed to investigate the involvement of BMPR2 in the protective effect of fluoxetine against monocrotaline (MCT)-induced endothelial apoptosis in rats. Models of pulmonary arterial hypertension in rats were established by a single intraperitoneal injection of MCT (60 mg/kg). Fluoxetine (2 and 10 mg/kg) was intragastrically administered once a day. After 21 days, MCT caused pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling and significantly reduced the BMPR2 expression in lungs and pulmonary arteries. Fluoxetine dose-dependently inhibited MCT-induced pulmonary arterial hypertension and effectively protected the lungs against MCT-induced endothelial apoptosis, reduction in the number of alveolar sacs, and loss of the pulmonary small vessels. Fluoxetine reversed the expression of cyclic guanosine 3',5'-monophosphate-dependent kinase ?, BMPR2, phospho-Smad1, β-catenin, and reduced the expression of caspase 3 in rat lungs. These findings suggest that BMPR2 is probably involved in the protective effect of fluoxetine against MCT-induced endothelial apoptosis in rats.     

Sildenafil improves long-term effect of endothelial progenitor cell-based treatment for monocrotaline-induced rat pulmonary arterial hypertension

Background aimsWe hypothesized that the long-term therapeutic effect of combined sildenafil and bone marrow-derived endothelial progenitor cells (BMDEPCs) on monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH) is superior to either treatment alone.MethodsMale Sprague-Dawley rats (n = 40) were equally divided into normal controls, MCT (65 mg/kg, subcutaneously) only, MCT + sildenafil (25 mg/kg/day, orally), MCT + BMDEPCs (2.0 × 10⁶ autologous cells, intravenously) and MCT + sildenafil+ BMDEPCs. BMDEPCs and sildenafil were given on day 21 after MCT administration. Animals were sacrificed by day 90 after MCT administration.ResultsThe apoptotic (caspase 3, Bax) and inflammatory (tumor necrosis factor-α, matrix metalloproteinase-9) biomarkers in right ventricle and lung and pulmonary expressions of fibrotic biomarkers (transforming growth factor-β, p-Smad3) and connexin 43 protein were lower in monotherapy groups (i.e., MCT + sildenafil and MCT + BMDEPCs) and further decreased in normal controls and combined treatment groups (i.e., MCT + sildenafil + BMDEPCs) compared with untreated animals (i.e., MCT only) (all P < 0.01). Expressions of anti-fibrotic biomarkers (bone morphogenetic protein-2, p-Smad1/5) and numbers of alveolar sacs and arterioles in lung were higher in monotherapy groups and further increased in normal controls and combined treatment groups compared with untreated animals (all P < 0.005). In right ventricle, connexin 43 and α-myosin heavy chain (MHC) expressions were higher in the monotherapy groups and further elevated in normal controls and combined treatment groups compared with untreated animals, whereas β-MHC exhibited the opposite pattern (all P < 0.01). Right ventricular systolic pressure and weight were lower in the monotherapy animals and further reduced in normal controls and combined treatment groups compared with untreated animals (all P < 0.0001).ConclusionsCombined therapy with BMDEPCs and sildenafil was superior to either treatment alone in attenuating rodent MCT-induced PAH.     

Inactivation of p53 Is Sufficient to Induce Development of Pulmonary Hypertension in Rats

Objective

Pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterial hypertension (PAH) show similarities to cancer cells. Due to the growth-suppressive and pro-apoptotic effects of p53 and its inactivation in cancer, we hypothesized that the p53 pathway could be altered in PAH. We therefore explored the involvement of p53 in the monocrotaline (MCT) rat model of pulmonary hypertension (PH) and the pathophysiological consequences of p53 inactivation in response to animal treatment with pifithrin-α (PFT, an inhibitor of p53 activity).

Methods and Results

PH development was assessed by pulmonary arterial pressure, right ventricular hypertrophy and arterial wall thickness. The effect of MCT and PFT on lung p53 pathway expression was evaluated by western blot. Fourteen days of daily PFT treatment (2.2 mg/kg/day), similar to a single injection of MCT (60 mg/kg), induced PH and aggravated MCT-induced PH. In the first week after MCT administration and prior to PH development, p53, p21 and MDM2 protein levels were significantly reduced; whereas PFT administration effectively altered the protein level of p53 targets. Anti-apoptotic and pro-proliferative effects of PFT were revealed by TUNEL and MTT assays on cultured human PA-SMCs treated with 50 μM PFT.

Conclusions

Pharmacological inactivation of p53 is sufficient to induce PH with a chronic treatment by PFT, an effect related to its anti-apoptotic and pro-proliferative properties. The p53 pathway was down-regulated during the first week in the rat MCT model. These in vivo experiments implicate the p53 pathway at the initiation stages of PH pathogenesis.     

HMGB1 Promotes the Development of Pulmonary Arterial Hypertension in Rats

Rationale

Pulmonary 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.

Objectives

To 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.

Methods

Male 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.

Results

HMGB1 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.

Conclusions

Our 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.     

Eglin-c prevents monocrotaline-induced ventilatory dysfunction

Lai, Y. L., and K.-R. Zhou. Eglin-c preventsmonocrotaline-induced ventilatory dysfunction. J. Appl. Physiol. 82(1): 324-328, 1997.The presentstudy was carried out to investigate the relationship between elastaseand monocrotaline (MCT)-induced ventilatory dysfunction in rats. Toaccomplish this, we used an elastase inhibitor eglin-c to suppress theactivity of endogenous elastase. Thirty-five young Sprague-Dawley ratswere randomly divided into six groups: control, MCT, eglin-c (1),eglin-c (2), eglin-c (1)+MCT, and eglin-c (2)+MCT.Rats in the control group received no treatment. Each MCT rat receiveda single subcutaneous injection of MCT (60 mg/kg) 1 wk before thefunctional test. Each eglin-c (1) rat was intratracheallyinstilled with eglin-c (9 mg/rat) twice in 1 wk. Each eglin-c (2)rat was intratracheally instilled with eglin-c (9 mg/rat) five times in1 wk. Both eglin-c+MCT groups were treated with the combination ofeglin-c (1) or eglin-c (2) and MCT. In the MCT group, therewere significant decreases in dynamic respiratory compliance, maximalexpiratory flow rate at 50% total lung capacity, and the slopes of themaximal expiratory flow-%total lung capacity curve and the maximalexpiratory flow-static recoil pressure curve. However, in theeglin-c (1)+MCT and eglin-c (2)+MCT groups, all of theabove-mentioned MCT-induced changes were prevented. All ventilatoryvalues of the eglin-c (1) and eglin-c (2) groups were notsignificantly different from those of the control group. These resultsdemonstrate that eglin-c treatment prevents MCT-induced ventilatorydysfunction and suggest that endogenous elastase may play an importantrole in MCT-induced inflammation-mediated ventilatory abnormality.

     

alpha-Difluoromethylornithine attenuates monocrotaline-induced airway/lung dysfunction.

On the basis of the previous findings that alpha-difluoromethylornithine (DFMO, an inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme in polyamine biosynthesis) treatment prevents monocrotaline-(MCT) induced pulmonary hypertension and that ventilatory dysfunction precedes pulmonary hypertension in MCT-treated rats, we hypothesize that MCT-induced changes in airway/lung function are polyamine dependent. To evaluate this hypothesis, in phase 1, 48 young Sprague-Dawley rats were evenly divided into four groups: control, DFMO, MCT, and DFMO + MCT. Each DFMO rat received DFMO in its drinking water (2%) for 11 days, with additional injections (400 mg/kg sc) on the 5th day. Each MCT rat received a single injection of MCT (60 mg/kg sc) 1 wk before the functional study. Each DFMO + MCT rat received the same DFMO and MCT treatments as above, and MCT was administered on the 5th day of the DFMO treatment. In the MCT group, there were marked rightward shifts in pressure-volume and maximal flow-static recoil (MFSR) curves and significant decreases in dynamic and quasi-static compliance, the maximal expiratory flow, slope of the MFSR curve, and the carbon monoxide diffusing capacity, as well as a significant increase in alveolar wall thickness. However, in rats treated with DFMO + MCT, most of MCT-induced changes were significantly attenuated. To evaluate whether MCT causes bronchoconstriction, a bronchodilator, terbutaline (0.2 mg/kg i.v.), was administered to control (n = 7) and MCT (n = 11) rats in phase 2. Terbutaline significantly reversed MCT-induced decreases in maximal expiratory flow and slope of the MFSR curve, whereas it did not alter these parameters in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Rosuvastatin provides pleiotropic protection against pulmonary hypertension, right ventricular hypertrophy, and coronary endothelial dysfunction in rats

We recently reported that increased vascular endothelial nitric oxide production could protect against the development of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and right ventricular hypertrophy (RVH) in rats (32). The present study investigated whether the pleiotropic action of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors in upregulating endothelial function could also protect against the MCT-induced end-organ damages. Rosuvastatin (2 mg kg(-1) day(-1) via oral gavage) or placebo was initiated 1 wk before or 1 wk after MCT (60 mg/kg ip) administration. One month after MCT, significant PAH developed in the placebo rats, which were accompanied by histological evidence of pulmonary vascular thickening and right ventricular hypertrophy. The coronary endothelial vasodilatory function, assessed with endothelial/nitric oxide-dependent responses to acetylcholine and N(G)-nitro-L-arginine methyl ester (L-NAME), was depressed, while the constrictory responses to known coronary constrictors was enhanced. In rats that received rosuvastatin treatment 1 wk before MCT administration, a significantly reduced PAH and RVH was observed, as well as reduced pulmonary vascular and right ventricular remodelings. Rosuvastatin 1-wk posttreatment had no effect on PAH, but inhibited RVH. Right coronary endothelial dysfunction, which was shown in placebo rats, was effectively prevented by both pre- and postrosuvastatin treatment, while this effect was more dramatic in the pretreated group. Left coronary endothelial function, which was not affected by MCT, also showed an upregulation by rosuvastatin. Taken together, our results demonstrated the pleiotropic protection of rosuvastatin against the development of PAH and RVH and confirmed our previous finding that the targeted preservation of coronary endothelial function and vasoactivity may provide a novel approach to protect against cardiac remodeling.     

Deficiency of cold-inducible RNA-binding protein exacerbated monocrotaline-induced pulmonary artery hypertension through Caveolin1 and CAVIN1

Cold-inducible RNA-binding protein (CIRP) was a crucial regulator in multiple diseases. However, its role in pulmonary artery hypertension (PAH) is still unknown. Here, we first established monocrotaline (MCT)-induced rat PAH model and discovered that CIRP was down-regulated predominantly in the endothelium of pulmonary artery after MCT injection. We then generated Cirp-knockout (Cirp-KO) rats, which manifested severer PAH with exacerbated endothelium damage in response to MCT. Subsequently, Caveolin1 (Cav1) and Cavin1 were identified as downstream targets of CIRP in MCT-induced PAH, and the decreased expression of these two genes aggravated the injury and apoptosis of pulmonary artery endothelium. Moreover, CIRP deficiency intensified monocrotaline pyrrole (MCTP)-induced rat pulmonary artery endothelial cells (rPAECs) injuries both in vivo and in vitro, which was counteracted by Cav1 or Cavin1 overexpression. In addition, CIRP regulated the proliferative effect of conditioned media from MCTP-treated rPAECs on rat pulmonary artery smooth muscle cells, which partially explained the exceedingly thickened pulmonary artery intimal media in Cirp-KO rats after MCT treatment. These results demonstrated that CIRP acts as a critical protective factor in MCT-induced rat PAH by directly regulating CAV1 and CAVIN1 expression, which may facilitate the development of new therapeutic targets for the intervention of PAH.     

Disease severity–related alterations of cardiac microRNAs in experimental pulmonary hypertension

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart‐relevant microRNAs, that is myomiRs (miR‐1, miR‐133a, miR‐208, miR‐499) and miR‐214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT‐qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR‐1/miR‐133a/miR‐208a/miR‐499) were decreased and the expression of miR‐214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR‐214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.     

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

Hill, Nicholas S., Rod R. Warburton, Linda Pietras, andJames R. Klinger. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats.J. Appl. Physiol. 83(4):1209-1215, 1997.Endothelin-1 (ET-1), a potent vasoactive andmitogenic peptide, has been implicated in the pathogenesis ofseveral forms of pulmonary hypertension. We hypothesized thatnonspecific blockade of ET receptors would blunt the development ofmonocrotaline (MCT)-induced pulmonary hypertension in rats. Asingle dose of the nonspecific ET blocker bosentan (100 mg/kg) given tointact rats by gavage completely blocked the pulmonary vasoconstrictoractions of Big ET-1 and partially blunted hypoxic pulmonaryvasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavagedonce daily with bosentan (200 mg/kg) had lower right ventricular (RV)systolic pressure (RVSP), RV-to-body weight (RV/BW) andRV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratiosand less percent medial thickness of small pulmonary arteries thancontrol MCT-injected rats. Lower dose bosentan (100 mg/kg) had noeffect on these parameters after MCT or saline injection. Bosentanraised 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 weightratios 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. Weconclude that ET-1 contributes to the pathogenesis of MCT-inducedpulmonary hypertension and acts mainly during the later inflammatoryrather than the acute injury phase after injection.

     

Astragaloside IV attenuates inflammatory response mediated by NLRP-3/calpain-1 is involved in the development of pulmonary hypertension

Inflammation eventually leads to pulmonary arterial hypertension (PAH). Astragaloside IV(AS-IV) has a protective effect on pulmonary hypertension, but the specific protective mechanism has been unclear until now. Therefore, in this study, our aim was to investigate the mechanisms underlying the effects of AS-IV on PAH. In vivo, male Sprague-Dawley (SD) rats were injected intraperitoneally with monocrotaline (MCT, 60 mg/kg) and treated with AS-IV (40 mg/kg, 80 mg/kg), MCC950 and MDL-28170. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with monocrotaline pyrrole (MCTP, 60 μg/mL). The protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1β and calpain-1 were measured in vivo and/or in vitro. The results showed that AS-IV decreased the protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1β and calpain-1 in vivo and/or vitro. In conclusion, in this study the results suggested that AS-IV could inhibit monocrotaline-induced pulmonary arterial hypertension via the NLRP-3/calpain-1 pathway.     

Nicorandil attenuates monocrotaline-induced vascular endothelial damage and pulmonary arterial hypertension

Background

An antianginal K_ATP channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats.

Materials and Methods

Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg⁻¹·day⁻¹) alone; or nicorandil as well as either a K_ATP channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N ^ω-nitro-l-arginine methyl ester (l-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs).

Results

Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression.

Conclusions

Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.     

Oxygen radicals and substance P in perinatal hypoxia-exaggerated, monocrotaline-induced pulmonary hypertension

Perinatal hypoxia has been observed to cause more aggressive pulmonary hypertension in human. Several mediators such as reactive oxygen species (ROS) and substance P are believed to be crucial in the mechanism of inducing pulmonary hypertension. This study was designed to test whether substance P and ROS play a role in perinatal hypoxia-exaggerated, monocrotaline (MCT)-induced pulmonary hypertension. Normoxic Wistar rats (weighing 258 ± 9 g, n = 31) were divided into two groups: control (n = 16) and MCT (n = 15). Perinatal hypoxia Wistar rats (weighing 260 ± 19 g, n = 49) were divided into six groups: Hypoxia (n = 8), Hypoxia+MCT (n = 8), Hypoxia+capsaicin (CP)+MCT (n = 7), Hypoxia+MCT+1,3-dimethyl-2-thiourea (DMTU)E (n = 10), Hypoxia+MCT+DMTUL (n = 9), and Hypoxia+MCT+ hexa(sulfobutyl) fullerenes (HSF) (n = 7). Rats in the control group received saline injections. MCT (60 mg/kg, s.c.) was given three weeks prior to the functional examination. Chronic capsaicin pretreatment was performed to deplete substance P. Hydroxyl radical scavenger DMTU (500 mg/kg) was intraperitoneally (i.p.) injected early (DMTUE ) or late (DMTUL ) after MCT. Antioxidant HSF (10 mg/kg, i.p.) was given once daily for three weeks following MCT. MCT treatment caused significant increases in pulmonary arterial pressure (Ppa) and substance P level in lung tissue in normoxic rats. The MCT-induced increase in pulmonary arterial blood pressure was exaggerated by perinatal hypoxia, but this exaggeration was attenuated by either capsaicin pretreatment or antioxidant administrations. These results suggest that both ROS and substance P are involved in perinatal hypoxia-augmented, MCT-induced pulmonary hypertension.     

Fluoxetine protects against monocrotaline-induced pulmonary arterial remodeling by inhibition of hypoxia-inducible factor-1α and vascular endothelial growth factor

The selective serotonin re-uptake inhibitor fluoxetine has been shown to protect against monocrotaline (MCT)-induced pulmonary hypertension in rats. To investigate the possible role of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in mediating this protective effect, MCT-treated rats were administered fluoxetine by gavage, at doses of 2?mg/kg body mass or 10?mg/kg once daily for 3 weeks. Changes in pulmonary hemodynamic parameters, pulmonary artery morphologies, and expressions of HIF-1α and VEGF were assessed. Fluoxetine at the 10?mg/kg dose, but not at the 2?mg/kg dose, attenuated the effects of MCT on pulmonary artery pressure, right ventricle index, and medial wall thickness. In addition, 10?mg/kg fluoxetine mitigated the MCT-induced up-regulation of HIF-1α and VEGF protein and reactive oxygen species (ROS) in the lungs. This dosage also decreased pERK1/2 levels and inhibited proliferation of pulmonary arterial smooth muscle cells in MCT-treated rats. In conclusion, fluoxetine can protect against MCT-induced pulmonary arterial remodeling, which linked to reduced ROS generation and decreased HIF-1α and VEGF protein levels via the ERK1/2 phosphorylation pathway.