Monocrotaline-induced Pulmonary Arterial Hypertension Research Articles

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

Celastrol attenuates the remodeling of pulmonary vascular and right ventricular in monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension is a progressive angio-proliferative disease associated with high morbidity and mortality rates. Although the histopathology of pulmonary arterial hypertension is well described, its therapeutic option remains unsatisfactory. This study investigated the effect of celastrol treatment on right ventricular dysfunction, remodeling, and pulmonary vascular remodeling in pulmonary arterial hypertension rats as well as its possible mechanisms. Pulmonary arterial hypertension was induced in male Sprague-Dawley rats by a single subcutaneously injection of monocrotaline. After daily delivery of celastrol (1 mg/kg) or vehicle via intraperitoneal injection for 4 weeks, the effects of celastrol on right ventricular function, fibrosis, and pulmonary vascular remodeling were assessed. The infiltration of macrophages, the expression of inflammatory cytokines, including MCP-1, IL-1β, IL-6, and IL-10, and the expression of NF-κB signaling pathway-associated proteins, IκBα, p-IKKα/β and p65 were further detected. Finally, the effect of celastrol on human pulmonary artery smooth cells proliferation under hypoxia was studied in vitro. Rats with pulmonary arterial hypertension had decreased right ventricular function, increased right ventricular fibrosis and pulmonary arteries with interstitial thickening and prominent media hypertrophy. Treatment with celastrol improved right ventricular function, attenuated right ventricular fibrosis and pulmonary vascular remodeling. Significantly decreased macrophage infiltration, reduced levels of pro-inflammatory cytokines, increased level of anti-inflammatory cytokine and inhibited NF-κB signaling pathway were observed in the lung tissues of rats treated with celastrol. Moreover, celastrol significantly suppressed the proliferation of human pulmonary artery smooth cells under hypoxia. We showed that in rats with pulmonary arterial hypertension, celastrol could improve right ventricular function, attenuate right ventricular and pulmonary vascular remodeling, and inhibit human pulmonary artery smooth cells proliferation under hypoxia. Suppression of the nuclear factor-κB (NF-κB) signaling pathway may be a part of the protective mechanism.

Protective Effect of Triclosan in Monocrotaline-Induced Pulmonary Arterial Hypertension: FASN Inhibition a Novel Approach.

Novel pharmacological approaches are needed to improve the outcomes of patients with idiopathic pulmonary hypertension. Fatty acid synthase (FASN) inhibitors have shown beneficial effects in preclinical models of pulmonary arterial hypertension (PAH), because of their role in the regulation of pulmonary artery vasoconstrictor tone and remodeling. We compared a Triclosan (FASN inhibitor), for the first time with the dual endothelin receptor antagonist, macitentan, in a monocrotaline-induced rat pulmonary hypertension model. Different methods (hemodynamics, histology of right ventricle and pulmonary vessels, and circulating biomarkers) showed consistently that 30 mg/kg daily of Triclosan (FASN inhibitor) and 10 mg/kg daily of macitentan slowed the progression of PAH both at the functional and structural levels. Treatments started on day 14 after monocrotaline injection and lasted 14 days. The findings of all experimental methods show that the FASN inhibitor has more similar effects as compared to macitentan. Our study reveals that inhibition of FAS decreases RV hypertrophy and improves cardiac function associated with PAH with the regulation of metabolic functions and governs further studies to establish "FASN inhibitor as a potential therapeutic approach" for the management of PAH.

18β-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling.

Endoplasmic reticulum stress (ERS)-induced inflammation participates in the occurrence of pulmonary arterial hypertension (PAH) by promoting pulmonary vascular remodeling, which involved in the activation of PERK/eIF2α/NF-κB signaling pathway. 18β-Glycyrrhetinic acid (18β-GA) has been found efficacious for attenuating PAH through its anti-remodeling effects in our previous research and it remains unclear whether 18β-GA has an effect on the remodeling caused by ERS-induced inflammation. In this study, we made observations in monocrotaline-induced PAH rats and found improvement of hemodynamic and histopathological parameters, decreases in the right ventricular hypertrophy index, and alleviation of pulmonary vascular remodeling after 18β-GA administration in vivo. Moreover, 18β-GA could significantly inhibit the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells (HPASMCs) induced by platelet-derived growth factor BB. At the cellular and molecular levels, we found that 18β-GA could significantly reduce the accumulation of misfolded protein in rat lung tissue, inhibit ERS activation, reduce the expression of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65, and increase IκB protein expression. 18β-GA could inhibit the migration of NF-κB into the nucleus, reduce the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant of HPASMCs, and reduce GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, increase IκB protein expression in HPASMCs. According to what we observed, this study indicated that 18β-GA could treat PAH, which is related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.

Targeted delivery of baicalein-p53 complex to smooth muscle cells reverses pulmonary hypertension

Pulmonary arterial hypertension (PAH) is an uncommon and deadly cardiopulmonary disease. PAH stems essentially from pulmonary artery (PA) remodeling induced predominantly by over-proliferation of PA smooth muscle cells (PASMCs) and inflammation. However, effective treatments are still missing in the clinic because the available drugs consisting of vasodilators are aimed to attenuate PAH symptoms rather than inhibit the remodeling process. Here, we aimed to specifically co-deliver apoptotic executor gene p53 and anti-inflammatory baicalein to PASMCs to alleviate PAH. The targeted co-delivery system was prepared through a carrier-free approach, which was prepared by loading the conjugate, NLS (nuclear localization signal) peptide-p53 gene, onto the baicalein pure crystals, followed by coating with glucuronic acid (GA) for targeting the glucose transport-1 (GLUT-1). The co-delivery system developed has a 200-nm diameter with a rod shape and a drug-loading capacity of 62% (w/w). The prepared system was shown to target PASMCs in vitro and enabled effective gene transfection, efficient apoptosis, and inflammation suppression. In vivo, via targeting the axis lung-PAs-PASMCs, the co-delivery reversed monocrotaline-induced PAH by reducing pulmonary artery pressure, downregulating the proinflammatory cytokine TNF-α, and inhibiting remodeling of both PAs and right ventricular. The potent efficacy may closely correlate with the activation of the signaling axis Bax/Bcl-2/Cas-3. Overall, our results indicate that the co-delivery system holds a significant potential to target the axis of lung-PAs-PASMCs and treat PAH.

Effects of Crocin on CCL2/CCR2 Inflammatory Pathway in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats.

Pulmonary arterial hypertension (PAH) is a malignant cardiopulmonary disease, in which pulmonary arterial remodeling is regarded as the prominent pathological feature. So far, the mechanism of PAH is still unclear, so its treatment remains a challenge. However, inflammation plays an important part in the occurrence and progression of PAH. It is well known that crocin has anti-inflammatory properties, so we investigated whether crocin could be a potential drug for the treatment of PAH rat models. Rats injected subcutaneously with monocrotaline (MCT) were treated with crocin via a gastric tube daily for four weeks. The results showed that crocin treatment significantly reduced the right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) in the PAH rat models. Moreover, crocin treatment reduced the proliferation of pulmonary arteriole smooth muscle cells (PASMCs). In addition, crocin treatment not only relieved inflammatory cell infiltration and collagen fiber hyperplasia in the lung and right ventricle, but also decreased the expression of the CCL2/CCR2 inflammatory pathway in the lung of PAH rat models. Furthermore, crocin treatment reduced the inflammatory cytokines and oxidative stress responses. In summary, crocin may play a protective role in MCT-induced PAH rats by alleviating inflammatory response, improving pulmonary arterial remodeling, and preventing PAH. Therefore, crocin as a new treatment for PAH may be quite worthy of consideration.

Abstract 11090: Therapeutic Effect of VEGF- and SDF-Nanoparticles in the Rat Monocrotaline Model of Pulmonary Hypertension

Introduction: Pulmonary vascular endothelial cell injury, dysfunction, and apoptosis are associated with the development of pulmonary arterial hypertension (PAH). Improving endothelial function may be therapeutically beneficial in this disorder. In the present study, we examined the effect of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), proteins that support endothelial repair and tissue regeneration, in rats with monocrotaline-induced PAH. A polysaccharide nanoparticle was used as a carrier for VEGF and SDF-1α delivery, the formulation of which extends the retention time of the proteins in the lung by ~17-fold compared with free protein. Method: A crosslinked dextran sulfate-chitosan nanoparticle (XDSCS NP) was prepared as the carrier. Recombinant human VEGF and SDF-1α were incorporated in the XDSCS NP to generate VEGF-NP and SDF-NP, respectively. Athymic nude rats were injected with monocrotaline (50 mg/kg, IP) to induce PAH. Two weeks after the injection, VEGF-NP (8 μg/rat) and SDF-NP (4 μg/rat) were delivered to the lungs of rats via intratracheal aerosolization. At 4 weeks, catheterization of right and left heart was performed, and lung tissue collected for histological analysis. Results: Monocrotaline induced PAH in the nude rats, leading to right ventricular systolic pressure (RVSP) of 71±8 mmHg (mean±SD, n=8) vs. 29±2 mmHg in the untreated control rats. VEGF-NP and SDF-NP treatment reduced the RVSP to 48±13 mmHg. The Fulton indices of the three groups were 0.45±0.07, 0.22±0.01, and 0.25±0.06, respectively. The pulmonary vascular resistance indices of the groups were 3.2±0.7, 0.6±0.3, and 2.1±1.0 mmHg.min/ml/kg, respectively. Histological analysis showed monocrotaline-treated rats had prominent muscularization of precapillary vessels, which was markedly reduced by the VEGF-NP and SDF-NP treatment. Aerosolization of free VEGF and SDF-1α or of empty XDSCS NP carrier had no effect on monocrotaline-induce PAH. Conclusion: These data indicate that the delivery of VEGF and SDF to the lungs of rats in a NP-bound form had beneficial therapeutic effects in monocrotaline-induced PAH, and offer the potential for a novel therapeutic strategy for human pulmonary hypertensive disorders.

Abstract 13792: Integration of Proteomic and Transcriptomic Signatures in the Failing Right Ventricle of Rats With Monocrotaline Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by cellular remodeling in the pulmonary circulation. Pulmonary vascular obstruction, stiffening and constriction elevate pulmonary artery pressure and right ventricular (RV) afterload leading to failure (RVF). In a model of PAH induced by injection of monocrotaline (MCT, 60mg/kg sc) RV decompensation was confirmed. Then the RV proteome was analyzed using Mass Spectrometry (LC-MSMS). Proteomics revealed 1,277 differentially regulated proteins. Integration of this proteome with our previously published transcriptomic study of RVs harvested from an independent group of rats with MCT-induced PAH revealed 410 common targets, 15 of which were both highly enriched at protein (corrected q -values <0.05) and mRNA level (corrected p-adjust <0.05 ). Most regulated pairs were increased in RVF, including: Atrial natriuretic peptide protein (7.55-fold, transcript = 4.19-fold), Thrombospondin-4 protein (5.56-fold, transcript = 4.4-fold), Periostin (protein = 3.83-fold, transcript = 4.56-fold), Latent transforming growth factor beta binding protein 2 (2.67-fold, transcript = 4.16-fold), Heme oxygenase 1 (4.59-fold, transcript = 2.05-fold), Transforming Growth Factor Beta-1-Induced Transcript 4 Protein (3.82-fold, transcript = 2.1-fold), Transmembrane glycoprotein NMB (3.21-fold, transcript = 2.35-fold), S100 calcium-binding protein A4 (2.43-fold, transcript = 1.93-fold), Collagen XVIII, alpha 1 (2.59-fold, transcript = 1.61-fold), Ankyrin repeat domain 2 (4.04-fold, transcript = 2.49-fold), Procollagen-Lysine 2-Oxoglutarate 5-Dioxygenase 1, (6.89-fold, transcript = 2.3-fold), Tenascin-C (5.18-fold, transcript = 2.53-fold). In contrast, ATPase Na+/K+ Transporting Subunit Alpha 2 3.47 (protein = 3.47-fold, transcript = 2.23-fold) and Glutathione S-Transferase Zeta 1 (protein = 1.97-fold, transcript = 2.38-fold) were downregulated in RVF. A functional analysis of this list of regulated protein/transcript pairs revealed enriched functions include mitochondrial metabolism, cellular respiration and purine metabolism. Our integrated multiomic approach is validated by independent datasets and provides a molecular context for RVF in MCT-induced PAH.

Abstract 10338: A Junctophilin-2-Mitofusin-2 Interaction Links T-Tubule Remodeling to Mitochondrial Dysfunction in Pulmonary Arterial Hypertension-Mediated Right Ventricular Failure

Introduction: Junctophilin-2 (JPH2) plays a crucial role in t-tubule structure/function and gene regulation in cardiomyocytes. In addition, JPH2 downregulation causes mitochondrial dysfunction and mitofusin-2 (MFN2) dysregulation, but the link between JPH2 and mitochondria is undetermined. Here, we investigated the hypothesis that a direct JPH2-MFN2 interaction is important for mitochondrial regulation and subsequent right ventricular (RV) function in pulmonary arterial hypertension (PAH). Methods and Results: Confocal microscopy revealed JPH2 and MFN2 colocalized at t-tubules in isolated cardiomyocytes. Immunoprecipitation demonstrated JPH2 and MFN2 interacted in RV extracts. Pulldown experiments showed JPH2 directly bound MFN2, and that the MFN2 binding domain was localized to the N-terminal third of JPH2. To probe the importance of the JPH2-MFN2 interaction for RV function, we compared the effects of treatment with AAV9-JPH2 to AAV9-GFP in monocrotaline-induced PAH. Consistent with its known role in t-tubule homeostasis, AAV9-JPH2 normalized RV t-tubule architecture. Interestingly, transmission electron microscopy showed AAV9-JPH2 corrected RV mitochondrial morphology. Immunoblots of RV extracts demonstrated AAV9-JPH2 increased JPH2 and MFN2 expression, which may underlie the restoration of mitochondrial morphology. At the organ level, AAV9-JPH2 reduced RV cardiomyocyte hypertrophy and RV fibrosis. AAV9-JPH2 increased tricuspid annular plane systolic excursion and RV free wall thickening as quantified by echocardiography. Pressure-volume loop analysis showed AAV9-JPH2 augmented RV ejection fraction and right ventricular-pulmonary arterial coupling and lowered RV end diastolic pressure and tau. Importantly, the improvements in RV systolic and diastolic function were independent of altering PAH severity as RV systolic pressure, effective arterial elastance, and pulmonary arterial small vessel remodeling were not altered with AAV9-JPH2. Conclusions: Disruption of the JPH2-MFN2 interaction may link altered t-tubule structure to mitochondrial dysfunction in the compromised RV. Strategies to increase JPH2 expression could have therapeutic value for RV failure.

Congestive Hepatopathy Secondary to Right Ventricular Hypertrophy Related to Monocrotaline-Induced Pulmonary Arterial Hypertension.

Heart dysfunction and liver disease often coexist. Among the types of cardiohepatic syndrome, Type 2 is characterized by the chronic impairment of cardiac function, leading to chronic liver injury, referred to as congestive hepatopathy (CH). In this study, we aimed to establish a rat model of CH secondary to right ventricular hypertrophy (RVH) related to monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Fifty male Wistar rats were divided into four groups and randomly assigned to control and experimental groups. Three experimental groups were submitted to intraperitoneal MCT inoculation (60 mg/kg) and were under its effect for 15, 30 and 37 days. The animals were then sacrificed, obtaining cardiac and hepatic tissues for anatomopathological and morphometric analysis. At macroscopic examination, the livers in the MCT groups presented a nutmeg-like appearance. PAH produced marked RVH and dilatation in the MCT groups, characterized by a significant increase in right ventricular free wall thickness (RVFWT) and chamber area. At histological evaluation, centrilobular congestion was the earliest manifestation, with preservation of the hepatocytes. Centrilobular hemorrhagic necrosis was observed in the groups exposed to prolonged MCT. Sinusoidal dilatation was markedly increased in the MCT groups, quantified by the Sinusoidal Lumen Ratio (SLR). The Congestive Hepatic Fibrosis Score and the Centrilobular Fibrosis Ratio (CFR) were also significantly increased in the MCT30 group. Hepatic atrophy, steatosis, apoptotic bodies and, rarely, hydropic swelling were also observed. SLR correlated strongly with CFR and RVFWT, and CFR correlated moderately with RVFWT. Our rat model was able to cause CH, related to monocrotaline-induced PAH and RVH; it was feasible, reproducible, and safe.

Oral delivery of ambrisentan-loaded lipid-core nanocapsules as a novel approach for the treatment of pulmonary arterial hypertension

Ambrisentan (AMB) is an orphan drug approved for oral administration that has been developed for the treatment of pulmonary arterial hypertension (PAH), a chronic and progressive pathophysiological state that might result in death if left untreated. Lipid-core nanocapsules (LNCs) are versatile nanoformulations capable of loading lipophilic drugs for topical, vaginal, oral, intravenous, pulmonary, and nasal administration. Our hypothesis was to load AMB into these nanocapsules (LNCamb) and test their effect on slowing or reducing the progression of monocrotaline-induced PAH in a rat model, upon oral administration. LNCamb displayed a unimodal distribution of diameters (around 200 nm), negative zeta potential (–11.5 mV), high encapsulation efficiency (78%), spherical shape, and sustained drug release (50–60% in 24 h). The in vivo pharmacodynamic effect of the LNCamb group was evaluated by observing the echocardiography, hemodynamic, morphometric, and histological data, which showed a significant decrease in PAH in this group, as compared to the control group (AMBsolution). LNCamb showed the benefit of reversing systolic dysfunction and preventing vascular remodeling with greater efficacy than that observed in the control group. The originality and contribution of our work reveal the promising value of this nanoformulation as a novel therapeutic strategy for PAH treatment.

MiR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis.

Pulmonary artery hypertension (PAH) pathology involves extracellular matrix (ECM) remodeling in cardiac tissues, thus promoting cardiac fibrosis progression. miR-29a-3p reportedly inhibits lung progression and liver fibrosis by regulating ECM protein expression; however, its role in PAH-induced fibrosis remains unclear. In this study, we aimed to investigate the role of miR-29a-3p in cardiac fibrosis progression in PAH and its influence on ECM protein thrombospondin-2 (THBS2) expression. The diagnostic and prognostic values of miR-29a-3p and THBS2 in PAH were evaluated. The expressions and effects of miR-29a-3p and THBS2 were assessed in cell culture, monocrotaline-induced PAH mouse model, and patients with PAH. The levels of circulating miR-29a-3p and THBS2 in patients and mice with PAH decreased and increased, respectively. miR-29a-3p directly targets THBS2 and regulates THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis. The circulating levels of miR-29a-3p and THBS2 were correlated with PAH diagnostic parameters, suggesting their independent prognostic value. miR-29a-3p targeted THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis, indicating miR-29a-3p acts as a messenger with promising therapeutic effects.

Dysregulated zinc and sphingosine-1-phosphate signaling in pulmonary hypertension: Potential effects by targeting of bone morphogenetic protein receptor type 2 in pulmonary microvessels.

Recently identified molecular targets in pulmonary artery hypertension (PAH) include sphingosine-1-phosphate (S1P) and zinc transporter ZIP12 signaling. This study sought to determine linkages between these pathways, and with BMPR2 signaling. Lung tissues from a rat model of monocrotaline-induced PAH and therapeutic treatment with bone marrow-derived endothelial-like progenitor cells transduced to overexpress BMPR2 were studied. Multifluorescence quantitative confocal microscopy (MQCM) was applied for analysis of protein expression and localization of markers of vascular remodeling (αSMA and BMPR2), parameters of zinc homeostasis (zinc transporter SLC39A/ZIP family members 1, 10, 12 and 14; and metallothionein MT3) and S1P extracellular signaling (SPHK1, SPNS2, S1P receptor isoforms 1, 2, 3, 5) in 20-200 µm pulmonary microvessels. ZIP12 expression in whole lung tissue lysates was assessed by western blot. Spearman nonparametric correlations between MQCM readouts and hemodynamic parameters, Fulton index (FI), and right ventricular systolic pressure (RVSP) were measured. In line with PAH status, pulmonary microvessels in monocrotaline-treated animals demonstrated significant (p < .05, n = 6 per group) upregulation of αSMA (twofold) and downregulation of BMPR2 (20%). Upregulated ZIP12 (92%), MT3 (57.7%), S1PR2 (54.8%), and S1PR3 (30.3%) were also observed. Significant positive and negative correlations were demonstrated between parameters of zinc homeostasis (ZIP12, MT3), S1P signaling (S1PRs, SPNS2), and vascular remodeling (αSMA, FI, RVSP). MQCM and western blot analysis showed that monocrotaline-induced ZIP12 upregulation could be partially negated by BMPR2-targeted therapy. Our results indicate that altered zinc transport/storage and S1P signaling in the monocrotaline-induced PAH rat model are linked to each other, and could be alleviated by BMPR2-targeted therapy.

MicroRNA-663 prevents monocrotaline-induced pulmonary arterial hypertension by targeting TGF-β1/smad2/3 signaling

ObjectivePulmonary vascular remodeling due to excessive growth factor production and pulmonary artery smooth muscle cells (PASMCs) proliferation is the hallmark feature of pulmonary arterial hypertension (PAH). Recent studies suggest that miR-663 is a potent modulator for tumorigenesis and atherosclerosis. However, whether miR-663 involves in pulmonary vascular remodeling is still unclear. Methods and resultsBy using quantitative RT-PCR, we found that miR-663 was highly expressed in normal human PASMCs. In contrast, circulating level of miR-663 dramatically reduced in PAH patients. In addition, in situ hybridization showed that expression of miR-663 was decreased in pulmonary vasculature of PAH patients. Furthermore, MTT and cell scratch-wound assay showed that transfection of miR-663 mimics significantly inhibited platelet derived growth factor (PDGF)-induced PASMCs proliferation and migration, while knockdown of miR-663 expression enhanced these effects. Mechanistically, dual-luciferase reporter assay revealed that miR-663 directly targets the 3’UTR of TGF-β1. Moreover, western blots and ELISA results showed that miR-663 decreased PDGF-induced TGF-β1 expression and secretion, which in turn suppressed the downstream smad2/3 phosphorylation and collagen I expression. Finally, intratracheal instillation of adeno-miR-663 efficiently inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy in monocrotaline (MCT)-induced PAH rat models. ConclusionThese results indicate that miR-663 is a potential biomarker for PAH. MiR-663 decreases PDGF-BB-induced PASMCs proliferation and prevents pulmonary vascular remodeling and right ventricular hypertrophy in MCT-PAH by targeting TGF-β1/smad2/3 signaling. These findings suggest that miR-663 may represent as an attractive approach for the diagnosis and treatment for PAH.

Integrated analysis of m6A mRNA methylation in rats with monocrotaline-induced pulmonary arterial hypertension.

Background: N6-methyladenosine (m6A) modification is one of the most common chemical modifications of eukaryotic mRNAs, which play an important role in tumors and cardiovascular disease through regulating mRNA stability, splicing and translation. However, the changes of m6A mRNA and m6A-related enzymes in pulmonary arterial hypertension (PAH) remain largely unexplored.Methods: MeRIP-seq was used to identify m6A methylation in lung tissues from control and MCT-PAH rats. Western blot and immunofluorescence were used to evaluate expression of m6A-related enzymes.Results: Compared with control group, m6A methylation was mainly increased in lung tissues from MCT-PAH rats. The up-methylated coding genes in MCT-PAH rats were primarily enriched in processes associated with inflammation, glycolysis, ECM-receptor interaction and PDGF signal pathway, while genes with down-methylation were enriched in processes associated with TGF-β family receptor members. The expression of FTO and ALKBH5 downregulated, METTL3 and YTHDF1 increased and other methylation modification-related proteins was not significantly changed in MCT-PAH rats lung tissues. Immunofluorescence indicated that expression of FTO decreased and YTHDF1 increased in small pulmonary arteries of MCT-PAH rats.Conclusion: m6A levels and the expression of methylation-related enzymes were altered in PAH rats, in which FTO and YTHDF1 may play a crucial role in m6A modification.

Stable Gastric Pentadecapeptide BPC 157 Therapy for Monocrotaline-Induced Pulmonary Hypertension in Rats Leads to Prevention and Reversal.

Background. Monocrotaline selectively injures the lung’s vascular endothelium and induces pulmonary arterial hypertension. The stable gastric pentadecapeptide BPC 157 acts as a prototype cytoprotective agent that maintains endothelium, and its application may be a novel therapy. Besides, BPC 157 prevents and reverses thrombosis formation, maintains platelet function, alleviates peripheral vascular occlusion disturbances, and has anti-arrhythmic and anti-inflammatory effects. Monocrotaline-induced pulmonary arterial hypertension in rats (wall thickness, total vessel area, heart frequency, QRS axis deviation, QT interval prolongation, increase in right ventricle systolic pressure and bodyweight loss) can be counteracted with early or delayed BPC 157 therapy. Methods and Results. After monocrotaline (80 mg/kg subcutaneously), BPC 157 (10 μg/kg or 10 ng/kg, days 1–14 or days 1–30 (early regimens), or days 14–30 (delayed regimen)) was given once daily intraperitoneally (last application 24 h before sacrifice) or continuously in drinking water until sacrifice (day 14 or 30). Without therapy, the outcome was the full monocrotaline syndrome, marked by right-side heart hypertrophy and massive thickening of the precapillary artery’s smooth muscle layer, clinical deterioration, and sometimes death due to pulmonary hypertension and right-heart failure during the 4th week after monocrotaline injection. With all BPC 157 regimens, monocrotaline-induced pulmonary arterial hypertension (including all disturbed parameters) was counteracted, and consistent beneficial effects were documented during the whole course of the disease. Pulmonary hypertension was not even developed (early regimens) as quickly as the advanced pulmonary hypertension was rapidly attenuated and then completely eliminated (delayed regimen). Conclusions. Thus, pentadecapeptide BPC 157 prevents and counteracts monocrotaline-induced pulmonary arterial hypertension and cor pulmonale in rats.

Profiling and Molecular Mechanism Analysis of Long Non-Coding RNAs and mRNAs in Pulmonary Arterial Hypertension Rat Models.

Pulmonary arterial hypertension (PAH) is an immune-mediated disease with poor prognosis and associated with various inflammatory immune diseases. In fact, its pathogenesis is far from clear. Although long non-coding RNAs (lncRNAs) have been implicated in PAH, the molecular mechanisms remain largely unknown. For the first time, in lungs of monocrotaline-induced PAH rat models, we simultaneously detected the expression profiles of lncRNAs and mRNAs by high-throughput sequencing, and explored their roles with bioinformatics analysis and cell assay to discover more potential pathogenesis about PAH. Our data identified that a total of 559 lncRNAs and 691 mRNAs were differentially expressed in lungs during the pathogenesis of PAH. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that these dysregulated lncRNAs and mRNAs participated in important biological processes and pathways of PAH, among which inflammatory and immune responses represented the chief enriched pathway. The lncRNA-mRNA co-expression network was developed to uncover the hidden interactions between lncRNAs and mRNAs. Further, the expression levels of lncRNAs (NONRATT018084.2, NONRATT009275.2, NONRATT007865.2, and NONRATT026300.2) and mRNAs (LGALS3, PDGFC, SERPINA1, and NFIL3) were confirmed using quantitative real-time PCR. In the end, lncRNA NONRATT009275.2 could facilitate macrophage polarization to M2 type and be involved in inflammatory immune response. In conclusion, this study provided candidate drug targets and potential roles on lncRNAs in the pathogenesis of PAH, and several key regulatory genes were identified, which laid the initial foundation for further mechanism study in PAH.

Lumican deficiency promotes pulmonary arterial remodeling

Pulmonary arterial hypertension (PAH) is caused by progressive extracellular matrix disorganization and increased pulmonary vascular cell proliferation. Lumican is a member of the small leucine-rich proteoglycan family that controls cell proliferation, and is a potential endogenous modulator of TGF-β signaling pathway. We show that the decreased lumican protein levels in pulmonary arterial smooth muscle cells (PASMCs) is related to the vascular remodeling and stiffening observed in PAH. The role of lumican in PASMC accumulation and activation in response to pulmonary vascular remodeling remains unclear and we hypothesized that the loss of lumican in PASMCs promotes the development of PAH. Our aim was to establish that lumican plays a pivotal role in modulating pathological vascular remodeling in humans using a rat model of monocrotaline-induced PAH and chronically hypoxic mice. We found that mice with a homozygous deletion of lumican (Lum-/-) showed severe pulmonary arterial remodeling and right ventricular hypertrophy in response to hypoxia, and these effects in mice with chronic hypoxia-induced pulmonary hypertension were successfully treated by the administration of a lumican C-terminal peptide (LumC13C-A, lumikine). We identified a mechanistic link by which lumican signaling prevents the activation of phosphorylated AKT, resulting in the suppression of PASMC proliferation. Lumican deficiency promotes pulmonary arterial remodeling. Administration of lumikine reverses the PAH pathogenesis caused by hypoxia-induced experimental PAH. Lumican is an antiproliferative target that functions to suppress pAKT activation during pathogenesis.

Downregulation of Soluble Guanylate Cyclase and Protein Kinase G in the Pulmonary Artery Leads to the Sensitization to Thromboxane A2 in the Monocrotaline‐induced Pulmonary Hypertensive Rats

Thromboxane A2(TXA2) relates to physiological and pathophysiological conditions of pulmonary artery (PA). Here, we examined the role of TXA2 receptor (TP)-mediated signaling in the pathophysiology of pulmonary arterial hypertension (PAH). The responsiveness to contractile agonist in PA could be determined by signals related to vasodilation such as nitric oxide (NO), soluble guanylate cyclase (sGC) and cGMP-dependent kinase (PKG) pathway. In this study, the TP receptor agonist (U46619)-induced contraction and modulatory role of NO/cGMP signaling were investigated in a monocrotaline-induced PAH rat model (PAH-MCT). PA from PAH-MCT exhibited higher sensitivity to U46619, that is decreased EC50. Lower expression of eNOS, sGC, and PKG were observed while higher expression of RhoA-dependent kinase 2 (ROCK2) was revealed by immunoblot analysis in the PA from PAH-MCT and control. The higher sensitivity to U46619 in PAH-MCT was ameliorated by 8-Br-cGMP, a membrane-permeable cGMP analogue, but not by NO donor, sodium nitroprusside (SNP 30 µM). In contrast, in the control PA, sGC inhibition by 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) 10 µM lowered the threshold of U46619-induced contraction. In the presence of ODQ, SNP treatment did not present its regulatory effect whereas the addition of 8-Br-cGMP lowered the sensitivity to U46619. The inhibition of ROCK by Y-27632 also lowered the sensitivity to U46619 in both control and PAH-MCT. The study suggests that the attenuation of NO/cGMP signaling and the upregulation of ROCK2 increase the sensitivity to TXA2 in the PAH animal, which might have pathophysiological implications in patients with PAH.

Dual-Functional MN-08 Attenuated Pulmonary Arterial Hypertension Through Vasodilation and Inhibition of Pulmonary Arterial Remodeling.

[Figure: see text].