Hypoxia-induced Pulmonary Hypertension Research Articles

Celastrol Ameliorates Hypoxia-Induced Pulmonary Hypertension by Regulation of the PDE5-cGMP-PKG Signaling Pathway.

Pulmonary hypertension (PH) is a severe pulmonary vascular disease characterized by poor clinical outcomes and limited therapeutic options. Celastrol (CEL), a natural product derived from Tripterygium wilfordii Hook F, has shown therapeutic potential in PH models, although its mechanisms are not fully understood. This study aims to investigate the role of CEL in PH and explore its potential underlying mechanisms. This study investigates the role of CEL in PH and explores its underlying mechanisms. We evaluated the effects of CEL in a chronic hypoxia-induced PH rat model and hypoxia-stimulated human pulmonary arterial smooth muscle cells (HPASMCs). Bioinformatics and network pharmacology were employed to identify potential targets and pathways, which were then validated through mechanistic and functional analyses. CEL significantly reduced right ventricular systolic pressure, hypertrophy, fibrosis, and dysfunction in hypoxia-induced PH rats. It also decreased proliferating cell nuclear antigen expression and promoted apoptosis in pulmonary arterioles. Our findings suggest that CEL's therapeutic effects are mediated through the modulation of phosphodiesterase 5 (PDE5) and the activation of the cGMP-PKG signaling pathway. In HPASMCs, CEL treatment mirrored the invivo results, and PDE5 overexpression negated CEL's antiproliferative, antimigratory, and pro-apoptotic effects. CEL ameliorates pulmonary vascular remodeling and right ventricular dysfunction in PH, potentially through the PDE5-cGMP-PKG signaling pathway. These findings position CEL as a promising candidate for PH therapy.

Obesity and hypoxia have differential effects on myocardial innervation in the right ventricle of the male mouse heart.

Obesity, along with hypoxia, is known to be a risk factor for pulmonary hypertension (PH), which can lead to right ventricular hypertrophy and eventually heart failure. Both obesity and PH influence the autonomic nervous system (ANS), potentially aggravating changes in the right ventricle (RV). This study investigates the combined effects of obesity and hypoxia on the autonomic innervation of the RV in a mouse model. Male C57BL/6N mice were subjected to a control diet (CD) or a high-fat diet (HFD) for 30 weeks, with subsets of the mice exposed to chronic normobaric hypoxia (13% O2) during the final 3 weeks. Light and electron microscopic stereology was used to quantify various parameters of nerve fibres innervating the RV myocardium. HFD-induced obesity significantly increased the total length of nerve fibres and axons in the RV under normoxic conditions, indicating hyperinnervation. Quantitatively, the length density of nerve fibres per unit volume of RV (unit: x10-3 µm-2) was similar in CD (0.158 ± 0.04), CD-Hyp (0.176 ± 0.06) and HFD-Hyp (0.147 ± 0.05). In contrast, in HFD the length density of nerve fibres showed higher values 0.206 ± 0.054. The total length of nerve fibres increased by 67% from 2.61 m ± 0.77 m in CD to 4.37 m ± 1.51 m in HFD. The total length of axons increased by 80% from 8.87 m ± 2.75 m to 15.95 m ± 4.62 m. However, when obesity was combined with hypoxia, the total axon length was significantly reduced by 27% in HFD-Hyp compared with HFD. In addition, the mean number of axon profiles per nerve fibre profile decreased from 3.44 ± 0.68 in HFD to 2.95 ± 0.43 in HFD-Hyp. Interestingly, chronic hypoxia alone did not significantly alter RV innervation but led to RV hypertrophy, independent of the diet. The attenuation of obesity-induced hyperinnervation by hypoxia suggests a complex and potentially antagonistic interaction between these conditions. In conclusion, obesity induced by a HFD caused hyperinnervation of the RV, whereas chronic hypoxia alone did not significantly alter RV innervation. Surprisingly, chronic hypoxia attenuated the obesity-induced changes in RV innervation. These findings indicate that the effects of obesity and hypoxia-induced PH on RV innervation are distinct and potentially antagonistic.

Specialized pericyte subtypes in the pulmonary capillaries.

Pericytes are essential for capillary stability and homeostasis, with impaired pericyte function linked to diseases like pulmonary arterial hypertension. Investigating pericyte biology has been challenging due to the lack of specific markers, making it difficult to distinguish pericytes from other stromal cells. Using bioinformatic analysis and RNAscope, we identified Higd1b as a unique gene marker for pericytes and subsequently generated a knock-in mouse line, Higd1b-CreERT2, that accurately labels pericytes in the lung and heart. Single-cell RNA sequencing revealed two distinct Higd1b+ pericyte subtypes: while Type 1 pericytes support capillary homeostasis, Type 2 pericytes accumulate in arterioles, and co-express smooth muscle markers and higher levels of vimentin under hypoxic conditions. Lastly, healthy human lung pericytes with upregulation of vimentin exhibited increased adhesion, migration, and higher expression levels of the smooth muscle marker SM22 in vitro. These findings highlight the specialization of pulmonary pericytes and their contribution to vascular remodeling during hypoxia-induced pulmonary hypertension.

L-Citrulline in Neonates: From Bench to Bed Side.

L-citrulline (L-CIT), a precursor to L-arginine (L-ARG), is a key contributor to the nitric oxide (NO) signaling pathway. Endothelial dysfunction, characterized by deficient nitric oxide synthesis, is implicated in the pathogenesis of various neonatal conditions such as necrotizing enterocolitis (NEC) and bronchopulmonary dysplasia (BPD) associated pulmonary hypertension (PH). This review summarizes the current evidence around the possible role of L-CIT supplementation in the treatment of these conditions. Detoxification of endogenously produced superoxide radicals is inadequate in preterm infants due to immature antioxidants that leads to the production of peroxynitrite, a reactive oxygen-free radical that is cytotoxic and causes damage to organelles and cellular membranes, further disrupting the coupling of endothelial NO synthase enzyme and the generation of high levels of reactive nitrogen and oxygen species. Animal studies in lipopolysaccharide-induced models of chorioamnionitis and hyperoxia- and inflammation-induced BPD-PH in rodent lung models revealed that L-CIT supplementation significantly mitigated structural changes in the pulmonary vasculature, preserved alveolar growth, and increased vascular endothelial growth factor gene expression, highlighting the anti-inflammatory and antioxidant effects of L-CIT supplementation. Similar benefits were noted in newborn piglet models of chronic hypoxia-induced PH and NEC. Pharmacokinetic studies in neonates have shown doses of 100-300 mg/kg/day to be safe and well tolerated. A few studies have shown the beneficial effects of L-CIT supplementation in pulmonary hypertension secondary to congenital heart disease, but evidence of efficacy in the neonatal population is lacking. While L-CIT shows promise in the treatment of various neonatal conditions, adequately powered studies to evaluate the safety and efficacy of L-CIT supplementation post-surgical NEC and BPD ± PH in the extremely preterm population are needed to translate this novel therapy to clinical practice.

Nbeal2 Knockout Mice Are Not Protected Against Hypoxia-Induced Pulmonary Vascular Remodeling and Pulmonary Hypertension.

Inflammation drives the initiation and progression of pulmonary hypertension (PH). Platelets, increasingly recognized as immune cells, are activated and increased in the lungs of patients with PH. Platelet activation leads to the release of α-granule chemokines, many of which are implicated in PH. We hypothesized that hypoxia-induced secretion of platelet α-granule stored proteins and PH would be prevented in Nbeal2 -/-, α-granule deficient mice. WT and Nbeal2 -/- mice were maintained in normoxia or exposed to 10% hypobaric hypoxia for 3, 14, 21, or 35 days. We observed macrothrombocytopenia, increased circulating neutrophils and monocytes, and increased lung interstitial macrophages in Nbeal2 -/- mice at baseline. Hypoxia-induced platelet activation was attenuated, and hypoxia-induced increase in lung PF4 and platelets was delayed in Nbeal2 -/- mice compared to WT mice. Finally, although pulmonary vascular remodeling (PVR) and PH were attenuated at day 21, Nbeal2 -/- mice were not protected against hypoxia-induced PVR and PH at day 35. While this mutation also impacted circulating monocytes, neutrophils, and lung IMs, all of which are critical in the development of experimental PH, we gained further support for the role of platelets and α-granule proteins, such as PF4, in PH progression and pathogenesis and made several observations that expand our understanding of α-granule deficient mice in chronic hypoxia.

LncRNA H19 Promotes Angiogenesis in Mouse Pulmonary Artery Endothelial Cells by Regulating the HIF-1α/VEGF Signaling Pathway.

Persistent pulmonary hypertension of the newborn (PPHN) is a syndrome of acute respiratory failure characterized by systemic hypoxemia and elevated pulmonary arterial pressure, which leads to pathological changes in pulmonary vascular remodeling and endothelial cell function. Long non-coding RNA (lncRNA) H19 has been shown to be involved in the regulation of arterial endothelial cell function, but its regulatory role in PPHN is not fully understood. In the present study, mouse pulmonary artery endothelial cells (MPAECs) were cultured in a hypoxic conditions. Subsequently, the regulatory function of lncRNA H19 on MPAECs was explored by constructing adenoviruses knocking down and overexpressing lncRNA H19. The results revealed that the hypoxic conditions could induce the proliferation and migration of MPAECs, as well as the high expression of lncRNA H19 in MPAECs. Knockdown of lncRNA H19 expression in MPAECs reversed hypoxic environment-induced functional changes in endothelial cells, whereas overexpression of lncRNA H19 further enhanced the proliferation and migration of MPAECs. In addition, lncRNA H19 upregulated the hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway through sponge of miNA-20a-5p, which in turn promoted changes in endothelial cell function. LncRNA H19 may interfere with vascular remodeling in hypoxia-induced pulmonary hypertension by upregulating the expression of HIF-1α and VEGF in vascular endothelial cells.

T follicular helper cell is essential for M2 macrophage polarization and pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension

BackgroundHypoxia-induced pulmonary hypertension (HPH) is a subgroup of type 3 pulmonary hypertension that may cause early right ventricular failure and eventual cardiac failure, which lacks potential therapeutic targets. Our previous research demonstrated that T follicular helper (TFH) cells that produce IL-21 were involved in HPH. However, the molecular mechanisms of TFH/IL-21-mediated pathogenesis of HPH have been elusive. Here we investigate the role of TFH cells and IL-21 in HPH.MethodsStudies were performed in C57BL/6 mice or IL-21 knockout mice exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in mouse lung and pulmonary arterial smooth muscle cells (PASMCs). M2 signature gene (Fizz1), M1 signature genes (iNos, IL-12β and MMP9), GC B cell and its marker GL-7, caspase-1, M2 macrophages, TFH cells, Bcl-6 and IL-21 level were measured. Proliferation rate of PASMCs was measured by EdU. Pyroptosis was assessed using Hoechst 33,342/PI double fluorescent staining.ResultsIn response to chronic hypoxia exposure-induced pulmonary hypertension, IL-21−/− mice or downregulation of TFH cells in WT mice developed blunted pulmonary hypertension, attenuated pulmonary vascular remodelling. Furthermore, chronic hypoxia exposure significantly increased the germinal center (GC) B cell responses, which were not present in IL-21−/− mice or downregulation of TFH cells in WT mice. Importantly, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Consistently, significantly enhanced expression of M2 macrophage marker Fizz1 were detected in the bronchoalveolar lavage fluid of HPH mice. Moreover, alveolar macrophages that had been cultivated with IL-21 promoted PASMCs proliferation and pyroptosis in vitro, while a selective CX3CR1 antagonist, AZD8797 (AZD), significantly attenuated the proliferation and pyroptosis of the PASMCs. Finally, ECM1 knockdown promoted IL-2–STAT5 signaling and inhibited Bcl-6 signaling to inhibit TFH differentiation in HPH.ConclusionsTFH/IL-21 axis amplified pulmonary vascular remodelling in HPH. This involved M2 macrophage polarization, PASMCs proliferation and pyroptosis. These data suggested that TFH/IL-21 axis may be a novel therapeutic target for the treatment of HPH.

RO4929097 inhibits NICD3 to alleviate pulmonary hypertension via blocking Notch3/HIF-2α/FoxM1 signaling pathway.

Pulmonary hypertension (PH) is a condition in which the smooth muscle cells (SMCs) in the pulmonary arteries multiply excessively, causing the arteries to narrow. This can ultimately result in right heart failure and premature death. Notch3 is an important factor involved in pulmonary vascular remodeling in PH. RO4929097, as a γ-secretase inhibitor that inhibits Notch3 signaling pathway, may be a potential drug for the treatment of PH, but its feasibility and related mechanism of action need to be further investigated. In vitro modeling by hypoxic incubation of human pulmonary artery SMCs (HPASMCs). RO4929097 and plasmids including overexpression-NICD3 (oe-NICD3) and NICD3 small interfering RNA (siRNA) were used to alter the expression of NICD3, and HIF-2α inhibitor PT-2385 was used to alter the expression of HIF-2α. Western blot, EdU incorporation assay was used to investigate the alteration of NICD3, HIF-2α, FoxM1 protein expression, and cell proliferation. The severity of PH in rats was assessed by measuring the weight ratio of right ventricle (RV) to left ventricle (LV) and septum (S) (RV/[LV + S]) and hematoxylin-eosin (H&E) staining of lung tissues in a hypoxia-induced PH rat model. We first determined that hypoxia induction for 48h had the strongest induction of NICD3 and Notch3 in HPASMCs, and the strongest inhibition by 10μM RO4929097. Treatment of HPASMCs under hypoxic conditions with RO4929097 inhibited hypoxia-induced expression of NICD3, HIF-2α, FoxM1, and proliferation of HPASMCs. The inhibitory effect of RO4929097 was reversed after overexpression of NICD3 in HPASMCs. Further, we found that PT-2385 reversed the promotional effect of overexpression of NICD3 on the proliferation of HPASMCs. In vivo experiments, hypoxia-induced PH rats treated with RO4929097 showed a reduction in right ventricular hypertrophy index (RVHI) and a return to normal pulmonary artery morphology, indicating a reduction in the severity of PH. Our data suggest that RO4929097 regulates the Notch3/HIF-2α/FoxM1 signaling pathway by inhibiting the expression of NICD3, thereby inhibiting hypoxia-induced proliferation of HPASMCs. In vivo experiments also confirmed that RO4929097 could alleviate PH as a potential therapeutic strategy.

Peli1 Deficiency in Macrophages Attenuates Pulmonary Hypertension by Enhancing Foxp1-Mediated Transcriptional Inhibition of IL-6.

The infiltration of macrophages into the lungs is a common characteristic of perivascular inflammation, contributing to vascular remodeling in pulmonary hypertension (PH). Peli1 (pellino E3 ubiquitin-protein ligase 1) plays a critical role in regulating the production of proinflammatory cytokines and the polarization of macrophages in various diseases. However, the role of Peli1 in PH remains to be investigated. The expression and biological function of Peli1 were investigated in both human and experimental models of PH. Peli1-deficient mice and bone marrow transplant mice were utilized to explore the roles of Peli1 in macrophages in vivo. Proteomic analysis and molecular biology techniques were used to uncover the underlying mechanisms. The upregulation of Peli1 in the lungs and alveolar macrophages was observed in hypoxia-treated mice. Peli1 knockout mice and myeloid Peli1-deficient mice significantly ameliorated hypoxia-induced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Mechanistically, Peli1 facilitated the ubiquitination and subsequent proteasomal degradation of Foxp1 (forkhead box p1), thereby alleviating its suppression of IL (interleukin)-6 transcription and contributing to macrophage activation. Furthermore, myeloid Foxp1 deficiency partially eliminates the protective effect of myeloid Peli1 deficiency in hypoxia-induced PH mice. Our findings demonstrate that the Peli1-Foxp1-IL-6 pathway plays a crucial role in macrophage activation and recruitment during the development of PH, underscoring the potential of Peli1 as a therapeutic target for PH.

Promotor Hypomethylation Mediated Upregulation of VCAN Targets Twist1 to Promote EndMT in Hypoxia-Induced Pulmonary Hypertension.

Hypoxia-induced pulmonary hypertension (HPH) is a severe vascular disorder that is characterized by the involvement of endothelial-to-mesenchymal transition (EndMT) in its pathogenesis. Our previous research has suggested that the gene versican may have a crucial role in the development of HPH. However, the exact function of versican in HPH requires further investigation. The expression of versican and markers of EndMT was assessed using Western blot, immunohistochemistry, and immunofluorescence. Vascular remodeling and right ventricular hypertrophy in patients with HPH and mice were evaluated through hematoxylin and eosin staining, Masson's staining, and hemodynamic measurements. Protein interactions were validated using co-immunoprecipitation, and the DNA methylation level of versican was examined using methylation-specific polymerase chain reaction. Compared with the control, EndMT was observed in patients with HPH, HPH mouse models, and hypoxia-treated human pulmonary artery endothelial cells, accompanied by a significant increase of versican. Endothelium-specific knockdown of versican reversed HPH progression and effectively prevented EndMT in mouse models and human pulmonary artery endothelial cells. We further confirmed that versican participated in EndMT by targeting the key transcription factor Twist1. Additionally, the upregulation of versican may be attributed to promoter hypomethylation, which was mediated by reduced DNA methyltransferases activity under hypoxic conditions. This study provides the initial evidence showcasing the role of promoter hypomethylation-mediated versican upregulation in promoting EndMT by targeting Twist1, which facilitates vascular remodeling and the progression of HPH. These findings offer a promising new target for the treatment of HPH.

Abstract 4138674: Long-acting CRF2 receptor agonist, COR-1389, improves cardiopulmonary function in the rat model of Sugen plus hypoxia-induced pulmonary hypertension and right heart failure

Introduction: Urocortin-2 (UCN-2), a peptide which is part of the corticotropin-releasing factor (CRF) family, functions as an autocrine and paracrine factor, exerting its effects on cardiac and pulmonary function through agonism of CRF2 receptors. Although acute administration of UCN-2 has shown promise by improving heart and lung function in conditions like heart failure (HF) and pulmonary hypertension (PH), its limited stability impedes its chronic therapeutic use. Hypothesis: In this study, we explored the efficacy of COR-1389, a potent, selective and long-acting CRF2 agonist peptide, in the Sugen 5416 (VEGFR2 inhibitor, Su) combined with hypoxia (Hx) rat model of PH and right heart failure (RHF). Methods: To this aim, male adult Sprague Dawley rats were divided into three groups: Control rats (normoxia) were compared with rats injected subcutaneously with 20 mg/kg Sugen 5416 and exposed to chronic hypoxia for 3 weeks, followed by 2 weeks of normoxia. At 5 weeks, control rats and one SuHx group received subcutaneously vehicle (control and SuHx), while the third group received COR-1389 at a dose of 100 μg/kg every 4 days subcutaneously for 3 weeks (SuHx + COR-1389). At 8 weeks, cardiac and pulmonary hemodynamic functions of the three groups were evaluated using echocardiography and right heart catheterization, and heart and lung tissue were evaluated by histology and immunohistochemistry. Results: Compared to controls (n=10), SuHx exhibited increased mean pulmonary arterial pressure (mPAP), right ventricle (RV) hypertrophy (Fulton Index/body weight), muscularization of distal pulmonary arteries, RV fibrosis and cardiomyocyte hypertrophy, alongside reduced RV systolic function (Tricuspid Annular Plane Systolic Excursion, TAPSE) and cardiac output (CO). Conversely, compared to the SuHx + vehicle group (n=11), curative treatment with COR-1389 for 3 weeks in SuHx rats (n=13) led to enhanced RV systolic function (TAPSE) and CO, together with reductions in mPAP, RV hypertrophy, muscularization of pulmonary arteries, RV fibrosis and cardiomyocyte hypertrophy. Systolic blood pressure remained unchanged across all groups. Conclusion: These findings indicate that COR-1389 ameliorates the deteriorating cardiopulmonary parameters observed in the SuHx model and represents a promising approach for the treatment of PH and RHF.

Abstract 4117657: Targeting GPR39 for Hypoxia-induced Pulmonary Hypertension in Mice

Background: Primary pulmonary arterial hypertension (PAH) is a disease affecting young subjects. It has very poor prognosis and optimal treatment is not available. 15-hydroxyeicosatetraenoic acid (15-HETE), a potent vasoconstrictor, has been implicated in the pathogenesis of PAH. Elevated levels of 15-HETE have been shown to be associated with worse prognosis in patients with PAH. Oral ingestion of 15-HETE leads to development of PAH in rodents. We recently showed that 15-HETE is the endogenous agonist for the G-protein coupled receptor 39 (GPR39) present in vascular smooth muscle cells. Hypothesis: We, therefore, hypothesized that global deletion of GPR39 (knock-out [KO] mice) would protect from PAH by removing the receptor through which 15-HETE causes vasoconstriction. Methods: Accordingly, 13 wild-type (WT) and 16 KO mice were placed in a hypoxia chamber (10% O 2 ). Litter-mate controls (7 WT and 13 KO) were subjected to normoxia (room air, 21% O 2 ). At the end of 4 weeks heart rate as well as aortic and right ventricular (RV) pressures were measured (latter using micromanometer-tipped catheter), and the animals were then euthanized for measurement of RV wall thickness and RV size from which RV wall stress was calculated. Results: Heart rate and systolic blood pressure were not different between the 4 groups . WT hypoxic animals developed PAH with peak RV systolic pressures (RVSP) being significantly higher than normoxic WT and hypoxic WT and hypoxic KO mice. (Figure 1) Similar results were noted for RV end-diastolic pressure (Figure 2) and RV wall stress (Figure 3). Conclusions: By deleting GPR39, the target for 15-HETE, we were able to prevent hypoxia induced PAH and RV dysfunction. Pharmacological inhibition of GPR39 may open new frontiers in the treatment of PAH.

The HIF2α-dependent upregulation of SETDB1 facilitates hypoxia-induced functional and phenotypical changes of pulmonary microvascular endothelial cells.

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. 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. 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 patients with idiopathic pulmonary arterial hypertension (IPAH), comparing with 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 that 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. 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.NEW & NOTEWORTHY Abnormal histone modification plays vital role in pulmonary hypertension (PH). This study reports the regulation and role of a histone 3 lysine 9 (H3K9) methyltransferase, SETDB1, in primarily cultured rat pulmonary microvascular endothelial cells (PMVECs). Hypoxia induces significant upregulation of SETDB1 at both mRNA and protein levels, in a HIF2α-dependent manner. The hypoxic upregulation of SETDB1 leads to significant apoptosis, senescence, and endothelial-to-mesenchymal transition in PMVECs. Treatment of a specific inhibitor of histone methyltransferase, Chaetocin, effectively attenuates the disease pathogenesis of PH rat model induced by SU5416/hypoxia.

A bovine model of hypoxia-induced pulmonary hypertension reveals a gradient of immune and matrisome response with a complement signature found in circulation.

Pulmonary hypertension (PH) is a progressive vascular disease characterized by vascular remodeling, stiffening, and luminal obstruction, driven by dysregulated cell proliferation, inflammation, and extracellular matrix (ECM) alterations. Despite the recognized contribution of ECM dysregulation to PH pathogenesis, the precise molecular alterations in the matrisome remain poorly understood. In this study, we employed a matrisome-focused proteomics approach to map the protein composition in a young bovine calf model of acute hypoxia-induced PH. Our findings reveal distinct alterations in the matrisome along the pulmonary vascular axis, with the most prominent changes observed in the main pulmonary artery. Key alterations included a strong immune response and wound repair signature, characterized by increased levels of complement components, coagulation cascade proteins, and provisional matrix markers. In addition, we observed upregulation of ECM-modifying enzymes, growth factors, and core ECM proteins implicated in vascular stiffening, such as collagens, periostin, tenascin-C, and fibrin(ogen). Notably, these alterations correlated with increased mean pulmonary arterial pressure and vascular remodeling. In the plasma, we identified increased levels of complement components, indicating a systemic inflammatory response accompanying the vascular remodeling. Our findings shed light on the dynamic matrisome remodeling in early-stage PH, implicating a wound-healing trajectory with distinct patterns from the main pulmonary artery to the distal vasculature. This study provides novel insights into the immune cell infiltration and matrisome alterations associated with PH pathogenesis and highlights potential biomarkers and therapeutic targets within the matrisome landscape.NEW & NOTEWORTHY Extensive immune cell infiltration and matrisome alterations associated with hypoxia-induced pulmonary hypertension in a large mammal model. Matrisome components correlate with increased resistance to identify candidate alterations that drive biomechanical manifestations of the disease.

Nr1d1 inhibition mitigates intermittent hypoxia-induced pulmonary hypertension via Dusp1-mediated Erk1/2 deactivation and mitochondrial fission attenuation

Intermittent hypoxia (IH) precipitates pulmonary vasoconstriction, culminating in the onset of pulmonary hypertension (PH) among individuals afflicted with sleep apnea. While Nuclear receptor subfamily 1 group D member 1 (Nr1d1) is progressively recognized as pivotal regulator of cellular physiology, the role in the pathogenesis of IH-induced PH remains largely uncharted. The expression of Nr1d1 was examined in IH-induced rodent PH and in IH-treated PASMCs. To elucidate the contribution of Nr1d1 to the development of IH-induced PH, we employed siRNA to modulate Nr1d1 expression in vitro and employed serotype 1 adeno-associated virus (AAV1) in vivo. Nr1d1 levels were elevated in IH-induced rodents PH lung tissues and IH-treated PASMCs. Knocking down Nr1d1 by AAV1 effectively inhibited PH progression in chronic IH-induced PH models. Mechanistic investigations identified dual specificity phosphatase 1 (Dusp1), as a direct target that Nr1d1 trans-repressed, mediating Nr1d1’s regulatory influence on Erk1/2/Drp1 signaling. Nr1d1 deficiency ameliorates mitochondrial dysfunction and fission by restoring Dusp1 dysregulation and Drp1 phosphorylation. Activation of Erk1/2 with PMA reversed the Dusp1-mediated regulation of Drp1 phosphorylation, indicating the involvement of the Erk1/2 pathway in Drp1 phosphorylation controlled by Dusp1. Meanwhile, intermittent hypoxia induced more severe PH in Dusp1 knockout mice compared with wild-type mice. Our data unveil a novel role for Nr1d1 in IH-induced PH pathogenesis and an undisclosed Nr1d1-Dusp1 axis in PASMCs mitochondrial fission regulation.

PEDF upregulated in Ngfr-positive cells suppresses proliferation of pulmonary artery smooth muscle cells

Abstract Background Pulmonary arterial hypertension (PAH) is a disease with poor prognosis that causes right heart failure due to progressive pulmonary artery remodeling. Although existing pulmonary vasodilators contribute to pulmonary artery reverse remodeling by reducing share stress, the development of therapeutic agents that directly target pulmonary vascular remodeling is desired. Nerve growth factor receptor (Ngfr) is involved in the inflammatory reaction and repair process of damaged tissues. We have previously reported that Ngfr-positive cells are increased in the peripheral blood (PB) of PAH patients and are associated with disease progression. We also found that Ngfr inhibited the pathogenetic progression of pulmonary hypertension in a model of hypoxia-induced pulmonary hypertension. However, it remains unclear how Ngfr positive cells are involved in the pathology of PAH. Purpose In this study, we investigate how Ngfr-positive cells affect the pathogenesis of PAH. Methods & Results To evaluate the localization of Ngfr-positive cells in lung tissue, WT mice (C57BL/6) transplanted with bone marrow (BM) of GFP-Tg mice were kept under hypoxia for 3 weeks to create hypoxia-induced pulmonary hypertension (PH) model. Immunostaining of lung tissue sections indicated that double positive cells (Ngfr+GFP+) were present in the interstitial tissue around the small pulmonary artery. To examine the cellular characteristics of Ngfr-positive cells, RNA sequencing of Ngfr-positive and Ngfr-negative cells in PB was performed. Significantly 78 genes were upregulated in Ngfr-positive cells, including 11 secreted proteins. Among these, we focused on pigment epithelium-derived factor (PEDF), a secreted protein involved in angiogenesis. PEDF mRNA expression was decreased in the lung of Ngfr-KO mice compared to WT mice. We next examined the effect of PEDF on pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF)-induced cell proliferation of PASMC was inhibited by PEDF stimulation. Furthermore, PEDF stimulation significantly inhibited PDGF-induced mRNA expressions of TNF and MMP9 in PASMCs. Conclusion In this study, we show that PEDF expression is upregulated in Ngfr-positive cells and that PEDF inhibits PDGF-induced hPASMC proliferation, suggesting that NGFR-positive cells may suppress pulmonary vascular remodeling through PEDF-mediated paracrine effects. PEDF might be a new therapeutic target for PH.

Enhanced lung endothelial glycolysis is implicated in the development of severe pulmonary hypertension in type 2 diabetes.

Metabolic abnormalities in pulmonary endothelial cells are implicated in pulmonary hypertension (PH) while increasing evidence shows the influence of diabetes on progressing PH. In this study, we examined the effect of type 2 diabetes on hypoxia-induced PH and investigated its molecular mechanisms using hypoxia-induced diabetic male mice. Chronic hypoxia led to a more severe PH in type 2 diabetic mice than in control mice. Next, we compared gene expression patterns in isolated pulmonary endothelial cells (MPECs) from control mice in normoxia (CN), diabetic mice in normoxia (DN), control mice exposed to hypoxia (CH), and diabetic mice exposed to hypoxia (DH). The results showed that expression levels of 27 mRNAs, out of 92 mRNAs, were significantly different among the four groups. Two glycolysis-related proteins, GAPDH and HK2, were increased in MPECs of DH mice compared to those in DN or CH mice. In addition, the levels of pyruvate and lactate (glycolysis end products) were significantly increased in MPECs of DH mice, but not in CH mice, compared to MPECs of CN mice. Augmentation of glycolysis by terazosin exacerbated hypoxia-induced PH in CH mice but not in DH mice. On the contrary, inhibiting GAPDH (a key enzyme of the glycolytic pathway) by koningic acid ameliorated hypoxia-induced PH in DH mice but had no effect in CH mice. These data suggest that enhanced glycolysis in diabetic mice is involved in severe hypoxia-induced PH, and glycolysis inhibition is a potential target to reduce the severe progression of PH in diabetic patients.

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.

SOX9 promotes hypoxic pulmonary hypertension through stabilization of DPP4 in pulmonary artery smooth muscle cells

Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by pulmonary vascular remodeling (PVR), primarily due to the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This study aimed to investigate the role and molecular mechanism of SOX9 in hypoxic PH in rats. The findings revealed that SOX9 was upregulated in the pulmonary arteries and PASMCs of hypoxia-exposed rats. SOX9 knockdown inhibited hypoxia-induced proliferation and migration of PASMCs, reduced PVR, and subsequently alleviated hypoxia-induced PH in rats, suggesting that SOX9 plays a critical role in PH. Further investigation demonstrated that SOX9 interacted with DPP4, preventing its ubiquitin degradation in hypoxia-exposed PASMCs. DPP4 knockdown inhibited hypoxia-induced PASMC proliferation and migration, and administration of the DPP4 inhibitor sitagliptin (5 mg/kg) significantly reduced PVR and alleviated hypoxia-induced PH in rats, indicating that SOX9 contributes to PH by stabilizing DPP4. The results also showed that hypoxia induced YAP1 expression and dephosphorylation, leading to YAP1 nuclear localization. YAP1 knockdown promoted the degradation of HIF-1α in hypoxia-exposed PASMCs and inhibited hypoxia-induced proliferation and migration of PASMCs. Additionally, HIF-1α, as a transcription factor, promoted SOX9 expression by binding to the SOX9 promoter in hypoxia-exposed PASMCs. In conclusion, hypoxia promotes the proliferation and migration of PASMCs through the regulation of the YAP1/HIF-1α/SOX9/DPP4 signaling pathway, leading to PH in rats. These findings suggest that SOX9 may serve as a potential prognostic marker and therapeutic target for PH.