Pulmonary Hypertension In Rats Research Articles

C-C motif chemokine receptor-2 blockade ameliorates pulmonary hypertension in rats and synergizes with a pulmonary vasodilator

Abstract Aims We investigated whether the disruption of C-C motif chemokine receptor (CCR) 2 may attenuate the development of pulmonary arterial hypertension (PAH) in any rat models with the reversal of the associated pro-inflammatory state and vascular dysfunction, and synergize with a conventional pulmonary vasodilator. Methods and Results Using Ccr2(-/-) rats generated by CRISPR/Cas9, we investigated pulmonary hypertension (PH) in Ccr2(+/+) or Ccr2(-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx) and chronic hypoxia (CH). Ccr2(-/-) decreased the right ventricular systolic pressure, an index of right ventricular hypertrophy and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (interleukin-6, tumor necrosis factor-α, C-C motif chemokine receptor (CCL)-2, interleukin-1β, transforming growth factor-β) in rats 3weeks after MCT injection, but not in SuHx or CH models. Consistently, Ccr2(-/-) decreased indices of pulmonary vascular diseases (PVD) and perivascular macrophage infiltration, as well as reversed impaired bone morphogenetic protein receptor type 2 signaling, increased endothelial apoptosis and impaired nitric oxide signaling and decreased phosphodiesterase-5 (PDE5) expression in lungs in MCT-treated rats. Gene expression of receptors for prostaglandin I2 and endothelin was not changed by Ccr2(-/-) in MCT-treated rats. In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2(-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. The whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2(-/-) PASMCs, which are related to regulation of cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2(-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2(-/-) rats but not in Ccr2(+/+) rats. Tadalafil further improved survival in MCT-treated Ccr2(-/-) rats. Conclusion The present findings demonstrated that CCR2 disruption ameliorated PAH in MCT-treated rats, which was associated with the reversal of dysregulated inflammatory pathways and vascular dysfunction and synergized with tadalafil. These findings suggest that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

Abstract 4123631: Significance of collagen triple helix repeat-containing protein 1 as a right ventricular biomarker in pulmonary hypertension

Background: Right ventricular failure is a major prognostic determinant of pulmonary hypertension and progresses with myocardial fibrosis. Extracellular matrix proteins play a major role in myocardial fibrosis. Right ventricular biomarkers for right ventricular function and fibrosis are poorly examined in pulmonary hypertension. This study investigated the significance of collagen triple helix repeat-containing protein 1 (CTHRC1), one of the extracellular matrix proteins, as a right ventricular biomarker. Methods and Results: A monocrotaline-induced pulmonary hypertension rat model was used in this study. CTHRC1 expression in the right ventricle was higher in rats with monocrotaline-induced pulmonary hypertension than in controls. CTHRC1 was associated with fibrosis in the rat's right ventricle. Next, human circulating CTHRC1 levels were evaluated in controls (n = 20), pulmonary arterial hypertension (PAH, n = 46), and chronic thromboembolic pulmonary hypertension (CTEPH) patients (n = 64). Human circulating CTHRC1 levels were higher in PAH (P = 0.006) and CTEPH patients (P = 0.011) than in controls (Figure). Human circulating CTHRC levels were correlated with tricuspid lateral annular peak systolic velocity (R = -0.213, P = 0.029), right ventricular fractional area change (R = -0.225, P = 0.017), and right ventricular global longitudinal strain evaluated by magnetic resonance imaging (R = 0.429, P = 0.009). In addition, human circulating CTHRC1 levels were decreased after balloon pulmonary angioplasty (P < 0.001) in CTEPH patients. Conclusions: CTHRC1 could be a novel biomarker associated with right ventricular failure and fibrosis in pulmonary hypertension patients.

Abstract 4137240: MHCII hi LYVE1 lo CCR2 hi Interstitial Macrophages Promote Medial Fibrosis in Pulmonary Arterioles and Contribute to Pulmonary Hypertension

Background: Pulmonary hypertension (PH) is a lethal disease characterized in part by progressive pulmonary arteriole (PA) remodeling. Excessive PA fibrosis and macrophage infiltration are often present in PH, but potential association is obscure. We invesitgated the link between interstitial macrophage (iMΦ) infiltration and PA fibrosis in PH and idiopathic pulmonary arterial hypertension (IPAH). Methods: Lung tissue samples from patients with IPAH (n=11) and experimental PH (sugen-5416 and hypoxia, hypoxia, or monocrotaline, n=5) animals (adult male and female SD rats) were obtained to analyze the extent of fibrosis in PA adventitia and media and their correlation to disease severity. Single-cell RNA sequencing, immunohistological analyses, iMΦs and PA smooth muscle cells (PASMCs) coculture, and transgenic animal experiments were used to investigate the cell heterogeneity and molecular mechanisms by which iMΦs promote PA medial fibrosis. Data in this study were analyzed with unpaired two-tailed t-tests with Welch’s correction, Kruskal-Wallis one-way ANOVA followed by Dunn’s test, RM ANOVA with Geisser-Greenhouse correction, or Pearson correlation test, and p < 0.05 was considered statistically significant. Results: We found that increased collagen deposition and fibrosis in the PA media were correlated with PH severity, and iMΦ medial infiltration may be involved in these pathological processes. Single-cell transcriptomics suggested that ligand-receptor associations between MHCII hi LYVE1 lo CCR2 hi iMΦ and fibroblast-like vascular SMC subclusters contributed to PA medial fibrosis. Mechanistically, MHCII hi LYVE1 lo CCR2 hi iMΦs regulated PASMC phenotypic transition and collagen production through the wingless member 11 (WNT11)/planar cell polarity (PCP) pathway. Deletion of iMΦ-enriched Wnt11 in myeloid cells of PH rats ( Itgam CreERT2 ; Wnt11 fl/fl ) normalized the fibrotic PASMC phenotype and decreased PA medial fibrosis, thereby protecting against PH. Moreover, myeloid-specific C-C motif chemokine receptor 2 ( Ccr2 ) deficiency ( Itgam CreERT2 ; Ccr2 fl/fl ) in PH-PAs inhibited MHCII hi LYVE1 lo CCR2 hi iMΦ infiltration. Conclusions: PA medial fibrosis is a causative factor of PH, and the inhibition of MHCII hi LYVE1 lo CCR2 hi iMΦ pathogenicity or infiltration reverses PA medial fibrosis and thus alleviates PH.

The HIF2α-dependent Upregulation of SETDB1 Facilitates Hypoxia-induced Functional and Phenotypical Changes of Pulmonary Microvascular Endothelial Cells.

Background: Emerging studies have reported the vital role of histone modification in the dysfunction of pulmonary vascular endothelial cells, which acts as the key reason to drive the hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension (PH). This study aims to investigate the role of a histone 3 lysine 9 (H3K9) methyltransferase, SET domain bifurcated 1 (SETDB1), in hypoxia-induced functional and phenotypical changes of pulmonary vascular endothelial cells. Methods: Primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as cell model. Specific knockdown and overexpression strategies were used to systematically determine the molecular regulation and function of SETDB1 in PMVECs. Results: SETDB1 is highly expressed and significantly upregulated in the pulmonary vascular endothelium of lung tissue isolated from SU5416/hypoxia-induced PH (SuHx-PH) rats, and also in pulmonary arterial endothelial cells (PAECs) from idiopathic pulmonary arterial hypertension (IPAH) patients, comparing to their respective controls. In primarily cultured rat PMVECs, treatment of hypoxia or CoCl2 induces significant upregulation of HIF2α, SETDB1 and H3K9me3. Specific knockdown and overexpression strategies indicate the hypoxia- or CoCl2-induced upregulation of SETDB1 is mediated through a HIF2α-dependent mechanism. Knockdown of SETDB1 significantly inhibits the hypoxia- or CoCl2-induced apoptosis, senescence and endothelial to mesenchymal transition (EndoMT) in rat PMVECs. Moreover, treatment of the specific inhibitor of histone methyltransferase, Chaetocin, effectively attenuates the disease pathogenesis of SuHx-PH in rat. Conclusions: Our results suggest that the HIF2α-dependent upregulation of SETDB1 facilitates hypoxia-induced functional and phenotypical changes of PMVECs, potentially contributing to the hypoxia-induced pulmonary vascular remodeling and PH.

Honokiol and Nicotinamide Adenine Dinucleotide Improve Exercise Endurance in Pulmonary Hypertensive Rats Through Increasing SIRT3 Function in Skeletal Muscle.

Pulmonary hypertension (PH) significantly impairs exercise capacity and the quality of life in patients, which is influenced by dysfunctions in multiple organ systems, including the right ventricle, lungs, and skeletal muscles. Recent research has identified metabolic reprogramming and mitochondrial dysfunction as contributing factors to reduced exercise tolerance in PH patients. In this study, we investigated the therapeutic potential of enhancing mitochondrial function through the activation of the mitochondrial deacetylase SIRT3, using SIRT3 activator Honokiol combined with the SIRT3 co-factor nicotinamide adenine dinucleotide (NAD), in a Sugen/Hypoxia-induced PH rat model. Our results show that Sugen/Hypoxia-induced PH significantly impairs RV, lung, and skeletal muscle function, leading to reduced exercise capacity. Treatment with Honokiol and NAD notably improved exercise endurance, primarily by restoring SIRT3 levels in skeletal muscles, reducing proteolysis and atrophy in the gastrocnemius, and enhancing mitochondrial complex I levels in the soleus. These effects were independent of changes in cardiopulmonary hemodynamics. We concluded that targeting skeletal muscle dysfunction may be a promising approach to improving exercise capacity and overall quality of life in PH patients.

Cathepsin L Promotes Pulmonary Hypertension via BMPR2/GSDME-Mediated Pyroptosis.

Pulmonary hypertension (PH) is a fatal progressive disease characterized by pulmonary endothelial injury and occlusive pulmonary vascular remodeling. Lysosomal protease cathepsin L degrades essential molecules to participate in the human pathophysiological process. BMPR2 (bone morphogenetic protein type II receptor) deficiency, an important cause of PH, results from mutational inactivation or excessive lysosomal degradation and induces caspase-3-mediated cell death. Given recent evidence that pyroptosis, as a new form of programmed cell death, is induced by caspase-3-dependent GSDME (gasdermin E) cleavage, we hypothesized that cathepsin L might promote PH through BMPR2/caspase-3/GSDME axis-mediated pyroptosis. Cathepsin L expression was evaluated in the lungs and plasma of patients with pulmonary arterial hypertension. The role of cathepsin L in the progression of PH and vascular remodeling was assessed in vivo. Small interfering RNA, specific inhibitors, and lentiviruses were used to explore the mechanisms of cathepsin L on human pulmonary arterial endothelial cell dysfunction. Cathepsin L expression is elevated in pulmonary artery endothelium from patients with idiopathic pulmonary arterial hypertension and experimental PH models. Genetic ablation of cathepsin L in PH rats relieved right ventricular systolic pressure, pulmonary vascular remodeling, and right ventricular hypertrophy, also restoring endothelial integrity. Mechanistically, cathepsin L promotes caspase-3/GSDME-mediated endothelial cell pyroptosis and represses BMPR2 signaling activity. Cathepsin L degrades BMPR2 via the lysosomal pathway, and restoring BMPR2 signaling prevents the pro-pyroptotic role of cathepsin L in PAECs and experimental PH models. These results show for the first time that cathepsin L promotes the development of PH by degrading BMPR2 to induce caspase-3/GSDME-mediated endothelial pyroptosis.

LncRNA VELRP Modulates Pulmonary Arterial Smooth Muscle Cell Proliferation and Promotes Vascular Remodeling in Pulmonary Hypertension.

Pulmonary hypertension is a devastating vascular disorder characterized by extensive pulmonary vascular remodeling, ultimately leading to right ventricular failure and death. Activation of PDGF (platelet-derived growth factor) signaling promotes the hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs), thus contributing to the pulmonary vascular remodeling. However, the molecular mechanisms that govern hyperproliferation of PASMCs induced by PDGF remain largely unknown, including the contribution of long noncoding RNAs (lncRNAs). In this study, we aimed to identify a novel lncRNA regulated by PDGF implicated in PASMC proliferation in pulmonary vascular remodeling. RNA-sequencing analysis was conducted to identify a novel lncRNA named vessel-enriched lncRNA regulated by PDGF-BB (VELRP). Functional investigations of VELRP were performed using knockdown and overexpression strategies along with RNA sequencing. Validation of the function and potential mechanisms of VELRP were performed through Western blot, RNA immunoprecipitation, and chromatin immunoprecipitation assays. We identified a novel vessel-enriched lncRNA with an increased response to PDGF-BB stimulus. VELRP was identified as an evolutionarily conserved RNA molecules. Modulation of VELRP in PASMCs significantly altered cell proliferation. Mechanistically, VELRP enhances trimethylation of H3K4 by interacting with WDR5 (WD repeat-containing protein 5), leading to increased expression of CDK (cyclin-dependent kinase) 1, CDK2, and CDK4 and consequent hyperproliferation of PASMCs. The pathological relevance of VELRP upregulation in pulmonary artery was confirmed using rat pulmonary hypertension models in vivo, as well as in PASMCs from patients with idiopathic pulmonary arterial hypertension patients. Specific knockdown of VELRP in smooth muscle cells using adeno-associated virus type 9 SM22α (smooth muscle protein 22α) promoter-shRNA-mediated silencing of VELRP resulted in a significant decrease in right ventricular systolic pressure and vascular remodeling in rat pulmonary hypertension model. VELRP, as an lncRNA upregulated by PDGF-BB, mediates PASMC proliferation via WDR5/CDK signaling. In vivo studies demonstrate that targeted intervention of VELRP in smooth muscle cells can prevent the development of pulmonary hypertension.

Current Overview of the Biology and Pharmacology in Sugen/Hypoxia-Induced Pulmonary Hypertension in Rats.

The Sugen 5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension (PAH) demonstrates most of the distinguishing features of PAH in humans, including increased wall thickness and obstruction of the small pulmonary arteries along with plexiform lesion formation. Recently, significant advancement has been made describing the epidemiology, genomics, biochemistry, physiology, and pharmacology in Su/Hx challenge in rats. For example, there are differences in the overall reactivity to Su/Hx challenge in different rat strains and only female rats respond to estrogen treatments. These conditions are also encountered in human subjects with PAH. Also, there is a good translation in both the biochemical and metabolic pathways in the pulmonary vasculature and right heart between Su/Hx rats and humans, particularly during the transition from the adaptive to the nonadaptive phase of right heart failure. Noninvasive techniques such as echocardiography and magnetic resonance imaging have recently been used to evaluate the progression of the pulmonary vascular and cardiac hemodynamics, which are important parameters to monitor the efficacy of drug treatment over time. From a pharmacological perspective, most of the compounds approved clinically for the treatment of PAH are efficacious in Su/Hx rats. Several compounds that show efficacy in Su/Hx rats have advanced into phase II/phase III studies in humans with positive results. Results from these drug trials, if successful, will provide additional treatment options for patients with PAH and will also further validate the excellent translation that currently exists between Su/Hx rats and the human PAH condition.

Hypoxic Preconditioned Tibetan Mesenchymal Stem Cell-derived Exosomes Ameliorate Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension Rats

BackgroundPulmonary hypertension associated with hypoxia frequently manifests in a range of long-standing lung disorders. A strategy to augment the therapeutic efficacy of mesenchymal stem cells (MSCs) involves subjecting them to hypoxic preconditioning, which bolsters the emission of exosomes exhibiting enhanced bioactivities through paracrine mechanisms. The objective of this research is to explore the healing impact of preconditioning with hypoxia on MSC-extracted exosomes in relation to vascular structural changes within the lungs of rodents afflicted by hypoxia-induced pulmonary arterial hypertension. Methods and ResultsThis research entailed the successful isolation, cultivation of mesenchymal stem cells from Tibetan umbilical cords, alongside the extraction of their exosomes. Our results highlight that exosomes derived from hypoxia-conditioned Tibetan cord mesenchymal stem cells (H-Tibetan-exo) demonstrated superior efficacy in suppressing pulmonary vascular structural changes compared to those from normoxic conditions (N-Tibetan-exo) in a rat model of hypoxia-induced pulmonary hypertension (HPH).In an effort to replicate pulmonary hypertension conditions within a laboratory setting, we subjected rat pulmonary arterial smooth muscle cells (rPASMCs) to a low oxygen environment. Our findings revealed that exosomes derived from high-altitude Tibetans (H-Tibetan-exo) showed a superior capacity in suppressing the growth of rPASMCs compared to those obtained from normoxic Tibetans (N-Tibetan-exo). Additionally, the Transwell assay revealed a more significant inhibition of rPASMCs migration by H-Tibetan-exo. The investigation of PI3K-AKT-mTOR pathway protein expression through western blot analysis revealed that under hypoxic conditions, there was an elevation in the phosphorylation status of these proteins in both cellular and tissue settings. Furthermore, the H-Tibetan-exo exhibited more effective inhibition of rPASMCs proliferation and a more significant downregulation of PI3K-AKT-mTOR pathway protein phosphorylation levels compared to the N-Tibetan-exo. ConclusionsH-Tibetan-exo significantly ameliorates pulmonary vascular remodeling in hypoxic pulmonary hypertension more effectively than N-Tibetan-exo. The enhancement observed could be linked to reduced activity of the PI3K-AKT-mTOR pathway. This discovery sheds fresh light on managing hypoxic pulmonary hypertension, highlighting the potential therapeutic benefits of H-Tibetan-exo in mitigating its development.

Magnesium Sulfate, Rosuvastatin, Sildenafil and Their Combination in Chronic Hypoxia-Induced Pulmonary Hypertension in Male Rats.

Previous experimental findings have led to considerable interest in the beneficial effects on pulmonary hypertension (PH) produced by sildenafil and in the pleiotropic effects of rosuvastatin and their positive role in the process of pulmonary angiogenesis. However, magnesium sulfate, the most abundant intracellular cation, is essential in vascular endothelial functionality due to its anti-inflammatory and vasodilatory effects. Therefore, the present study aims to assess these treatment regimens and how they could potentially provide some additional benefits in PH therapy. Fourteen days after chronic-hypoxia PH was induced, rosuvastatin, sildenafil and magnesium sulfate were administered for an additional fourteen days to male Wistar rats. The Fulton Index, right ventricle (RV) anterior wall thickness, RV internal diameter and pulmonary arterial (PA) acceleration time/ejection time were evaluated, and another four biochemical parameters were calculated: brain natriuretic peptide, vascular endothelial growth factor, nitric oxide metabolites and endothelin 1. The present study demonstrates that sildenafil and rosuvastatin have modest effects in reducing RV hypertrophy and RV systolic pressure. The drug combination of sildenafil + rosuvastatin + magnesium sulfate recorded statistically very highly significant results on all parameters; through their positive synergistic effects on vascular endothelial function, oxidative stress and pathological RV remodeling, they attenuated PH in the chronic hypoxia pulmonary hypertension (CHPH) rat model.

Long-term swimming exercise attenuates right ventricular hypertrophy via modulating meta-inflammation in monocrotaline-induced pulmonary hypertensive rats

Pulmonary arterial hypertension (PAH) is a complex disease characterized by elevated pulmonary vascular resistance, resulting in right ventricular (RV) hypertrophy and, eventually, failure, which remains the primary cause of mortality in PAH patients. While current PAH therapies primarily target vascular abnormalities, most fail to address RV dysfunction. Therefore, improving RV function is a critical treatment goal. Exercise has emerged as an effective intervention for PAH, but the specific impact of swimming exercise on this disease and its associated pathological changes has been less extensively studied. In this study, we investigated the effects of swimming training (60 min/day, 5 days/week for 4 weeks) on monocrotaline (MCT; 60 mg/kg, i. p.)-induced PAH in rats. Our findings demonstrate that swimming significantly attenuates RV hypertrophy and reduces mean pulmonary arterial pressure (MPAP), mitigating the detrimental effects of PAH. Furthermore, we observed structural remodeling in the right ventricle, including increased myocardial necrosis, collagen deposition, and fibrosis-related protein expression. Swimming exercise training was found to reduce these pathological changes, suggesting a protective effect on the right ventricle. Mechanistically, our study revealed the crucial role of meta-inflammation in PAH and the anti-PAH effects of exercise. Swimming training attenuated macrophage accumulation, reduced serum inflammatory cytokines, and improved systemic and RV insulin sensitivity, highlighting its potential to modulate meta-inflammatory processes. In summary, our study suggests that swimming training exerts a beneficial effect on RV function and hypertrophy in MCT-induced PAH rats by targeting meta-inflammation. These results underscore the potential value of exercise-based rehabilitation as a complementary therapy for PAH patients.

β-sitosterol alleviates pulmonary arterial hypertension by altering smooth muscle cell phenotype and DNA damage/cGAS/STING signaling

BackgroundPulmonary arterial smooth muscle cells (PASMCs) have a neoplastic phenotype characterized by hyperproliferative and anti-apoptotic features that contribute to pulmonary hypertension (PH) development. DNA-sensing adapter protein stimulator of interferon genes (STING) regulate the phenotypic switch of vessel smooth muscle cells. β-sitosterol (SITO) is a nutrient derived from plants that inhibits vascular smooth muscle cell proliferation without notable toxicity. However, the effect of SITO on cancer-like PH-associated pulmonary vascular remodeling and the specific mechanism has not yet be studied. PurposeThis study investigated the in vitro and in vivo effects of SITO against PH, and its underlying mechanisms. MethodsThe therapeutic efficacy of SITO was assessed, and its underlying mechanisms were explored in hypoxia-induced and platelet-derived growth factor (PDGF)-BB-stimulated primary PASMCs and in a monocrotaline (MCT)-induced preclinical PH rat model. SITO or sildenafil (SID) were administered after the MCT intraperitoneal injection. Pulmonary parameters, right heart function, morphology, and PASMCs were cultured for verification. The expression levels of DNA damage/cyclic GMP–AMP synthase (cGAS)/STING were determined using immunofluorescence and Western blotting. STING agonists that interfere with PASMCs were used to determine whether STING mediates the effects of SITO. ResultsSITO prevented PASMCs proliferation, promoted apoptosis and suppressed phenotypic switching in a dose-dependent manner in vitro and in vivo. In vivo results in rats demonstrated that four weeks of intragastric SITO administration effectively mitigated the MCT-induced elevation of hemodynamic parameters, improved right cardiac function, and reduced pulmonary arteries remodeling. Mechanistically, DNA damage and cGAS/STING/nuclear factor kappa-B signaling activation were observed in rats with PH and cultured PASMCs. SITO exhibited protective effects by suppressing the DNA damage, potentially via inhibiting the expression level of the cGAS/STING signaling pathway. Pharmacological overexpression of STING abolished the anti-proliferative effects of SITO treatment in hypoxia-induced and PDGF-stimulated PASMCs by downregulating PCNA. ConclusionSITO may be an attractive agent for PH vascular remodeling by inhibiting proliferation and modulating the phenotypic switch in PASMCs via the DNA damage/cGAS/STING signaling pathway. This study provides a novel therapeutic agent and mediator of the pathological development of PASMCs and PH.

LncRNA-536 and RNA Binding Protein RBM25 Interactions in Pulmonary Arterial Hypertension.

Hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the essential features of the maladaptive inward remodeling of the pulmonary arteries in pulmonary arterial hypertension (PAH). In this study, we define the mechanistic association between long-noncoding RNA: ENST00000495536 (Lnc-536) and anti-proliferative HOXB13 in mediating smooth muscle hyperplasia. Antisense oligonucleotide-based GapmeRs or plasmid overexpressing lnc-536 were used to evaluate the role of lnc-536 in mediating hyperproliferation of PDGF-treated or idiopathic PAH (IPAH) PASMCs. Further, we pulled down lnc536 to identify the proteins directly interacting with lnc536. The in-vivo role of lnc-536 was determined in Sugen-hypoxia and HIV-transgenic pulmonary hypertensive rats. Increased levels of lnc-536 in PDGF-treated or IPAH PASMCs promote hyperproliferative phenotype by downregulating the HOXB13 expression. Knockdown of lnc-536 in-vivo prevented increased RVSP, Fulton Index, and pulmonary vascular remodeling in Sugen-Hypoxia rats. The lncRNA-536 pull-down assay demonstrated the interactions of RNA binding protein: RBM25 with SFPQ, a transcriptional regulator that has a binding motif on HOXB13 exon Further, The RNA-IP experiment using the SFPQ antibody showed direct interaction of RBM25 with SFPQ and knockdown of RBM25 resulted in increased interactions of SFPQ and HOXB13 mRNA while attenuating PASMC proliferation. Finally, we examined the role of lnc-536 and HOXB13 axis in the PASMCs exposed to the dual hit of HIV and a stimulant: cocaine as well. lnc-536 acts as a decoy for RBM25, which in turn sequesters SFPQ, leading to the decrease in HOXB13 expression and hyperproliferation of smooth muscle cells associated with PAH development.

Parthenolide attenuates hypoxia-induced pulmonary hypertension through inhibiting STAT3 signaling

BackgroundPulmonary hypertension (PH) is a chronic lung disease characterized by the progressive pulmonary vascular remodeling with increased pulmonary arterial pressure and right ventricular failure. Pulmonary vascular remodeling involves the proliferation, migration, and resistance to apoptosis of pulmonary artery smooth cells (PASMCs). Parthenolide (PTN) is a bioactive compound derived from a traditional medical plant feverfew (Tanacetum parthenium), and it has been studied for treatment of pulmonary fibrosis, lung cancer, and other related ailments. However, the function of PTN in the treatment of PH has not been studied. PurposeThis study aimed to evaluate the anti-proliferation and pro-apoptosis effects of PTN on PH and investigate its potential mechanisms. MethodsAn in vivo hypoxia-induced pulmonary hypertension (HPH) model was established by maintaining male rats in a hypoxia chamber (10% O2) for 3 weeks, and PTN was intraperitoneally administered at the dose of 10 or 30 mg/kg. We assessed the impact of PTN on mean pulmonary arterial pressure (mPAP), pulmonary vascular remodeling, and right ventricular hypertrophy. In vitro, we evaluated hypoxia-induced cellular proliferation, migration, and apoptosis of rat PASMCs. Proteins related to the STAT3 signaling axis were analyzed by western blotting and immunofluorescence assays. Recovery experiments were performed using the STAT3 activator, colivelin TFA. ResultsPTN significantly alleviated the symptoms of HPH rats by attenuating pulmonary arterial remodeling. It also prevented the proliferation and migration of PASMCs. PTN also induced the apoptosis of PASMCs. PTN could directly interact with STAT3 and markedly inhibited STAT3 phosphorylation and nuclear translocation. In vitro, and in vivo experiments demonstrated that overexpression of STAT3 partially suppressed the effect of PTN. ConclusionOur study indicated that PTN alleviated hypoxia-induced pulmonary hypertension in rats by suppressing STAT3 activity.

Au-modified ceria nanozyme prevents and treats hypoxia-induced pulmonary hypertension with greatly improved enzymatic activity and safety

BackgroundDespite recent advances the prognosis of pulmonary hypertension remains poor and warrants novel therapeutic options. Extensive studies, including ours, have revealed that hypoxia-induced pulmonary hypertension is associated with high oxidative stress. Cerium oxide nanozyme or nanoparticles (CeNPs) have displayed catalytic activity mimicking both catalase and superoxide dismutase functions and have been widely used as an anti-oxidative stress approach. However, whether CeNPs can attenuate hypoxia-induced pulmonary vascular oxidative stress and pulmonary hypertension is unknown.ResultsIn this study, we designed a new ceria nanozyme or nanoparticle (AuCeNPs) exhibiting enhanced enzyme activity. The AuCeNPs significantly blunted the increase of reactive oxygen species and intracellular calcium concentration while limiting proliferation of pulmonary artery smooth muscle cells and pulmonary vasoconstriction in a model of hypoxia-induced pulmonary hypertension. In addition, the inhalation of nebulized AuCeNPs, but not CeNPs, not only prevented but also blunted hypoxia-induced pulmonary hypertension in rats. The benefits of AuCeNPs were associated with limited increase of intracellular calcium concentration as well as enhancement of extracellular calcium-sensing receptor (CaSR) activity and expression in rat pulmonary artery smooth muscle cells. Nebulised AuCeNPs showed a favorable safety profile, systemic arterial pressure, liver and kidney function, plasma Ca2+ level, and blood biochemical parameters were not affected.ConclusionWe conclude that AuCeNPs is an improved reactive oxygen species scavenger that effectively prevents and treats hypoxia-induced pulmonary hypertension.Graphical

A Non-Metallic Nanozyme Ameliorates Pulmonary Hypertension Through Inhibiting ROS/TGF-β1 Signaling.

Pulmonary hypertension (PH) is a life-threatening cardiovascular disease with a lack of effective treatment options. Nanozymes, though promising for PH therapy, pose safety risks due to their metallic nature. Here, a non-metallic nanozyme is reported for the treatment of monocrotaline (MCT)-induced PH with a therapeutic mechanism involving the ROS/TGF-β1 signaling. The synthesized melanin-polyvinylpyrrolidone-polyethylene glycol (MPP) nanoparticles showcaseultra-small size, excellent water solubility, high biocompatibility, and remarkable antioxidant capacity. The MPP nanoparticles are capable of effectively eliminating ROS in isolated pulmonary artery smooth muscle cells (PASMCs) from PH rats, and significantly reducePASMC proliferation and migration. In vivo results from a PH model demonstratethat MPP nanoparticles significantly increasepulmonary artery acceleration time, decreasewall thickening and PCNA expression in lung tissues, as evidenced by echocardiograpy, histology and immunoblot analysis. Additionally, MPP nanoparticles treatment improverunning capacity, decreaseFulton index, and attenuateright ventricular fibrosis in MCT-PH rats by using treadmill test, picrosirius red, and trichrome Masson staining. Further transcriptomic and biochemical analyses revealthat inhibiting ROS-driven activation of TGF-β1 in the PA is the mechanism by which MPP nanoparticles exerttheir therapeutic effect. This study providesa novel approach for treating PH with non-metallic nanozymes based on a well-understood mechanism.

Pulmonary artery smooth muscle cell pyroptosis promotes the proliferation of PASMCs by paracrine IL‑1β and IL‑18 in monocrotaline‑induced pulmonary arterial hypertensive rats.

Pulmonary arterial hypertension (PAH) is a common vascular disease, and pulmonary vascular remodeling is a pivotal pathophysiological mechanism of PAH. Major pathological changes of pulmonary arterial remodeling, including proliferation, hypertrophy and enhanced secretory activity, can occur in pulmonary artery smooth muscle cells (PASMCs). Multiple active factors and cytokines play important roles in PAH. However, the regulatory mechanisms of the active factors and cytokines in PAH remain unclear. The present study aimed to reveal the crucial role of PASMC pyroptosis in PAH and to elucidate the intrinsic mechanisms. To establish the PAH rat models, Sprague-Dawley rats were injected intraperitoneally with monocrotaline (MCT) at a dose of 60 mg/kg. The expression of proteins and interleukins were detected by western blotting and ELISA assay. The results indicated that the pyroptosis of PASMCs is significantly increased in MCT-induced PAH rats. Notably, pyroptotic PASMCs can secret IL-1β and IL-18 to promote the proliferation of PASMCs. On this basis, inhibiting the secretion of IL-1β and IL-18 can markedly inhibit PASMC proliferation. Collectively, the findings of the present study indicate a critical role for PASMC pyroptosis in MCT-induced PAH rats, prompting a new preventive and therapeutic strategy for PAH.

Identification of TFRC as a biomarker for pulmonary arterial hypertension based on bioinformatics and experimental verification

BackgroundPulmonary arterial hypertension (PAH) is a life-threatening chronic cardiopulmonary disease. However, there is a paucity of studies that reflect the available biomarkers from separate gene expression profiles in PAH.MethodsThe GSE131793 and GSE113439 datasets were combined for subsequent analyses, and batch effects were removed. Bioinformatic analysis was then performed to identify differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) and a protein-protein interaction (PPI) network analysis were then used to further filter the hub genes. Functional enrichment analysis of the intersection genes was performed using Gene Ontology (GO), Disease Ontology (DO), Kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA). The expression level and diagnostic value of hub gene expression in pulmonary arterial hypertension (PAH) patients were also analyzed in the validation datasets GSE53408 and GSE22356. In addition, target gene expression was validated in the lungs of a monocrotaline (MCT)-induced pulmonary hypertension (PH) rat model and in the serum of PAH patients.ResultsA total of 914 differentially expressed genes (DEGs) were identified, with 722 upregulated and 192 downregulated genes. The key module relevant to PAH was selected using WGCNA. By combining the DEGs and the key module of WGCNA, 807 genes were selected. Furthermore, protein–protein interaction (PPI) network analysis identified HSP90AA1, CD8A, HIF1A, CXCL8, EPRS1, POLR2B, TFRC, and PTGS2 as hub genes. The GSE53408 and GSE22356 datasets were used to evaluate the expression of TFRC, which also showed robust diagnostic value. According to GSEA enrichment analysis, PAH-relevant biological functions and pathways were enriched in patients with high TFRC levels. Furthermore, TFRC expression was found to be upregulated in the lung tissues of our experimental PH rat model compared to those of the controls, and the same conclusion was reached in the serum of the PAH patients.ConclusionsAccording to our bioinformatics analysis, the observed increase of TFRC in the lung tissue of human PAH patients, as indicated by transcriptomic data, is consistent with the alterations observed in PAH patients and rodent models. These data suggest that TFRC may serve as a potential biomarker for PAH.

Sex-dependent remodeling of right ventricular function in a rat model of pulmonary arterial hypertension.

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.

β-catenin mediates monocrotaline-induced pulmonary hypertension via glycolysis in rats

BackgroundMetabolic 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 β-catenin plays a crucial role in metabolic reprogramming. This study aimed to investigate the effect of β-catenin on macrophage glycolysis in PH.MethodsLPS-induced BMDMs were generated via in vitro experiments. A monocrotaline (MCT)-induced PH rat model was established, and the β-catenin inhibitor XAV939 was administered in vivo. The role of β-catenin in glycolysis was analysed. The degree of pulmonary vascular remodelling was measured.Resultsβ-catenin was significantly increased in both in vitro and in vivo models. In LPS-induced BMDMs, β-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.ConclusionsOur findings suggest that β-catenin is involved in the development of PH by promoting glycolysis and the inflammatory response in macrophages. Inhibition of β-catenin could improve the progression of PH.