ABSTRACT
Pulmonary arterial hypertension (PAH) is a chronic disorder characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary vascular resistance and right ventricle (RV) overload and failure. MicroRNA-146a (miR-146a) promotes vascular smooth muscle cell proliferation and vascular neointimal hyperplasia, both hallmarks of PAH. This study aimed to investigate the effects of miR-146a through pharmacological or genetic inhibition on experimental PAH and RV pressure overload animal models. Additionally, we examined the overexpression of miR-146a on human pulmonary artery smooth muscle cells (hPASMCs). Here, we showed that miR-146a genic expression was increased in the lungs of patients with PAH and the plasma of monocrotaline (MCT) rats. Interestingly, genetic ablation of miR-146a improved RV hypertrophy and systolic pressures in Sugen 5415/hypoxia (SuHx) and pulmonary arterial banding (PAB) mice. Pharmacological inhibition of miR-146a improved RV remodeling in PAB-wild type mice and MCT rats, and enhanced exercise capacity in MCT rats. However, overexpression of miR-146a did not affect proliferation, migration, and apoptosis in control-hPASMCs. Our findings show that miR-146a may play a significant role in RV function and remodeling, representing a promising therapeutic target for RV hypertrophy and, consequently, PAH.
Subject(s)
MicroRNAs , Pulmonary Arterial Hypertension , Pulmonary Artery , Ventricular Function, Right , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Rats , Humans , Mice , Male , Pulmonary Arterial Hypertension/genetics , Pulmonary Arterial Hypertension/metabolism , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Disease Models, Animal , Monocrotaline , Cell Proliferation/genetics , Myocytes, Smooth Muscle/metabolism , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/metabolism , Vascular Remodeling/genetics , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Metabolic abnormalities and immune inflammation are deeply involved in pulmonary vascular remodelling and the development of pulmonary hypertension (PH). However, the regulatory mechanisms of glycolysis in macrophages are still elusive. Cumulative evidence indicates that ß-catenin plays a crucial role in metabolic reprogramming. This study aimed to investigate the effect of ß-catenin on macrophage glycolysis in PH. METHODS: LPS-induced BMDMs were generated via in vitro experiments. A monocrotaline (MCT)-induced PH rat model was established, and the ß-catenin inhibitor XAV939 was administered in vivo. The role of ß-catenin in glycolysis was analysed. The degree of pulmonary vascular remodelling was measured. RESULTS: ß-catenin was significantly increased in both in vitro and in vivo models. In LPS-induced BMDMs, ß-catenin increased the levels of hexokinase 2 (HK2), phosphofructokinase (PFK), M2-pyruvate kinase (PKM2), lactate dehydrogenase (LDH), and lactate (LA) and the expression of inflammatory cytokines and promoted PASMC proliferation and migration in vitro. XAV939 decreased the level of glycolysis and downregulated the expression of inflammatory cytokines in vivo. MCT promoted pulmonary arterial structural remodelling and right ventricular hypertrophy, and XAV939 alleviated these changes. CONCLUSIONS: Our findings suggest that ß-catenin is involved in the development of PH by promoting glycolysis and the inflammatory response in macrophages. Inhibition of ß-catenin could improve the progression of PH.
Subject(s)
Disease Models, Animal , Glycolysis , Hypertension, Pulmonary , Macrophages , Monocrotaline , Pulmonary Artery , Rats, Sprague-Dawley , Vascular Remodeling , beta Catenin , Animals , Glycolysis/drug effects , beta Catenin/metabolism , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/physiopathology , Male , Vascular Remodeling/drug effects , Macrophages/metabolism , Macrophages/drug effects , Pulmonary Artery/metabolism , Pulmonary Artery/drug effects , Pulmonary Artery/physiopathology , Pulmonary Artery/pathology , Cell Proliferation/drug effects , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Signal Transduction , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/chemically induced , Inflammation Mediators/metabolism , Rats , Cell Movement/drug effectsABSTRACT
Indigo naturalis (IN), derived from the leaves of the indigo plant, is a traditional Chinese medicine that has historically been used for its anti-inflammatory properties in the treatment of various diseases, including ulcerative colitis (UC). However, long-term use of IN in UC patients is incontrovertibly associated with the onset of pulmonary arterial hypertension (PAH). To investigate the mechanisms by which IN induces PAH, we focused on the raw material of IN, indigo leaves (IL). Only the condition of long-term chronic (6 months) and high-dose (containing 5% IL in the control diet) administration of IL induced medial thickening in the pulmonary arteries without right ventricular hypertrophy in our rat model. IL administration for a month did not induce pulmonary arterial remodeling but increased endothelin-1 (ET-1) expression levels within endothelial cell (EC) layers in the lungs. Gene Expression Omnibus analysis showed that ET-1 is a key regulator of PAH and that the IL component indican and its metabolite IS induced ET-1 mRNA expression via reactive oxygen species-dependent mechanism. We identified the roles of indican and IS in ET-1 expression in ECs, which were linked to pulmonary arterial remodeling in an animal model.
Subject(s)
Endothelin-1 , Hypertrophy, Right Ventricular , Plant Leaves , Pulmonary Artery , Rats, Sprague-Dawley , Vascular Remodeling , Animals , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Male , Endothelin-1/metabolism , Vascular Remodeling/drug effects , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/physiopathology , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/metabolism , Rats , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Lung/drug effects , Lung/pathology , Lung/metabolismABSTRACT
Increased mitochondrial reactive oxygen species (ROS) formation is important for the development of right ventricular (RV) hypertrophy (RVH) and failure (RVF) during pulmonary hypertension (PH). ROS molecules are produced in different compartments within the cell, with mitochondria known to produce the strongest ROS signal. Among ROS-forming mitochondrial proteins, outer-mitochondrial-membrane-located monoamine oxidases (MAOs, type A or B) are capable of degrading neurotransmitters, thereby producing large amounts of ROS. In mice, MAO-B is the dominant isoform, which is present in almost all cell types within the heart. We analyzed the effect of an inducible cardiomyocyte-specific knockout of MAO-B (cmMAO-B KO) for the development of RVH and RVF in mice. Right ventricular hypertrophy was induced by pulmonary artery banding (PAB). RV dimensions and function were measured through echocardiography. ROS production (dihydroethidium staining), protein kinase activity (PamStation device), and systemic hemodynamics (in vivo catheterization) were assessed. A significant decrease in ROS formation was measured in cmMAO-B KO mice during PAB compared to Cre-negative littermates, which was associated with reduced activity of protein kinases involved in hypertrophic growth. In contrast to littermates in which the RV was dilated and hypertrophied following PAB, RV dimensions were unaffected in response to PAB in cmMAO-B KO mice, and no decline in RV systolic function otherwise seen in littermates during PAB was measured in cmMAO-B KO mice. In conclusion, cmMAO-B KO mice are protected against RV dilatation, hypertrophy, and dysfunction following RV pressure overload compared to littermates. These results support the hypothesis that cmMAO-B is a key player in causing RV hypertrophy and failure during PH.
Subject(s)
Hypertension, Pulmonary , Hypertrophy, Right Ventricular , Monoamine Oxidase , Reactive Oxygen Species , Animals , Male , Mice , Disease Models, Animal , Heart Failure/metabolism , Heart Failure/etiology , Heart Failure/genetics , Heart Failure/pathology , Heart Ventricles/pathology , Heart Ventricles/metabolism , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/pathology , Mice, Knockout , Monoamine Oxidase/genetics , Monoamine Oxidase/metabolism , Monoamine Oxidase/deficiency , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Reactive Oxygen Species/metabolism , Ventricular Dysfunction, Right/metabolism , Ventricular Dysfunction, Right/genetics , Ventricular Dysfunction, Right/etiology , Ventricular Dysfunction, Right/pathologyABSTRACT
Nur77 is a member of the NR4A subfamily of orphan nuclear receptors that is expressed and has a function within the immune system. This study aimed to investigate the role of Nur77 in hypoxic pulmonary hypertension. SPF male SD rats were exposed in hypobaric chamber simulating 5000 m high altitude for 0, 3, 7, 14, 21 or 28 days. Rat pulmonary artery smooth muscle cells (RPASMCs) were cultured under normoxic conditions (5% CO2-95% ambient air) or hypoxic conditions (5% O2 for 6 h, 12 h, 24 h, 48 h). Hypoxic rats developed pulmonary arterial remodeling and right ventricular hypertrophy with significantly increased pulmonary arterial pressure. The levels of Nur77, HIF-1α and PNCA were upregulated in pulmonary arterial smooth muscle from hypoxic rats. Silencing of either Nur77 or HIF-1α attenuated hypoxia-induced proliferation. Silencing of HIF-1α down-regulated Nur77 protein level, but Nur77 silence did not reduce HIF-1α. Nur77 was not con-immunoprecipitated with HIF-1α. This study demonstrated that Nur77 acted as a downstream regulator of HIF-1α under hypoxia, and plays a critical role in the hypoxia-induced pulmonary vascular remodeling, which is regulated by HIF-1α. Nur77 maybe a novel target of HPH therapy.
Subject(s)
Hypertension, Pulmonary , Hypoxia-Inducible Factor 1, alpha Subunit , Hypoxia , Nuclear Receptor Subfamily 4, Group A, Member 1 , Pulmonary Artery , Rats, Sprague-Dawley , Vascular Remodeling , Animals , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Vascular Remodeling/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/genetics , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Hypoxia/metabolism , Cell Proliferation , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Rats , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/pathology , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/genetics , Cells, CulturedABSTRACT
Right ventricular (RV) function is an important prognostic indicator for pulmonary arterial hypertension (PAH), a vasculopathy that primarily and disproportionally affects women with distinct pre- and postmenopausal clinical outcomes. However, most animal studies have overlooked the impact of sex and ovarian hormones on RV remodeling in PAH. Here, we combined invasive measurements of RV hemodynamics and morphology with computational models of RV biomechanics in sugen-hypoxia (SuHx)-treated male, ovary-intact female, and ovariectomized female rats. Despite similar pressure overload levels, SuHx induced increases in end-diastolic elastance and passive myocardial stiffening, notably in male SuHx animals, corresponding to elevated diastolic intracellular calcium. Increases in end-systolic chamber elastance were largely explained by myocardial hypertrophy in male and ovary-intact female rats, whereas ovariectomized females exhibited contractility recruitment via calcium transient augmentation. Ovary-intact female rats primarily responded with hypertrophy, showing fewer myocardial mechanical alterations and less stiffening. These findings highlight sex-related RV remodeling differences in rats, affecting systolic and diastolic RV function in PAH.NEW & NOTEWORTHY Combining hemodynamic and morphological measurements from male, female, and ovariectomized female pulmonary arterial hypertension (PAH) rats revealed distinct adaptation mechanisms despite similar pressure overload. Males showed the most diastolic stiffening. Ovariectomized females had enhanced myocyte contractility and calcium transient upregulation. Ovary-intact females primarily responded with hypertrophy, experiencing milder passive myocardial stiffening and no changes in myocyte shortening. These findings suggest potential sex-specific pathways in right ventricular (RV) adaptation to PAH, with implications for targeted interventions.
Subject(s)
Disease Models, Animal , Ovariectomy , Pulmonary Arterial Hypertension , Rats, Sprague-Dawley , Ventricular Function, Right , Ventricular Remodeling , Animals , Female , Male , Pulmonary Arterial Hypertension/physiopathology , Pulmonary Arterial Hypertension/metabolism , Pulmonary Arterial Hypertension/etiology , Sex Factors , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/pathology , Rats , Ventricular Dysfunction, Right/physiopathology , Ventricular Dysfunction, Right/metabolism , Ventricular Dysfunction, Right/etiology , Pulmonary Artery/physiopathology , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Models, Cardiovascular , Calcium Signaling , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/etiology , HemodynamicsABSTRACT
Pulmonary arterial hypertension (PAH) is a fatal disease featured by high morbidity and mortality. Although Cordycepin is known for its anti-inflammatory, antioxidant and immune-enhancing effects, its role in PAH treatment and the underlying mechanisms remain unclear. The therapeutic effects of Cordycepin on rats with PAH were investigated using a monocrotaline (MCT)-induced rat model. The metabolic effects of Cordycepin were assessed based on the plasma metabolome. The potential mechanisms of Cordycepin in PAH treatment were investigated through transcriptome sequencing and validated in pulmonary artery smooth muscle cells (PASMC). Evaluations included hematoxylin and eosin staining for pulmonary vascular remodeling, CCK-8 assay, EDU, and TUNEL kits for cell viability, proliferation, and apoptosis, respectively, and western blot for protein expression. Cordycepin significantly reduced right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) in PAH rats, and mitigated pulmonary vascular remodeling. Plasma metabolomics showed that Cordycepin could reverse the metabolic disorders in the lungs of MCT-induced PAH rats, particularly impacting linoleic acid and alpha-linolenic acid metabolism pathways. Transcriptomics revealed that the P53 pathway might be the primary pathway involved, and western blot results showed that Cordycepin significantly increased P53 and P21 protein levels in lung tissues. Integrated analysis of transcriptomics and metabolomics suggested that these pathways were mainly enriched in linoleic acid metabolism and alpha-linolenic acid metabolism pathway. In vitro experiments demonstrated that Cordycepin significantly inhibited the PDGFBB (PD)-induced abnormal proliferation and migration of PASMC and promoted PD-induced apoptosis. Meanwhile, Cordycepin enhanced the expression levels of P53 and P21 proteins in PD-insulted PASMC. However, inhibitors of P53 and P21 eliminated these effects of Cordycepin. Cordycepin may activate the P53-P21 pathway to inhibit abnormal proliferation and migration of PASMC and promote apoptosis, offering a potential approach for PAH treatment.
Subject(s)
Apoptosis , Cell Proliferation , Deoxyadenosines , Pulmonary Arterial Hypertension , Animals , Deoxyadenosines/pharmacology , Deoxyadenosines/therapeutic use , Rats , Male , Apoptosis/drug effects , Pulmonary Arterial Hypertension/drug therapy , Pulmonary Arterial Hypertension/metabolism , Pulmonary Arterial Hypertension/pathology , Cell Proliferation/drug effects , Transcriptome/drug effects , Metabolomics , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/drug effects , Monocrotaline , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Rats, Sprague-Dawley , Disease Models, Animal , Vascular Remodeling/drug effects , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Linoleic Acid/pharmacology , Hypertrophy, Right Ventricular/drug therapy , Hypertrophy, Right Ventricular/metabolism , Gene Expression ProfilingABSTRACT
Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance (PVR), right ventricular (RV) failure and premature death. Compounds with vasodilatory characteristics, such as ß-caryophyllene, could be promising therapeutics for PAH. This study aimed to determine the effects of free and nanoemulsified ß-caryophyllene in lung oxidative stress and heart function in PAH rats. Male Wistar rats (170 g, n = 6/group) were divided into four groups: control (CO), monocrotaline (MCT), monocrotaline + ß-caryophyllene (MCT-Bcar) and monocrotaline + nanoemulsion with ß-caryophyllene (MCT-Nano). PAH was induced by MCT (60 mg/kg i.p.), and 7 days later, treatment with ß-caryophyllene, either free or in a nanoemulsion (by gavage, 176 mg/kg/day) or vehicle was given for 14 days. Echocardiographic and hemodynamic measurements were performed, and after, the RV was collected for morphometry and the lungs for evaluation of oxidative stress, antioxidant enzymes, total sulfhydryl compounds, nitric oxide synthase (NOS) activity and endothelin-1 receptor expression. RV hypertrophy, increased PVR and RV systolic and diastolic pressures (RVSP and RVEDP, respectively) and increased mean pulmonary arterial pressure (mPAP) were observed in the MCT group. Treatment with both free and nanoemulsified ß-caryophyllene reduced RV hypertrophy, mPAP, RVSP and lipid peroxidation. The reduction in RVSP was more pronounced in the MCT-Nano group. Moreover, RVEDP decreased only in the MCT-Nano group. These treatments also increased superoxide dismutase, catalase and NOS activities and decreased endothelin-1 receptors expression. Both ß-caryophyllene formulations improved mPAP, PVR and oxidative stress parameters. However, ß-caryophyllene in a nanoemulsion was more effective in attenuating the effects of PAH.
Subject(s)
Hypertension, Pulmonary , Polycyclic Sesquiterpenes , Pulmonary Arterial Hypertension , Rats , Male , Animals , Pulmonary Arterial Hypertension/metabolism , Monocrotaline/toxicity , Monocrotaline/metabolism , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/metabolism , Rats, Wistar , Pulmonary Artery/metabolism , Hypertrophy, Right Ventricular/chemically induced , Hypertrophy, Right Ventricular/metabolismABSTRACT
Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine-histidine-purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.
Subject(s)
Disease Models, Animal , Hypertension, Pulmonary , Hypertrophy, Right Ventricular , Metabolomics , Proteomics , Animals , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/diagnostic imaging , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/diagnostic imaging , Ventricular Function, Right , Ventricular Remodeling , Sus scrofa , Swine , MaleABSTRACT
ABSTRACT: Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance (PVR), imposing overload on the right ventricle (RV) and imbalance of the redox state. Our study investigated the influence of treatment with sulforaphane (SFN), found in cruciferous vegetables, on RV remodeling and redox homeostasis in monocrotaline (MCT)-induced PAH. Male Wistar rats were separated into 4 groups: control (CTR); CTR + SFN; MCT; and MCT + SFN. PAH induction was implemented by a single dose of MCT (60 mg/kg intraperitoneally). Treatment with SFN (2.5 mg/kg/day intraperitoneally) started on the seventh day after the MCT injection and persisted for 2 weeks. After 21 days of PAH induction, echocardiographic, hemodynamic, and oxidative stress evaluation was performed. The MCT group showed an increase in RV hypertrophy, RV systolic area, RV systolic, mean pulmonary artery pressure, and PVR and exhibited a decrease in the RV outflow tract acceleration time/ejection time ratio, RV fractional shortening, and tricuspid annular plane systolic excursion compared to CTR ( P < 0.05). SFN-treated PAH attenuated detrimental changes in tricuspid annular plane systolic excursion, mean pulmonary artery pressure, and PVR parameters. Catalase levels and the glutathione/Glutathione disulfide (GSSG) ratio were diminished in the MCT group compared to CTR ( P < 0.05). SFN increased catalase levels and normalized the glutathione/GSSG ratio to control levels ( P < 0.05). Data express the benefit of SFN treatment on the cardiac function of rats with PAH associated with the cellular redox state.
Subject(s)
Disease Models, Animal , Isothiocyanates , Monocrotaline , Oxidation-Reduction , Oxidative Stress , Rats, Wistar , Sulfoxides , Ventricular Function, Right , Animals , Sulfoxides/pharmacology , Isothiocyanates/pharmacology , Male , Ventricular Function, Right/drug effects , Oxidative Stress/drug effects , Antioxidants/pharmacology , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/drug therapy , Homeostasis/drug effects , Ventricular Remodeling/drug effects , Myocardial Contraction/drug effects , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/chemically induced , Pulmonary Artery/drug effects , Pulmonary Artery/physiopathology , Pulmonary Artery/metabolism , Rats , Arterial Pressure/drug effects , Pulmonary Arterial Hypertension/drug therapy , Pulmonary Arterial Hypertension/physiopathology , Pulmonary Arterial Hypertension/metabolism , Ventricular Dysfunction, Right/physiopathology , Ventricular Dysfunction, Right/drug therapy , Ventricular Dysfunction, Right/metabolismABSTRACT
Purpose: Pulmonary arterial hypertension (PAH) is a devastating disease with little effective treatment. The proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by the nuclear factor-κB (NF-κB) signaling activation plays a pivotal role in the pathogenesis of PAH. Forsythoside B (FTSâ¢B) possesses inhibitory effect on NF-κB signaling pathway. The present study aims to explore the effects and mechanisms of FTSâ¢B in PAH. Methods: Sprague-Dawley rats received monocrotaline (MCT) intraperitoneal injection to establish PAH model, and FTSâ¢B was co-treated after MCT injection. Right ventricular hypertrophy and pulmonary artery pressure were measured by echocardiography and right heart catheterization, respectively. Histological alterations were detected by H&E staining and immunohistochemistry. FTSâ¢B's role in PASMC proliferation and migration were evaluated by CCK-8 and wound healing assay. To investigate the underlying mechanisms, Western blotting, immunofluorescence staining and ELISA were conducted. The NF-κB activator PMA was used to investigate the role of NF-κB in FTSâ¢B's protective effects against PAH. Results: FTSâ¢B markedly alleviated MCT-induced vascular remodeling and pulmonary artery pressure, and improved right ventricular hypertrophy and survival. FTSâ¢B also reversed PDGF-BB-induced PASMC proliferation and migration, decreased PCNA and CyclinD1 expression in vitro. The elevated levels of IL-1ß and IL-6 caused by MCT were decreased by FTSâ¢B. Mechanistically, MCT-triggered phosphorylation of p65, IκBα, IKKα and IKKß was blunted by FTSâ¢B. FTSâ¢B also reversed MCT-induced nuclear translocation of p65. However, all these protective effects were blocked by PMA-mediated NF-κB activation. Conclusion: FTSâ¢B effectively attenuates PAH by suppressing the NF-κB signaling pathway to attenuate vascular remodeling. FTSâ¢B might be a promising drug candidate with clinical translational potential for the treatment of PAH.
Subject(s)
Caffeic Acids , Glucosides , Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Rats , Animals , NF-kappa B/metabolism , Monocrotaline/adverse effects , Rats, Sprague-Dawley , Vascular Remodeling , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/pathology , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/drug therapy , Signal TransductionABSTRACT
Pulmonary arterial hypertension (PAH) is a progressive disease that affects both the lungs and heart. Right ventricle (RV) hypertrophy is a primary pathological feature of PAH; however, its underlying molecular mechanisms remain insufficiently studied. In this study, we employed tandem mass tag (TMT)-based quantitative proteomics for the integrative analysis of the proteome and phosphoproteome of the RV derived from monocrotaline-induced PAH model rats. Compared with control samples, 564 significantly upregulated proteins, 616 downregulated proteins, 622 downregulated phosphopeptides, and 683 upregulated phosphopeptides were identified (P < 0.05, abs (log2 (fold change)) > log2 1.2) in the MCT samples. The quantitative real-time polymerase chain reaction (qRT-PCR) validated the expression levels of top 20 significantly altered proteins, including Nppa (natriuretic peptides A), latent TGF-ß binding protein 2 (Ltbp2), periostin, connective tissue growth factor 2 (Ccn2), Ncam1 (neural cell adhesion molecule), quinone reductase 2 (Nqo2), and tropomodulin 4 (Tmod4). Western blotting confirmed the upregulation of Ncam1 and downregulation of Nqo2 and Tmod4 in both MCT-induced and hypoxia-induced PH rat models. Pathway enrichment analyses indicated that the altered proteins are associated with pathways, such as vesicle-mediated transport, actin cytoskeleton organization, TCA cycle, and respiratory electron transport. These significantly changed phosphoproteins were enriched in pathways such as diabetic cardiomyopathy, hypertrophic cardiomyopathy, glycolysis/gluconeogenesis, and cardiac muscle contraction. In summary, this study provides an initial analysis of the RV proteome and phosphoproteome in the progression of PAH, highlighting several RV dysfunction-associated proteins and pathways.
Subject(s)
Hypertension, Pulmonary , Rats , Animals , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/metabolism , Hypertrophy, Right Ventricular/metabolism , Proteome/genetics , Phosphopeptides , ProteomicsABSTRACT
Fine particulate matter (PM2.5) as an environmental pollutant is related with respiratory and cardiovascular diseases. Pulmonary arterial hypertension (PAH) was characterized by incremental pulmonary artery pressure and pulmonary arterial remodeling, leading to right ventricular hypertrophy, and finally cardiac failure and death. The adverse effects on pulmonary artery and the molecular biological mechanism underlying PM2.5-caused PAH has not been elaborated clearly. In the current study, the ambient PM2.5 exposure mice model along with HPASMCs models were established. Based on bioinformatic methods and machine learning algorithms, the hub genes in PAH were screened and then adverse effects on pulmonary artery and potential mechanism was studied. Our results showed that chronic PM2.5 exposure contributed to increased pulmonary artery pressure, pulmonary arterial remodeling and right ventricular hypertrophy in mice. In vitro, PM2.5 induced phenotypic switching in HPASMCs, which served as the early stage of PAH. In mechanism, we investigated that PM2.5-mediated mitochondrial dysfunction could induce phenotypic switching in HPASMCs, which was possibly through reprogramming lipid metabolism. Next, we used machine learning algorithm to identify ELK3 as potential hub gene for mitochondrial fission. Besides, the effect of DNA methylation on ELK3 was further detected in HPASMCs after PM2.5 exposure. The results provided novel directions for protection of pulmonary vasculature injury, against adverse environmental stimuli. This work also provided a new idea for the prevention of PAH, as well as provided experimental evidence for the targeted therapy of PAH.
Subject(s)
Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Animals , Mice , Cell Proliferation , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/metabolism , Hypertrophy, Right Ventricular/metabolism , Lipid Metabolism , Myocytes, Smooth Muscle/metabolism , Particulate Matter/metabolism , Pulmonary Arterial Hypertension/metabolism , Pulmonary Artery/metabolism , Vascular RemodelingABSTRACT
BACKGROUND: This study aimed to determine whether postnatal treatment with recombinant human IGF-1 (rhIGF-1)/binding peptide 3 (BP3) ameliorates lung injury and prevents pulmonary hypertension (PH) in bronchopulmonary dysplasia (BPD) models. METHODS: We used two models of BPD in this study: one model that was associated with chorioamnionitis (CA), stimulated by intra-amniotic fluid and exposure to lipopolysaccharide (LPS), whereas the other was exposed to postnatal hyperoxia. Newborn rats were treated with rhIGF-1/BP3 (0.2 mg/Kg/d) or saline via intraperitoneal injection. The study endpoints included the wet/dry weight (W/D) ratio of lung tissues, radial alveolar counts (RACs), vessel density, right ventricular hypertrophy (RVH), lung resistance, and lung compliance. Hematoxylin and eosin (H&E) and Masson staining were used to evaluate the degree of lung injury and pulmonary fibrosis. IGF-1 and eNOS expression were detected using western blotting or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The levels of SP-C, E-cadherin, N-cadherin, FSP1, and Vimentin in the lung tissues were detected by immunofluorescence. RESULTS: LPS and hyperoxia treatment increased lung injury and pulmonary fibrosis, enhanced RVH and total respiratory resistance, and decreased RAC, pulmonary vascular density and pulmonary compliance in young mice (all p < 0.01). Simultaneously, LPS and hyperoxia induced an increase in epithelial-mesenchymal transition (EMT) in airway epithelial cells. However, rhIGF-1/BP3 treatment reduced lung injury and pulmonary fibrosis, decreased RVH and total respiratory resistance, and enhanced RAC, pulmonary vascular density and pulmonary compliance, as well as inhibited EMT in airway epithelial cells in LPS and hyperoxia treated mice. CONCLUSION: Postnatal rhIGF-1/BP3 treatment relieved the effects of LPS or hyperoxia on lung injury and prevented RVH, providing a promising strategy for the treatment of BPD.
Subject(s)
Bronchopulmonary Dysplasia , Hyperoxia , Hypertension, Pulmonary , Lung Injury , Pulmonary Fibrosis , Infant, Newborn , Pregnancy , Female , Rats , Animals , Humans , Mice , Bronchopulmonary Dysplasia/metabolism , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/prevention & control , Hypertension, Pulmonary/metabolism , Lung Injury/metabolism , Hyperoxia/metabolism , Lipopolysaccharides/pharmacology , Pulmonary Fibrosis/pathology , Animals, Newborn , Insulin-Like Growth Factor I/metabolism , Rats, Sprague-Dawley , Lung/pathology , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/prevention & control , Hypertrophy, Right Ventricular/metabolism , Disease Models, AnimalABSTRACT
Right ventricular (RV) failure is the major determinant of outcome in pulmonary hypertension (PH). Calves exposed to 2-wk hypoxia develop severe PH and unlike rodents, hypoxia-induced PH in this species can lead to right heart failure. We, therefore, sought to examine the molecular and structural changes in the RV in calves with hypoxia-induced PH, hypothesizing that we could identify mechanisms underlying compensated physiological function in the face of developing severe PH. Calves were exposed to 14 days of environmental hypoxia (equivalent to 4,570 m/15,000 ft elevation, n = 29) or ambient normoxia (1,525 m/5,000 ft, n = 25). Cardiopulmonary function was evaluated by right heart catheterization and pressure volume loops. Molecular and cellular determinants of RV remodeling were analyzed by cDNA microarrays, RealTime PCR, proteomics, and immunochemistry. Hypoxic exposure induced robust PH, with increased RV contractile performance and preserved cardiac output, yet evidence of dysregulated RV-pulmonary artery mechanical coupling as seen in advanced disease. Analysis of gene expression revealed cellular processes associated with structural remodeling, cell signaling, and survival. We further identified specific clusters of gene expression associated with 1) hypertrophic gene expression and prosurvival mechanotransduction through YAP-TAZ signaling, 2) extracellular matrix (ECM) remodeling, 3) inflammatory cell activation, and 4) angiogenesis. A potential transcriptomic signature of cardiac fibroblasts in RV remodeling was detected, enriched in functions related to cell movement, tissue differentiation, and angiogenesis. Proteomic and immunohistochemical analysis confirmed RV myocyte hypertrophy, together with localization of ECM remodeling, inflammatory cell activation, and endothelial cell proliferation within the RV interstitium. In conclusion, hypoxia and hemodynamic load initiate coordinated processes of protective and compensatory RV remodeling to withstand the progression of PH.NEW & NOTEWORTHY Using a large animal model and employing a comprehensive approach integrating hemodynamic, transcriptomic, proteomic, and immunohistochemical analyses, we examined the early (2 wk) effects of severe PH on the RV. We observed that RV remodeling during PH progression represents a continuum of transcriptionally driven processes whereby cardiac myocytes, fibroblasts, endothelial cells, and proremodeling macrophages act to coordinately maintain physiological homeostasis and protect myocyte survival during chronic, severe, and progressive pressure overload.
Subject(s)
Heart Failure , Hypertension, Pulmonary , Ventricular Dysfunction, Right , Animals , Cattle , Hypertension, Pulmonary/metabolism , Endothelial Cells/metabolism , Mechanotransduction, Cellular , Proteomics , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/metabolism , Heart Ventricles , Disease Models, Animal , Hypoxia , Ventricular Remodeling , Ventricular Function, Right , Ventricular Dysfunction, Right/genetics , Ventricular Dysfunction, Right/complicationsABSTRACT
Hypoxia leads to hypoxic pulmonary hypertension (HPH), causing right ventricular hypertrophy (RVH). RVH becomes a significant and nonnegligible public health issue in the world. In our study, we successfully established the HPH rat model and found that RVH happened in HPH, and then we observed an increased inflammation response in the heart tissue of HPH-induced RVH rats. Moreover, increased N-deacetylase-N-sulfotransferase-1 (NDST1) and decreased nuclear localized protein 1 (NULP1) were found in the heart tissue of HPH-induced RVH rats. An in vitro cell experiment showed that inhibition of NDST1 expression enhanced cell viability, reduced cell apoptosis, alleviated cardiomyocyte hypertrophy, decreased inflammation and increased phosphorylated AKT level, however, over-expression of NDST1 had opposite effects on these aspects. NULP1 reversed the effects of NDST1 on these regulations. Finally, we found that up-regulated NDST1 reduced NULP1 expression and down-regulated NDST1 increased NULP1 expression. Our study confirmed that inhibition of the NDST1/NULP1 pathway might contribute to the attenuation of HPH-induced RVH, and the mechanism may be related to the reduction of inflammation, cardiomyocyte apoptosis, and AKT phosphorylation.
Subject(s)
Hypertension, Pulmonary , Hypertrophy, Right Ventricular , Animals , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/metabolism , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/metabolism , Hypoxia/complications , Hypoxia/metabolism , Inflammation , Proto-Oncogene Proteins c-akt , Rats , SulfotransferasesABSTRACT
PURPOSE: It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. METHOD: PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. RESULTS: FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. CONCLUSION: FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.
Subject(s)
Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Actins/metabolism , Animals , Autophagy , Disease Models, Animal , Familial Primary Pulmonary Hypertension , Forkhead Box Protein M1/metabolism , Forkhead Box Protein M1/therapeutic use , Hypertension, Pulmonary/drug therapy , Hypertrophy, Right Ventricular/chemically induced , Hypertrophy, Right Ventricular/metabolism , Ki-67 Antigen/metabolism , Monocrotaline/metabolism , Monocrotaline/toxicity , Phosphorylation , Pulmonary Arterial Hypertension/chemically induced , Pulmonary Artery , Rats , Rats, Sprague-DawleyABSTRACT
Nearly 1 in every 100 children born have a congenital heart defect. Many of these defects primarily affect the right heart causing pressure overload of the right ventricle (RV). The RV maintains function by adapting to the increased pressure; however, many of these adaptations eventually lead to RV hypertrophy and failure. In this study, we aim to identify the cellular and molecular mechanisms of these adaptions. We utilized a surgical animal model of pulmonary artery banding (PAB) in juvenile rats that has been shown to accurately recapitulate the physiology of right ventricular pressure overload in young hearts. Using this model, we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post-banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK). Single myocyte calcium and contractility recordings showed impaired contraction and relaxation in PAB RV myocytes, consistent with the loss of cMLCK. In the PAB myocytes, calcium transients were of smaller amplitude and decayed at a slower rate compared to controls. We also identified miR-200c, which has been shown to regulate cMLCK expression, as upregulated in the RV in response to pressure overload. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects.
Subject(s)
Myosin-Light-Chain Kinase , Ventricular Dysfunction, Right , Animals , Disease Models, Animal , Heart Ventricles/metabolism , Hypertrophy, Right Ventricular/metabolism , Myocytes, Cardiac/metabolism , Rats , Ventricular Dysfunction, Right/etiology , Ventricular Function, Right/physiology , Ventricular Pressure/physiologyABSTRACT
In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group-sacrificed 4 weeks after MCT administration and ptMCT group-prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr231 /Ser232 -RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr231 /Ser232 -RIP3 levels in both PAH subgroups were associated rather with GSDMD-mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH-induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr231 /Ser232 -RIP3 upregulation resulting in two different necrosis-like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.
Subject(s)
Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Animals , DNA-Binding Proteins , Disease Models, Animal , Familial Primary Pulmonary Hypertension , Hypertension, Pulmonary/metabolism , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/metabolism , Monocrotaline/toxicity , Necroptosis , Pyroptosis , Rats , Rats, WistarABSTRACT
For the first time, the present study unravels a cardiospecific therapeutic approach for Pulmonary Arterial Hypertension (PAH), a disease with a very poor prognosis and high mortality rates due to right ventricle (RV) dysfunction. We first established a new in vitro model of high-pressure-induced hypertrophy that closely resembles heart defects associated with PAH and validated our in vitro findings on a preclinical in vivo model of monocrotaline (MCT)-induced PAH. Our results showed the in vitro antihypertrophic effect of 1,8-cineole, a monoterpene widely found in several essential oils. Also, a decrease in RV hypertrophy and fibrosis, and an improvement in heart function in vivo was observed, when 1,8-cineole was applied topically. Furthermore, 1,8-cineole restored gap junction protein connexin43 distribution at the intercalated disks and mitochondrial functionality, suggesting it may act by preserving cardiac cell-to-cell communication and bioenergetics. Overall, our results point out a promising therapeutic compound that can be easily applied topically, thus paving the way for the development of effective cardiac-specific therapies to greatly improve PAH outcomes.