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.     

miR-181a/b-5p ameliorates inflammatory response in monocrotaline-induced pulmonary arterial hypertension by targeting endocan

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by vascular remodeling, endothelial cell (EC) dysfunction, and inflammation. The roles of microRNAs have received much critical attention. Thus, this study was attempted to show the biological function of miR-181a/b-5p (miR-181a/b) in monocrotaline (MCT)-induced PAH. Here, rats injected with MCT were used as PAH models. The expression of miR-181a/b and its effect on PAH pathologies were examined using miR-181a/b overexpression lentivirus. A luciferase reporter analysis was performed to measure the relationships between miR-181a/b and endocan. Additionally, primary rat pulmonary arterial endothelial cells (rPAECs) treated with tumor necrosis factor-α (TNF-α) were employed to further validate the regulatory mechanism of miR-181a/b in vitro. Our results showed that miR-181a/b expression was reduced in PAH, and its upregulation significantly attenuated the short survival period, right ventricular systolic pressure and mean pulmonary artery pressure increments, right ventricular remodeling, and lung injury. Furthermore, the increase of intercellular cell adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) in PAH rats was inhibited by miR-181a/b overexpression. Similarly, our in vitro results showed that inducing miR-181a/b suppressed TNF-α-stimulated increase of ICAM1 and VCAM1 in rPAECs. Importantly, the increased expression of endocan in PAH model or TNF-α-treated rPAECs was restored by miR-181a/b upregulation. Further analysis validated the direct targeting relationships between miR-181a/b and endocan. Collectively, this study suggests that miR-181a/b targets endocan to ameliorate PAH symptoms by inhibiting inflammatory states, shedding new lights on the prevention and treatment of PAH.     

Dexamethasone attenuates development of monocrotaline-induced pulmonary arterial hypertension

Immunity and inflammation are well established factors in the pathogenesis of pulmonary arterial hypertension (PAH). We aimed to investigate whether dexamethasone (Dex), a potent immunosuppressant, could prevent the development of monocrotaline (MCT)-induced PAH in rats as compared with pyrrolidine dithiocarbamate (PDTC) and its effect on the immune mechanism. PAH in rats (n = 66) was induced by MCT (50 mg/kg) injected intraperitoneally. Two days after MCT treatment, Dex (1.0 mg/kg) and PDTC (100 mg/kg) were administered once daily for 21 days. Samples were collected at 7, 14, and 21 days. Dex effectively inhibited MCT-induced PAH and reduced the T-helper (Th) 1 dominant cytokine response (interferon-γ) but up-regulated the Th2 one (interleukin 4). It increased the number of CD4+ T cells and decreased the number of CD8+ T cells around pulmonary arteries, upregulated the mRNA expression of fractalkine and downregulated that of CX3CR1 in the lung. Serum levels of interferon γ and interleukin 4 did not significantly differ from that of controls. Dex attenuated the process of MCT-induced PAH through its immunomodulatory property. Dex could be an appropriate therapy for PAH, although more studies are needed to define the appropriate treatment regimen.     

Activation of PPAR-γ ameliorates pulmonary arterial hypertension via inducing heme oxygenase-1 and p21: An in vivo study in rats

Aims

Our previous study has indicated that activation of PPAR-γ inhibits the proliferation of rat pulmonary artery smooth muscle cells (PASMCs) in vitro through inducing the expression of heme oxygenase-1 (HO-1), which in turn up-regulates the p21^WAF1 expression. In the present study, we intended to determine whether similar mechanisms have been involved in activation of PPAR-γ inhibition of development of rat PAH model.

Material and methods

Rat pulmonary arterial hypertension (PAH) model was established by subcutaneous injection of monocrotaline (MCT). Rosiglitazone was administered to activate PPAR-γ. Zinc protoporphyria IX (ZnPP-IX), was used to confirm the role of HO-1 in mediating PPAR-γ function. Parameters including the right ventricle systolic pressure (RVSP), the right ventricular hypertrophy (RVH) and the percentage of medial wall thickness were used to evaluate the development of PAH. Immunoblotting was used to determine the expression of HO-1 and p21^WAF1.

Key findings

Rosiglitazone significantly decreased the RVSP and inhibited the RVH in MCT-induced rat PAH model, and partially inhibited the pulmonary vascular remodeling. These effects were coupled with the sequential increase of HO-1 and p21^WAF1 expressions by rosiglitazone.

Significance

Activation of PPAR-γ benefits PAH by inhibiting proliferation of PASMCs and reducing pulmonary vascular remodeling. The present study suggests that enhancing PPAR-γ activity might have potential value in clinical treatment of PAH.     

POLYAMINE REGULATORY PROCESSES AND OXIDATIVE STRESS IN MONOCROTALINE-TREATED PULMONARY ARTERY ENDOTHELIAL CELLS

Alterations in polyamine metabolism may be a critical mechanism of monocrotaline (MCT)-induced structural remodeling of the pulmonary vasculature. In the present study, the hypothesis that MCT, through the induction of oxidative stress, modulates cellular polyamine regulatory mechanisms which in turn might be involved in the upregulation of fibronectin production in pulmonary artery endothelial cells (PAEC) was examined. A 24-h treatment with MCT significantly increased PAEC polyamine concentrations as compared to vehicle-treated cells. In addition, exposure to MCT caused an increase in abundance of ornithine decarboxylase (ODC) mRNA, upregulation of ODC activity and enhancement of spermidine import into PAEC. Inhibition ofde novopolyamine synthesis further increased spermidine uptake in MCT-treated cells. The depletion of cellular polyamine contents through the blockade of bothde novopolyamine biosynthesis and polyamine transport prevented MCT-induced increases in the medium level of fibronectin. In addition, PAEC treatment with MCT stimulated cellular oxidative stress as determined by increased levels of thiobarbituric acid reactive substances, enhanced dichlorofluorescein fluorescence and activation of NF-KB. A co-treatment with dimethylthiourea, an oxygen radical scavenger, prevented MCT-induced increases in cellular oxidation and attenuated disturbances in polyamine metabolism. These data suggest that MCT can stimulate polyamine regulatory processes in PAEC possibly through an increase in cellular oxidative stress. The present study may have significant implication in understanding mechanisms of MCT-induced pulmonary hypertension and remodeling of pulmonary vasculature.     

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.     

Paracrine Effects of Bone Marrow–Derived Endothelial Progenitor Cells: Cyclooxygenase-2/Prostacyclin Pathway in Pulmonary Arterial Hypertension

Background

Endothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH). Some paracrine factors secreted by bone marrow–derived endothelial progenitor cells (BMEPCs) have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT)-induced PAH via producing vasoprotective substances in a paracrine fashion.

Methods and Results

Bone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2) expression, prostacyclin (PGI₂) and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS) and its progenies PGI₂/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture.

Conclusions

Implantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI₂ and level of cAMP.     

野百合碱诱导大鼠肺动脉高压及肺源性心脏病的病理生理特征

目的:探讨野百合碱诱发肺动脉高压及肺源性心脏病模型的建立机制。方法:雄性Wistar大鼠20只,随机分为两组(n=10):正常组,模型组。模型组大鼠腹腔一次性注射野百合碱50 mg/kg,对照组注射同剂量的溶媒,28 d后测定大鼠血流动力学参数,硝酸盐还原酶法测定血清和肺组织中一氧化氮的含量;放射免疫法测定血浆中内皮素、脑钠素和肺组织中肿瘤坏死因子、内皮素的含量。结果:与对照组比较,右心室压力上升、心率和平均动脉压下降,血液和肺组织中肿瘤坏死因子、一氧化氮、内皮素-1、脑钠素含量上升,具有统计学意义。结论:野百合碱通过诱发肺血管和组织炎性损伤,升高体内肿瘤坏死因子、一氧化氮、内皮素-1的含量,建立肺动脉高压及肺源性心脏病模型。     

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.     

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.     

Enhanced store-operated Ca²+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that canonical transient receptor potential (TRPC) genes are upregulated and store-operated Ca(2+) entry (SOCE) is augmented in PASMCs of chronic hypoxic rats and patients of pulmonary arterial hypertension (PAH). Here we further examine the involvement of TRPC and SOCE in PH with a widely used rat model of monocrotaline (MCT)-induced PAH. Rats developed severe PAH, right ventricular hypertrophy, and significant increase in store-operated TRPC1 and TRPC4 mRNA and protein in endothelium-denuded pulmonary arteries (PAs) 3 wk after MCT injection. Contraction of PA and Ca(2+) influx in PASMC evoked by store depletion using cyclopiazonic acid (CPA) were enhanced dramatically, consistent with augmented SOCE in the MCT-treated group. The time course of increase in CPA-induced contraction corresponded to that of TRPC1 expression. Endothelin-1 (ET-1)-induced vasoconstriction was also potentiated in PAs of MCT-treated rats. The response was partially inhibited by SOCE blockers, including Gd(3+), La(3+), and SKF-96365, as well as the general TRPC inhibitor BTP-2, suggesting that TRPC-dependent SOCE was involved. Moreover, the ET-1-induced contraction and Ca(2+) response in the MCT group were more susceptible to the inhibition caused by the various SOCE blockers. Hence, our study shows that MCT-induced PAH is associated with increased TRPC expression and SOCE, which are involved in the enhanced vascular reactivity to ET-1, and support the hypothesis that TRPC-dependent SOCE is an important pathway for the development of PH.     

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。     

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.

     

Ventilatory dysfunction precedes pulmonary vascular changes in monocrotaline-treated rats

Rats with established monocrotaline (MCT)-induced pulmonary hypertension also exhibit a profound increase in lung resistance (RL) and a decrease in lung compliance. Because airway/lung dysfunction could precede and influence the evolution of MCT-induced pulmonary vascular disease, it is important to establish the temporal relationship between development of pulmonary hypertension and altered ventilatory function in MCT-treated rats. To resolve this issue, we segregated 47 young Sprague-Dawley rats into four groups: control (n = 13), MCT1 (n = 9), MCT2 (n = 11), and MCT3 (n = 14). Each MCT rat received a single subcutaneous injection of MCT (60 mg/kg) 1 MCT1), 2 (MCT2), or 3 (MCT3) wk before the functional study. At 1 wk after MCT, significant increases in RL and alveolar wall thickness were observed, as was a significant decrease in carbon monoxide diffusing capacity (DLCO). Medial thickness of pulmonary arteries (50-100 microns OD) and right ventricular hypertrophy were not observed until 2 and 3 wk post-MCT, respectively. Coincident with the right ventricular hypertrophy at 3 wk post-MCT were decreased DLCO and increased alveolar wall thickness and lung dry weight. Pressure-volume curves of air-filled and saline-filled lungs showed marked rightward shifts during the 1st and 2nd wk after MCT administration and then decreased at the 3rd wk. These data suggest that MCT-induced alterations in airway/lung function preceded those of pulmonary vasculature and, therefore, implicate airway/lung dysfunctions as potentially contributing to the later development of pulmonary vascular abnormalities.     

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.     

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.     

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.     

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.     

LncRNA H19 promotes the proliferation of pulmonary artery smooth muscle cells through AT<Subscript>1</Subscript>R via sponging let-7b in monocrotaline-induced pulmonary arterial hypertension

Background

Pulmonary arterial hypertension (PAH) is related to inflammation, and the lncRNA H19 is associated with inflammation. However, whether PDGF-BB-H19-let-7b-AT₁R axis contributes to the pathogenesis of PAH has not been thoroughly elucidated to date. This study investigated the role of H19 in PAH and its related mechanism.

Methods

In the present study, SD rats, C57/BL6 mice and H19?/? mice were injected with monocrotaline (MCT) to establish a PAH model. H19 was detected in the cytokine-stimulated pulmonary arterial smooth muscle cells (PASMCs), serum and lungs of rats/mice. H19 overexpression and knockdown experiments were also conducted. A dual luciferase reporter assay was used to explore whether let-7b is a sponge miRNA of H19, and AT₁R is a novel target of let-7b. A CCK-8 assay and flow cytometry were used to analyse cell proliferation.

Results

The results showed that H19 was highly expressed in the serum and lungs of MCT-induced rats/mice, and H19 was upregulated by PDGF-BB in vitro. H19 upregulated AT₁R expression via sponging miRNA let-7b following PDGF-BB stimulation. AT₁R is a novel target of let-7b. Moreover, the overexpression of H19 and AT₁R could facilitate PASMCs proliferation in vitro. H19 knockout protected mice from pulmonary artery remodeling and PAH following MCT treatment.

Conclusion

Our study showed that H19 is highly expressed in MCT-induced rodent lungs and upregulated by PDGF-BB. The H19-let-7b-AT₁R axis contributed to the pathogenesis of PAH by stimulating PASMCs proliferation. The H19 knockout had a protective role in the development of PAH. H19 may be a potential tar-get for the treatment of PAH.