Background This study presents an improved technique for precision-cut lung slices to overcome the challenge of identifying pulmonary arteries in hypoxic pulmonary vasoconstriction. Methods The innovation employs the connexin 40 immunofluorescence staining to precisely identify pulmonary arterial endothelium. Results The results of the experiments demonstrated that the refined protocol successfully differentiated between pulmonary vessels, trachea or bronchus, and bronchial arteries in precision-cut lung slices.This differentiation was accomplished by administrating a cell tracer into the pulmonary circulation to aid in identifying specific structure. Furthermore, this study accurately distinguished pulmonary arteries from veins using CX40 immunostaining. Conclusions This study validates the accuracy and reliability of the improved precision-cut lung slices method in evaluating hypoxic pulmonary vasoconstriction particularly in pulmonary arteries.

This study introduced a vacuum-stabilized lung window technique for intravital imaging, which for the first time enabled the simultaneous, high-resolution, real-time monitoring of both pulmonary arterial vascular reactivity and dynamic changes in intracellular calcium signaling smooth muscle cells during acute hypoxic pulmonary vasoconstriction (HPV) in spontaneously breathing mice. By integrating two-photon microscopy with intravascular fluorescent tracers (FITC, Evans Blue) and a smooth muscle-specific genetically encoded calcium indicator (GCaMP6f), this technique enabled stable imaging of pulmonary arteries within a diameter range of 13.95–80.16 μm and supported the systematic extraction and analysis of calcium signals from multiple regions of interest along the longitudinal axis of the vascular wall. Acute hypoxia (10

Pulmonary hypertension (PH) stands as a tumor paradigm cardiovascular disease marked by hyperproliferation of cells and vascular remodeling, culminating in heart failure. Complex genetic and epigenetic mechanisms collectively contribute to the disruption of pulmonary vascular homeostasis. In recent years, advancements in research technology have identified numerous gene deletions and mutations, in addition to bone morphogenetic protein receptor type 2, that are closely associated with the vascular remodeling process in PH. Additionally, epigenetic modifications such as RNA methylation, DNA methylation, histone modification, and noncoding RNAs have been shown to precisely regulate PH molecular networks in a cell-type-specific manner, emerging as potential biomarkers and therapeutic targets. This review summarizes and analyzes the roles and molecular mechanisms of currently identified genes and epigenetic factors in PH, emphasizing the pivotal role of long ncRNAs in its regulation. Additionally, it examines current clinical and preclinical therapies for PH targeting these genes and epigenetic factors and explores potential new treatment strategies.

BACKGROUND:HIMF (hypoxia-induced mitogenic factor) induces pulmonary hypertension; however, the molecular nature of its extracellular membrane receptor(s) remains unknown. METHODS:A combination of cross-immunoprecipitation and immunoblotting, yeast 2-hybrid assays, and proteomics analysis was performed to screen and identify the candidate receptors. The interaction of HIMF with these candidate(s) was further evaluated using overexpression, silencing, point mutation, and blocking peptide strategies in chronic hypoxia and sugen/hypoxia pulmonary hypertension rat models to decipher the underlying pathophysiologic mechanisms. RESULTS:Cross-immunoprecipitation and immunoblotting identified HIMF interaction with the BMPR2 (bone morphogenetic protein receptor 2) complex. Yeast 2-hybrid revealed HIMF binding to the DTLPF motif (Asp-Thr-leucine-Pro-Phe [aspartic acid-threonine-leucine-proline-phenylalanine]) at the 54 to 58 amino acids (aa) in the extracellular domain of the BMPR1A (bone morphogenetic protein receptor 1A), one partner of the BMPR2 heterodimeric complex, but not BMPR2 itself. HIMF and human counterpart RELM (resistin-like molecule)-β binding to BMPR1A disrupted the BMPR1A/BMPR2 complex and decreased BMPR2 activity as revealed by reduced downstream events including ≈43% to 56% decline in Smad1/5/9 (Sma- and Mad-related protein 1/5/9) phosphorylation and ≈41% to 60% decrease in Id-1 (inhibitor of DNA binding 1) expression. This dynamic induced pulmonary artery smooth muscle cell proliferation and pulmonary vascular remodeling leading to pulmonary hypertension. A mutated motif in rats or a blocking peptide targeting this motif restrained HIMF binding with BMPR1A, rescued BMPR2 activity by ≈25% increase in Smad/1/5/9 phosphorylation and ≈39% elevation in Id-1 expression, and attenuated chronic hypoxia or sugen/hypoxia-induced pulmonary hypertension. CONCLUSIONS:HIMF induced pulmonary hypertension by direct binding to BMPR1A ectodomain, subsequently disrupting its binding to BMPR2 and BMPR2 activity. This HIMF signaling pathway represents a potential therapeutic target by selectively interfering with BMPR1A binding.

This study introduced a novel dual fixation method for the pulmonary vasculature and lung tissue in pulmonary hypertension (PH) rats, addressing the limitations of traditional fixation methods that failed to accurately preserve the in vivo status of pulmonary vascular morphology. The modified method involved a dual fixation process, combining individualized ventilation support and vascular perfusion to simulate the respiratory motion, pulmonary artery pressure and right ventricular output of the rat under in vivo conditions. Utilizing a monocrotaline-induced PH rat model, this study compared the dual fixation with the traditional immersion fixation, focusing on the quantitative assessment of alveolar expansion degree, capillary patency, endothelial cell quantity and wall thickness of pulmonary vein and artery. The results demonstrated that the dual fixation is superior in maintaining the authenticity and integrity of lung tissue and more sensitive in the evaluation of pulmonary artery hypertrophy, providing a more reliable representation of pulmonary vascular remodeling associated with PH.

Pulmonary hypertension (PH) is a global health issue characterized by high mortality. The main targets for current therapies in PH focus on the prostacyclin, nitric oxide, and endothelin pathways. While the approaches targeting these pathways form the foundation of standard PH treatment, the challenge remains to develop more effective therapeutic strategies. Evidence of pathological characteristics in PH illustrates other cell signaling pathways that also participate in the proliferation, apoptosis, extracellular matrix remodeling, mitochondrial dysfunction, inflammation, endothelial-to-mesenchymal transition, ferroptosis, pyroptosis, and the intricate network of cell-cell interactions of endothelial cells, smooth muscle cells, fibroblasts, and macrophages. In this review, we explore the roles of twenty key signaling pathways in PH pathogenesis. Furthermore, the crosstalks among some pathways offer a more detailed understanding of the complex mechanisms of PH. Considering the crucial role of signaling pathways in PH progression, targeting these aberrant signaling or their hub molecules offers great potential for mitigating PH pathology. This review delves into a variety of therapeutic approaches for PH that target critical signaling pathways and network interactions, including gene therapy, cell therapy, and pharmacological interventions. Supported by evidence from both animal studies and clinical trials, these strategies aim to reverse pathological alterations in pulmonary vessels and restore their normal function, addressing the significant health challenges associated with PH.

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.

BACKGROUND: STIM1 (stromal interaction molecule 1) regulates store-operated calcium entry and is involved in pulmonary artery vasoconstriction and pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial hypertension (PAH). METHODS: Bioinformatics analysis and a 2-stage matched case-control study were conducted to screen for noncoding variants that may potentially affect STIM1 transcriptional regulation in 242 patients with idiopathic PAH and 414 healthy controls. Luciferase reporter assay, real-time quantitative polymerase chain reaction, western blot, 5-ethynyl-2’-deoxyuridine (EdU) assay, and intracellular Ca 2+ measurement were performed to study the mechanistic roles of those STIM1 noncoding variants in PAH. RESULTS: Five noncoding variants (rs3794050, rs7934581, rs3750996, rs1561876, and rs3750994) were identified and genotyped using Sanger sequencing. Rs3794050, rs7934581, and rs1561876 were associated with idiopathic PAH (recessive model, all P <0.05). Bioinformatics analysis showed that these 3 noncoding variants possibly affect the enhancer function of STIM1 or the microRNA (miRNA) binding to STIM1 . Functional validation performed in HEK293 and pulmonary artery smooth muscle cells demonstrated that the noncoding variant rs1561876-G ( STIM1 mutant) had significantly stronger transcriptional activity than the wild-type counterpart, rs1561876-A, by affecting the transcriptional regulatory function of both hsa-miRNA-3140-5p and hsa-miRNA-4766-5p. rs1561876-G enhanced intracellular Ca 2+ signaling in human pulmonary artery smooth muscle cells secondary to calcium-sensing receptor activation and promoted proliferation of pulmonary artery smooth muscle cells under both normoxia and hypoxia conditions, suggesting a possible contribution to PAH development. CONCLUSIONS: The potential clinical implications of the 3 noncoding variants of STIM1 , rs3794050, rs7934581, and rs1561876, are 2-fold, as they may help predict the risk and prognosis of idiopathic PAH and guide investigations on novel therapeutic pathway(s).

Pulmonary hypertension (PH) is a disease that affects the right ventricle and venous circulation. The need for improvement in the discovery of novel therapeutic targets and drugs focusing on the immunomodulatory for the treatment of pulmonary hypertension is very important. Abnormalities in the immune system may result in pulmonary hypertension, and macrophages and Treg may be important targets for the development of novel treatment of pulmonary hypertension. Certainly, the macrophages, Treg, and the immune system have the potential therapeutic benefit by effectively modulating the macrophage to benefit pro-resolving phenotype anti-inflammatory and repair. The multifaceted association of immune system homeostasis, macrophages, and Treg is still debatable. Here, in this review, we highlight the significance of macrophages, Treg, and other immune cells’ functions in pulmonary hypertension. We also considered the Treg and macrophages in the phosphatidyl inositol 3-kinase signaling pathway.

Background: Data are quite sparse on the comprehensive analyses of pulmonary hypertension (PH) clinical trials worldwide. Methods: Information including participating countries (developed or developing), intervention type, trial size, PH categories, sponsorship, study phase, design strategies, and participants’ demographic characteristics was extracted from PH trials registered on ClinicalTrials.gov from 1999 to 2021. Results: A total of 203 eligible clinical PH trials were screened, involving 23,402 participants, 67.8% of whom were females. Major clinical trials were designed to test drug interventions (95.6%), sponsored solely by industries in 59.5%, and targeting Group 1 PH patients in 76.3%. A large number of countries participated in PH clinical trials; however, most clinical trials were conducted in developed countries (84.2%). Developing countries were involved in clinical trials with larger sample sizes (P<0.01). Additionally, the differences between developed and developing countries centered on interventions, sponsors, PH groups, and design strategies. Furthermore, developing countries participated in multinational clinical trials with good quality, homogeneity, reliability, and data authenticity. All pediatric participants were diagnosed with Group 1 PH and were only involved in drug intervention trials. Children participated in far fewer clinical trials than adults (P<0.01), and most were enrolled in PH clinical trials in developed countries. Among the entire clinical trial population, younger patients with Group 1 PH had a much higher participation to prevalence ratio (PPR). There was no difference in women’s PPRs between developed and developing countries. However, developing countries had higher PPRs for PH Groups I and IV (1.28 vs. 1.22, P<0.01), while developed countries had a lower PPR for Group III (P=0.02). Conclusions: PH is attracting increasing global attention, which is not at the same level of progress in developed and developing countries. Women and children with this disease have unique characteristics and require more attention.

Aberrant gene expression is a key mechanism underlying pulmonary hypertension (PH) development. The alterations of genomic chromatin accessibility and their relationship with the aberrant gene expressions in PH are poorly understood. We used bulk Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) in pulmonary artery smooth muscle cells (PASMCs) of chronic hypoxia-exposed rats mimicking group 3 human PH. Adult Sprague Dawley rats were commercially obtained from Hunan SJA (Hunan SJA Laboratory Animal Co., Changsha, China) and randomizedly allocated into four groups exposing to nomobaric hypoxia or normoxia for 1 or 28 days respectively. After the assessment of pulmonary hemodynamics, smooth muscle cells were isolated from intralobular arteries and simultaneously subjected to bulk Assay of ATAC-seq and RNA-seq. Hypoxic exposure for continuous 28-days, but not for 1-day, induced established PH phenotypes in rats. ATAC-seq revealed a major distribution of differential accessibility regions (DARs) annotated to the genome in out-of-promoter regions, following 1-day or 28-days hypoxia. 1188 DAR-associated genes and 378 differentially expressed genes (DEGs) were identified in rats after exposure to 1-day hypoxia, while 238 DAR-associated genes and 452 DEGs for 28-days hypoxia. Most of the DAR-associated genes or DEGs in 1-day did not overlap with that of 28-days hypoxia. A Pearson correlation analysis indicated no significant correlation between ATAC-seq and RNA-seq. The alterations in genomic chromatin accessibility and genes expression of PASMCs in the initial stage of hypoxia are distinct from the established stage of hypoxia-induced PH. The genomic differential accessibility regions may not be the main mechanisms directly underlying the differentially expressed genes observed either in the initial or established stages of PH. Thus the time-course alterations of gene expression and their possible indirect link with genomic chromatin accessibility warrant more attention in mechanistic study of pulmonary hypertension.

Venous thromboembolism (VTE) is a multifactorial disease, and pulmonary hypertension (PH) is a serious condition characterized by pulmonary vascular remodeling leading with increased pulmonary vascular resistance, ultimately leading to right heart failure and death. Although VTE and PH have distinct primary etiologies, they share some pathophysiologic similarities such as dysfunctional vasculature and thrombosis. In both conditions there is solid evidence that EVs derived from a variety of cell types including platelets, monocytes, endothelial cells and smooth muscle cells contribute to vascular endothelial dysfunction, inflammation, thrombosis, cellular activation and communications. However, the roles and importance of EVs substantially differ between studies depending on experimental conditions and parent cell origins of EVs that modify the nature of their cargo. Numerous studies have confirmed that EVs contribute to the pathophysiology of VTE and PH and increased levels of various EVs in relation with the severity of VTE and PH, confirming its potential pathophysiological role and its utility as a biomarker of disease severity and as potential therapeutic targets.

Background: Pulmonary arterial hypertension is an incurable disease, in which the extracellular CaSR (calcium sensing receptor) is mechanistically important. This study was aimed to genetically link the CaSR gene and function to the disease severity. Methods: Sanger sequencing, Sugen/hypoxia pulmonary arterial hypertension rat model, CaSR mutated rat, transcriptional reporter assay and measurement of CaSR activity were used. Results: Sanger sequencing identified a significant association between the variant rs1042636(A>G), located in CaSR exon 7, and idiopathic pulmonary arterial hypertension (IPAH) formation in patients. The frequency of 2968G homozygotes was higher in patients with IPAH compared with healthy individuals (23.6% versus 17.5%; P=0.001, OR=1.864), and the minor alleles of rs6776158, rs1048213, and rs9883099, located in CaSR promoter, raised the IPAH odds ratio to 2.173. Patients with IPAH carrying heterozygotes or homozygotes genotype of rs1042636 showed markedly higher pulmonary artery pressure and reduced survival compared with individuals carrying the wild-type allele. The minor alleles of rs6776158, rs1048213, and rs9883099 increased CaSR expression in reporter assay. In Sugen/hypoxia pulmonary arterial hypertension rats, the point mutation replicating rs1042636 found in IPAH exacerbated pulmonary arterial hypertension severity by promoting the overexpression and the enhanced activity of CaSR. Conclusions: Our functional genomic analysis thus indicates that the CaSR minor alleles of rs1042636, rs6776158, rs1048213, and rs9883099 contribute to the development and severity of IPAH. These findings may benefit clinical prognosis and treatment for IPAH.

Dear editor, We demonstrated in this study that functional CAR peptide-labelled mitochondria were detectable in venous blood and in lungs of rats after enteric encapsulation and oral administration, and were able to attenuate pulmonary hypertension in two different experimental models. Pulmonary hypertension is characterised by pulmonary vasoconstriction and remodelling, resulting in increased pulmonary vascular resistance eventually leading to right heart failure and death. It is well known that hypoxia induces pulmonary vasoconstriction,1, 2 but causes systemic vessel vasodilation.3, 4 One explication for these discrepancies in terms of hypoxia response may be the function and structure heterogeneity of mitochondria in smooth muscle cells from pulmonary vessels compared to systemic vessels.5, 6 Our recent studies have shown that femoral artery smooth muscle cell-derived mitochondria via intravenous injection can be transplanted into pulmonary artery smooth muscle cells (PASMCs), a process attenuating pulmonary hypertension.5, 6 Mitochondria transplantation for conditions associated with mitochondrial dysfunction is emerging as a novel therapeutic strategy. Previous studies have conducted mitochondrial transplantation mainly by intravenous,5, 6 tissue injection7 and nebulization.8 If mitochondrial transplantation could be performed orally, it would greatly improve its safety and convenience. To address this issue and considering the biological specificity of mitochondria, we linked the CAR peptide, a cyclic peptide with cell-penetrating properties and lung targeting properties,9, 10 to mitochondria via the mitochondrial outer membrane localization peptide. Five mitochondrial outer membrane localization peptides were screened to link CAR peptide to the surface of the mitochondrial outer membrane, and then labelled with FITC (Figure 1A). We found higher labelling efficiency for peptides -1, -3, -5 rather than peptides -2, -4 (Figure 1B). Therefore, the peptides-1, -3, -5 were used in subsequent experiments. To assess whether CAR peptide-labelled mitochondria could be absorbed into the blood through the small intestine, they were encapsulated to prevent any damage caused by acidic gastric secretions and administered to rats by gavage (see supplement). Labelled mitochondria were detectable after 4 h in the blood, 8 h in the lungs and heart (Figure 1C–E), ∼20–24 h with very small amounts in liver, while they were undetectable in brain or kidneys (Figure 1F). We found that the number of labelled mitochondria in erythrocytes was much higher than that in plasma(∼4.64 fold) and white blood cells(∼8.91 fold, Figure 2A), and the number of mitochondria in venous erythrocytes increased significantly after oral administration of mitochondria labelled with peptides -1, -3, -5 compared with the vehicle group (Figure 2B). Furthermore, the number of erythrocytes containing mitochondria was higher in venous blood than in arterial blood, and also higher in unoxygenated venous blood (venous blood control) than in oxygenated venous blood (venous blood oxygenation) (Figure 2C). The same trends were observed in the mitochondria number in erythrocytes (Figure 2C). The number of mitochondria per erythrocyte containing mitochondria was greater in unoxygenated venous erythrocytes than in oxygenated venous erythrocytes, while no significant difference was observed between venous erythrocytes and arterial erythrocytes (Figure 2C). The number of erythrocytes containing mitochondria in venous blood and the total number of mitochondria per 105 venous erythrocytes were also superior at 8 h than at 48 h after gavage (Figure 2C). The transwell experiments on the erythrocytes charged with CAR-labelled mitochondria highlighted an increased mitochondria release from erythrocytes with increased oxygen partial pressure (Figure 2D). These results suggest that mitochondrial release from erythrocytes may be regulated at least in part by oxygen partial pressure, and the release of mitochondria from erythrocytes may be in an All-or-None process in vivo, while in vitro the mechanism is somewhat between "All-or-None" with "partial" release. CAR peptide-labelled mitochondria were also obtained by immunoprecipitation with FITC antibody and were then functionally analysed for ATP production, respiratory control rate and membrane potential. The combined results showed that exogenous mitochondria absorbed in venous blood and lungs were functional (Figure 2E). To determine the molecular mechanism of the absorption of mitochondria through the intestine, we screened intestinal protein candidates after interaction with exogenous mitochondria. A total of 2127 proteins were identified, and three of them were receptor proteins that have been known to mediate endocytosis or transcytosis, including TGF-beta receptor type-2 (TGFBR2), transferrin receptor protein 1 (TFRC) and low-density lipoprotein receptor-related protein 2 (LRP2). To explore which protein(s) mediates the transcytosis process of CAR-labelled mitochondria, we implemented transwell experiments on the rat intestinal villus epithelial cells with CAR-labelled mitochondria and found that LRP2, but not TFGBR2 or TFRC knockdown using shRNA, significantly reduced the transcytosis of CAR peptide-labelled mitochondria (Figure 2F). In rats under chronic hypoxia exposure or treated with a single intraperitoneal injection of monocrotaline (MCT), peptide-5-labelled mitochondria encapsulated with enteric capsules attenuated pulmonary hypertension as illustrated by the significant decrease of mPAP, PVR, RV/(LV+S) ratio and pulmonary artery wall thickness (Figure 3A–C). This improvement was associated with enhanced mitochondrial function in rat pulmonary arteries (Figure 3D,E). However, the benefits of CAR-labelled mitochondria were lost with LRP2 silencing. Molecular mechanisms underlying the benefits of CAR-labelling mitochondria also involved the reduction of extracellular calcium-sensing receptors expression and related calcium signalling (Figure 4A–C), an important mediator of pulmonary hypertension as revealed in recent studies including ours. To confirm the localization of exogenous mitochondria in pulmonary artery, immunogold electron microscopy was conducted to examine pulmonary artery smooth muscle cells in rats, and gold particles were clearly identified in some PASMCs after intragastric administration of enteric-coated capsules containing peptide 5-labelled mitochondria (Figure 4D). In conclusion, our data demonstrate that CAR peptide-labelled mitochondria are absorbed from the intestine into blood after oral administration. The mechanism of mitochondrial uptake is associated with LRP2-mediated transcytosis. We also showed that exogenous mitochondria absorbed into venous blood were mainly distributed into erythrocytes and were carried into the lungs. The release of mitochondria from erythrocyte was driven at least in part by increased oxygen partial pressure upon blood re-oxygenation in lungs. Oral administration of encapsulated CAR peptide-labelled mitochondria attenuated both hypoxia- and MCT-induced pulmonary hypertension in rats. Our study provides a novel approach for easy and effective treatment of pulmonary hypertension, and reveals a new mechanism underlying exogenous mitochondria transportation and delivery through erythrocytes as carriers. This work was supported by grants from the National Natural Science Foundation of China (82270060, 82130002, 82170068, 31771275, 81770055, 81861128024, 81922001, 81770052, 81800053 and 31800980), and Wuhan Department of Science and Technology (2020020601012233). The authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. Methods: MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. Results: MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. Conclusions: Cigarette smoke–enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10–NFκB–calcium-sensing receptor cascade.

Background: Metabolic reprogramming is a hallmark of pulmonary arterial hypertension. Platelet activation has been implicated in pulmonary arterial hypertension (PAH), whereas the role of platelet in the pathogenesis of PAH remains unclear. Methods: First, we explored the platelet function of semaxanib‚ a inhibitor of VEGF receptor (SU5416)/hypoxia mice and monocrotaline-injected rats PAH model. Then we investigated pulmonary arterial smooth muscle cell aerobic glycolysis after being treated with platelet supernatant. TGF (transforming growth factor)-βRI, pyruvate kinase muscle 2, and other antagonists were applied to identify the underlying mechanism. In addition, platelet-specific deletion TGF-β1 mice were exposed to chronic hypoxia and SU5416. Cardiopulmonary hemodynamics, vascular remodeling, and aerobic glycolysis of pulmonary arterial smooth muscle cell were determined. Results: Here, we demonstrate that platelet-released TGF-β1 enhances the aerobic glycolysis of pulmonary arterial smooth muscle cells after platelet activation via increasing pyruvate kinase muscle 2 expression. Mechanistically, platelet-derived TGF-β1 regulate spyruvate kinase muscle 2 expression through mTOR (mammalian target of rapamycin)/c-Myc/PTBP-1(polypyrimidine tract binding protein 1)/hnRNPA-1(heterogeneous nuclear ribonucleoprotein A1) pathway. Platelet TGF-β1 deficiency mice are significantly protected from SU5416 plus chronic hypoxia–induced PAH, including attenuated increases in right ventricular systolic pressure and less pulmonary vascular remodeling. Also, in Pf4cre+ Tgfb1fl/fl mice, pulmonary arterial smooth muscle cells showed lower glycolysis capacity and their pyruvate kinase muscle 2 expression decreased. Conclusions: Our data demonstrate that TGF-β1 released by platelet contributes to the pathogenesis of PAH and further highlights the role of platelet in PAH.

S-palmitoylation of protein is a posttranslational, reversible lipid modification; it was catalyzed by a family of 23 mammalian palmitoyl acyltransferases in humans. S-palmitoylation can impact protein function by regulating protein sorting, secretion, trafficking, stability, and protein interaction. Thus, S-palmitoylation plays a crucial role in many human diseases including mental illness and cancers. In this chapter, we systematically reviewed the influence of S-palmitoylation on protein performance, the characteristics of S-palmitoylation regulating protein function, and the role of S-palmitoylation in pulmonary inflammation and pulmonary hypertension and summed up the treatment strategies of S-palmitoylation-related diseases and the research status of targeted S-palmitoylation agonists/inhibitors. In conclusion, we highlighted the potential role of S-palmitoylation and depalmitoylation in the treatment of human diseases.

BACKGROUND:The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. METHODS:We developed a sensitive high-pressure liquid chromatography technique to quantify dietary GGPP and conducted proteomics, qualitative real-time polymerase chain reaction screening, and Western blot to determine signaling cascades, gene expression, protein-protein interaction, and protein membrane trafficking in wild-type and transgenic rats. RESULTS:GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2 (Rho associated coiled-coil containing protein kinase 2)/Rab10 (Ras-related protein rab-10) signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and human pulmonary artery smooth muscle cells upregulated the expression of Ca2+-sensing receptor (CaSR) and HIMF (hypoxia-induced mitogenic factor), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10 knockdown almost abrogated hypoxia-promoted CaSR membrane trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed a high GGPP diet, blood GGPP levels were increased. This abolished statin-lowering effects on plasma GGPP, and also on hypoxia-enhanced RhoA activity of blood monocytes that was rescued by garlic extracts. CONCLUSIONS:There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of RhoA-ROCK2 cascade activation and Rab10-faciliated CaSR membrane trafficking with subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.

Whole exome sequencing (WES) was used in the research of familial pulmonary arterial hypertension (FPAH). CAV1 and KCNK3 were found as two novel candidate genes of FPAH. However, few pathogenic genes were identified in idiopathic pulmonary arterial hypertension (IPAH). We conducted WES in 20 unrelated IPAH patients who did not carry the known PAH-pathogenic variants among BMPR2, CAV1, KCNK3, SMAD9, ALK1, and ENG. We found a total of 4,950 variants in 3,534 genes, including 4,444 single-nucleotide polymorphisms and 506 insertions/deletions (InDels). Through the comprehensive and multilevel analysis, we disclosed several novel signaling cascades significantly connected to IPAH, including variants related to cadherin signaling pathway, dilated cardiomyopathy, glucose metabolism, immune response, mucin-type O-glycosylation, phospholipase C (PLC)-activating G protein-coupled receptor (GPCR) signaling pathway, vascular contraction and generation, and voltage-dependent Ca2+ channels. We also conducted validation studies in five mutant genes related to PLC-activating GPCR signaling pathway potentially involved in intracellular calcium regulation through Sanger sequencing for mutation accuracy, qRT-PCR for mRNA stability, immunofluorescence for subcellular localization, Western blotting for protein level, Fura-2 imaging for intracellular calcium, and proliferation analysis for cell function. The validation experiments showed that those variants in CCR5 and C3AR1 significantly increased the rise of intracellular calcium and the variant in CCR5 profoundly enhanced proliferative capacity of human pulmonary artery smooth muscle cells. Thus, our study suggests that multiple genetically affected signaling pathways take effect together to cause the formation of IPAH and the development of right heart failure and may further provide new therapy targets or putative clues for the present treatments such as limited therapeutic effectiveness of Ca2+ channel blockers.