New biomarkers are needed to detect and follow individuals with World Health Organization group 1.1 pulmonary hypertension (idiopathic pulmonary arterial hypertension (IPAH)). As NOTCH3 cleavage occurs constitutively in the lungs of individuals with IPAH, we investigated whether the NOTCH3 extracellular domain (NOTCH3-ECD) shed into serum could be used as a robust biomarker for IPAH. In three geographically distinct cohorts comprising 341 individuals with IPAH (267 women, 74 men) and 376 healthy individuals (278 women, 98 men), serum NOTCH3-ECD levels were significantly higher in individuals with IPAH (mean ± s.d.: 19.9 ± 5.5 ng ml-1) compared to controls (10.5 ± 1.9 ng ml-1; P < 0.001), with consistent results among the three cohorts. NOTCH3-ECD levels correlated with mean right atrial pressure, pulmonary vascular resistance, mean pulmonary artery pressure, tricuspid regurgitant velocity, 6-min walk distance and the New York Heart Association class. The area under the receiver operating curve for diagnosis of IPAH, based on serum NOTCH3-ECD, was 0.96 (95% confidence interval, 0.95-0.98) with a 90% sensitivity and 93% specificity at a cutoff of 13.0 ng ml-1. The 3-year mortality risk for individuals with IPAH increased by 18% for each increase in 3 ng ml-1 of NOTCH3-ECD above the diagnostic cutoff. The addition of serum NOTCH3-ECD levels improved the performance of prognostic calculators for PAH, including REVEAL 2.0, REVEAL 2.0 Lite and COMPERA 2.0. Moreover, serum NOTCH3-ECD levels predicted the presence of IPAH in treatment-naive individuals and correlated with disease progression over a follow-up of 6 years. Measurement of serum NOTCH3-ECD can therefore provide a highly sensitive, specific and noninvasive test for predicting the presence, disease severity, progression and survival of individuals with IPAH.

Endothelial-to-mesenchymal transition (EndMT) is a biological process through which lung vascular endothelial cells (ECs) transdifferentiate into mesenchymal-like cells. EndMT has recently been implicated in the development and progression of pulmonary vascular remodeling in pulmonary hypertension (PH); however, its underlying regulatory mechanisms remain incompletely understood. MicroRNAs (miRNAs) are key post-transcriptional regulators of EC gene expression and cellular responses to various stimuli. Notably, microRNA-153 (miR-153) has been shown to directly target SNAI1 to modulate epithelial-to-mesenchymal transition (EMT), a process closely related to EndMT and extensively studied in cancer. Whether miR-153 also participates in EndMT regulation, however, remains unknown. In this study, we demonstrate that 72-hour hypoxic exposure induces SNAI1-mediated EndMT in human lung vascular ECs. Hypoxia also increased cell proliferation and disrupted intercellular junctions, leading to enhanced endothelial permeability. Reduced miR-153 expression was observed in both hypoxia- and TGF-β1-induced EndMT, as well as in ECs isolated from PH patients exhibiting an EndMT phenotype. Similar to hypoxia, TGF-β1 promoted EC permeability. Loss of miR-153 enhanced SNAI1-mediated EndMT, endothelial survival, and permeability under normoxic conditions, whereas miR-153 overexpression attenuated EndMT induced by hypoxia or TGF-β1. However, miR-153 restoration did not completely restore endothelial barrier integrity disrupted by these stimuli. In vitro findings were validated in experimental PH model. In conclusion, miR-153 serves as a critical regulator of EndMT, maintaining endothelial identity and barrier function. Therapeutic delivery of miR-153 may therefore represent a novel strategy to inhibit EndMT and attenuate pulmonary vascular remodeling in PH.

Mechanosensitive cation channels expressed in pulmonary arterial smooth muscle (SMC) and endothelial (EC) cells contribute to the regulation of vasoconstriction and vasodilation of the pulmonary vasculature. Here, we report that activating Piezo1, a mechanosensitive cation channel, with Yoda1 induced a mild increase in pulmonary arterial pressure (PAP) via vasoconstriction in the intact pulmonary vascular system. Inhibition of nitric oxide (NO) synthase (NOS) with l-NAME significantly potentiated Yoda1-mediated pulmonary vasoconstriction. The Yoda1-mediated vasoconstriction was inhibited by Gd3+, a non-selective blocker of cation channels, but not affected by nifedipine, a dihydropyridine blocker of voltage-dependent Ca2+ channels (VDCC). Alveolar hypoxia alone increased PAP via hypoxic pulmonary vasoconstriction (HPV); Yoda1-mediated pulmonary vasoconstriction overlaps with HPV in the isolated and perfused/ventilated lung. On top of the Yoda1-mediated increase in PAP, intrapulmonary perfusion of high K+ (25 mM) solution or phenylephrine (PE, 30 µM) induced an additional increase in PAP. These data indicate that, under normal conditions, the activation of Piezo1 (by Yoda1) caused pulmonary vasodilation due to Ca2+-associated activation of NOS in ECs and vasoconstriction due to Ca2+ influx in SMCs. In conclusion, Piezo1 or Ca2+ influx through Piezo1 in ECs and SMCs functions differently in the regulation of pulmonary vasculature. When the endothelium-derived relaxing factor (e.g., NO) is decreased due to EC injury and/or inflammation, Piezo1 or Ca2+ influx through Piezo1 channels is an important trigger for pulmonary vasoconstriction in the whole-lung vascular system.

BACKGROUND AND AIMS:The intricate balance between angiotensin-converting enzyme 1 (ACE1) and 2 (ACE2) in the pulmonary vasculature is pivotal for the pathogenesis of pulmonary arterial hypertension (PAH). Catalysing the K48-linked deubiquitination, ubiquitin carboxyl-terminal hydrolase 10 (USP10) is involved in tumour suppression, autophagy, and cell proliferation. This study aims to determine whether a positive feedback loop of USP10 and AMP-activated protein kinase (AMPK) in pulmonary endothelium is protective against PAH. METHODS:In silico data analyses and in vitro culture cell experiments were used to investigate the role of USP10 in human idiopathic PAH (IPAH) and rodent pulmonary hypertension (PH) as well as the underlying mechanism involving a positive feedback loop of AMPK and USP10 in lung endothelium. Endothelial cell (EC)-specific USP10 transgenic (Tg) mice and mice administered liraglutide were used to explore the efficacy of the AMPK/USP10 loop in mitigating PH in rodents. RESULTS:USP10 level was decreased in the lung endothelium of human IPAH and rodent PH. AMPK/USP10 loop activation increased ACE2 Ser-680 phosphorylation and Lys-788 deubiquitination, thus contributing to the homeostatic level of ACE2 and lung vascular patency. Mice with liraglutide administration phenocopied the mitigated PH in EC-specific USP10 Tg mice, in part because of the activated AMPK/USP10 loop in the pulmonary endothelium. CONCLUSIONS:Genetic or pharmacologic [via glucagon-like peptide-1 receptor agonists (GLP-1 RAs)] interventions in the AMPK/USP10 loop can augment ACE2 level in lung endothelium. This type of ACE2 enhancement garners protection against PAH in humans and PH in rodents, which provides a rationale for using GLP-1 RAs to alleviate PAH.

Background: Berberine (BBR) has been reported to exert cardiovascular protective effects, but its direct role in pulmonary vascular tone regulation and the underlying mechanisms remain insufficiently understood. This study aimed to investigate whether BBR promotes pulmonary vasorelaxation through the soluble guanylate cyclase/cyclic guanosine monophosphate (sGC/cGMP) pathway and downstream calcium signalling in pulmonary arterial smooth muscle cells (PASMCs).Methods: The therapeutic and vasorelaxant potential of BBR was evaluated in pulmonary hypertension (PH)-related models and isolated arterial rings. To elucidate the underlying mechanism, we integrated ex vivo pulmonary artery tension study, network pharmacology, molecular docking, surface plasmon resonance (SPR), and sGC/cGMP pathway validation (via NS-2028 and cGMP measurement), alongside assessing PASMCs intracellular calcium mobilization.Results: BBR exerted a significant therapeutic effect and induced relaxation of pulmonary arterial rings. Endothelium removal did not abolish BBR-induced vasorelaxation, indicating that its relaxant effect comprises both endothelium-dependent and endothelium-independent components. Network pharmacology analysis indicated that the nitric oxide (NO) signalling pathway may be involved in BBR-mediated vascular regulation. Molecular docking and SPR assays supported a direct interaction between BBR and sGC, while NS-2028 markedly attenuated BBR-induced vasorelaxation. BBR also increased cGMP levels, indicating activation of sGC/cGMP signalling. In addition, BBR reduced hypoxia-induced elevation of intracellular Ca2+ levels and inhibited store-operated calcium entry (SOCE). In addition, BBR suppressed CPA-induced calcium release from intracellular stores.Conclusion: These findings demonstrate that BBR promotes pulmonary vasorelaxation through a mechanism involving direct activation of sGC/cGMP signalling and subsequent regulation of PASMC calcium homeostasis, including inhibition of intracellular calcium release and SOCE.

This review focuses on describing the potential pathogenic roles of endothelial Ca2+ and K+ signaling in the development and progression of pulmonary hypertension through its putative regulation of cellular senescence and inflammasome activation. Ca2+ influx through mechanosensitive and receptor-operated cation channels and Ca2+ release from the endoplasmic reticulum are involved in upregulating the cell cycle inhibitors p53, p21, and p16 (which result in cellular senescence) by activating the AKT/mTORC1 (Ak strain transforming/mammalian target of rapamycin) pathway in lung vascular endothelial cells. A rise in cytosolic Ca2+ concentration, resulting from Ca2+ influx and release in lung vascular endothelial cells, is also necessary to activate both canonical (NLRP3 [nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3]) and noncanonical inflammasomes, thereby promoting vascular and perivascular inflammation. Furthermore, K+ efflux through multiple types of K+-permeable channels and pores (eg, K+ ionophores, toxin-formed pores/channels, nonselective cation channels, and Ca2+-activated K+ channels) is sufficient for canonical (NLRP3) inflammasome activation. The senescent endothelial cells release senescence-associated secretory phenotype factors that subsequently cause endothelial-to-mesenchymal transition in adjacent endothelial cells and promote cell proliferation/migration in adjacent smooth muscle cells and (myo)fibroblasts, leading to vascular remodeling and occlusive intimal lesions, and pulmonary hypertension.

Endothelial-to-mesenchymal transition (EndMT) is a biological process that converts endothelial cells to mesenchymal cells with increased proliferative and migrative abilities. EndMT has been implicated in the development of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), a fatal and progressive lung vascular disease. Transforming growth factor β1 (TGF-β1), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVECs). An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca2+ signaling is involved in EndMT. In this study, we tested the hypothesis that TGF-β1-induced EndMT in human LVEC is Ca2+-dependent. Treatment of LVEC with TGF-β1 for 5-7 days resulted in increase in SNAI1/2 expression, induction of EndMT, upregulation of STIM/Orai1, and enhancement of store-operated Ca2+ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca2+ with EGTA or BAPTA-AM, respectively, abolished EndMT in response to TGF-β1. Moreover, EGTA diminished TGF-β1-induced increase in SNAI in a dose-dependent manner. Knockdown of either STIM1 or Orai1 was sufficient to prevent TGF-β-mediated increase in SNAI1/2 and EndMT but did not rescue the continuous adherent junctions. Blockade of Orai1 channels by AnCoA4 inhibited TGF-β-mediated EndMT and restored PECAM1-positive continuous adherent junctions. In conclusion, intracellular Ca2+ signaling plays a critical role in TGF-β-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca2+ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of precapillary pulmonary hypertension.NEW & NOTEWORTHY EndMT has been reported to contribute to the pathogenesis of PAH. In this study, we aimed to determine the role of Ca2+ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β1 requires store-operated Ca2+ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time, we demonstrated that TGF-β1-induced EndMT is a Ca2+-dependent event, whereas inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β1.

Introduction: Pulmonary hypertension (PH) is a life-threatening disease that results in elevated pulmonary arterial pressure and heart failure if left untreated. The downregulated expression and activity of voltage-gated potassium (K + ) channels 1.5 (Kv1.5) in pulmonary arterial smooth muscle cells (PASMC) have been associated with the development of PH. Hypoxia induces PH and attenuates Kv1.5 channels; however, the precise mechanism is unknown. MicroRNA (miRNA) is a small non-coding RNA that binds to the 3’-untranslated regions (3’-UTR) of the mRNAs to regulate gene expression post-transcriptionally. We previously reported that miRNA-29b directly targets Kv1.5 in PASMC isolated from patients with PH. In this study, we hypothesized that the increased level of miRNA-29b is involved in hypoxia-induced downregulation of Kv1.5 channels to contribute to increased PASMC proliferation and the development of PH. Methods: Normal human PASMC was exposed to normoxia (21% O 2 ) or hypoxia (3% O 2 ) for 72 hours. MiRNA-29b or Kv1.5 expression was quantified by qPCR, Western blot, or immunostaining. EdU and Tunnel assays were used to assess PASMC proliferation and apoptosis, respectively. Cells were transfected with miR-29b mimic or inhibitor and exposed to normoxia or hypoxia, respectively for 72 hours. Results: Upregulated miRNA-29b was correlated with downregulated Kv1.5 level in PASMC exposed to hypoxia compared with normoxic cells. Additionally, hypoxia promoted PASMC survival by increasing proliferation and decreasing apoptosis. Transfection of cells with miRNA-29 mimic significantly increased expression of miRNA-29b compared with negative controls, while transfection of cells with miRNA-29 inhibitor dramatically reduced miRNA-29b level. Overexpression of miRNA-29b attenuated Kv1.5 channels under normoxic conditions to contribute to enhanced PASMC proliferation, whereas miRNA-29b inhibitor prevented Kv1.5 downregulation in response to hypoxia. Conclusions: Elevated miRNA-29b contributes to hypoxia-induced PASMC proliferation by targeting Kv1.5. Therefore, miRNA-29b inhibition can be a new therapeutic strategy to treat patients with hypoxia-induced PH or other types of PH. This study is supported by UMN-HI-Transformative Ideas Program (AB), Eagles Telethon Postdoctoral Fellowship (IE), and APS Summer Undergraduate Research Fellowship (KK). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the excessive accumulation of activated myofibroblasts that deposit extracellular matrix (ECM) protein, leading to progressive scar formation and mechanical stress. However, the cellular origin and fate of myofibroblasts remain controversial, and the mechanisms by which myofibroblasts sense mechanical cues in the lung are unclear. Here, we report that periostin (Postn) is a reliable and distinctive marker for pulmonary myofibroblasts, while ablation of Postn+ myofibroblasts after injury ameliorated lung fibrosis. PIEZO1 was highly expressed in Postn+ myofibroblast and played a vital role in mechanoactivation of Postn+ myofibroblast and development of lung fibrosis. Conditional deletion of Piezo1 in Postn+ myofibroblasts significantly inhibited lung fibrosis by suppressing myofibroblast activation and proliferation. Loss of Piezo1 led to disruption of actin organization and prevention of Yap/Taz nuclear localization, thus shifting the myofibroblasts from a proliferative state into a stressed and apoptotic state. Furthermore, myofibroblast-specific Yap/Taz deletion fully recapitulated the protective phenotypes of myofibroblast-Piezo1-KO mice. These findings show that periostin marks pulmonary myofibroblasts, and that PIEZO1-mediated mechanosensation is essential for myofibroblast activation in the lung. Targeting PIEZO1 in the periostin-expressing cells is a novel therapeutic option to interfere with fibrotic diseases such as IPF .

BACKGROUND:The attenuation of cellular phenotypic switchingdriving PAH vascular remodeling remains an unmet therapeutic need. As eNAMPT (nicotinamide phosphoribosyltransferase)/TLR4 signaling significantly contributes to PH pathobiology, an eNAMPT-neutralizing ALT-100 mAb was utilized to rescue monocrotaline (MCT) and hypoxia/Sugen (Hy/Su) preclinical PH rat models and to evaluate eNAMPT/TLR4 involvement in endothelial cell (EC), smooth muscle cell (SMC) and monocyte/macrophage phenotypic switching. METHODS:MCT-PH or Hy/Su-PH rats received IgG or ALT-100 mAb (subQ, beginning week 4) with measurements of PH severity and lung tissue scRNAseq at day 42. RESULTS:PH severity indices (hemodynamic, histologic, vascular remodeling) were significantly attenuated in MCT-PH and Hy/Su-PH rats receiving ALT-100 mAb. scRNAseq studies revealed Hy/Su exposure increased populations of ECs undergoing EC-to-mesenchymal cell transition (EndMT), proliferating SMCs, and monocytes undergoing macrophage differentiation. Cellular phenotypic switching was ameliorated in Hy/Su-mAb rats. CONCLUSIONS:Autocrine/paracrine eNAMPT/TLR4 signaling contributes to accelerated cellular phenotypic switching, a druggable strategy to reverse vascular remodeling.

Endothelial-to-mesenchymal transition (EndMT) is a biological process through which lung vascular endothelial cells (ECs) transdifferentiate into mesenchymal-like cells. EndMT has recently been implicated in the development and progression of pulmonary vascular remodeling in pulmonary hypertension (PH); however, its underlying regulatory mechanisms remain incompletely understood. MicroRNAs (miRNAs) are key post-transcriptional regulators of EC gene expression and cellular responses to various stimuli. Notably, microRNA-153 (miR-153) has been shown to directly target SNAI1 to modulate epithelial-to-mesenchymal transition (EMT), a process closely related to EndMT and extensively studied in cancer. Whether miR-153 also participates in EndMT regulation, however, remains unknown. In this study, we demonstrate that 72-hour hypoxic exposure induces SNAI1-mediated EndMT in human lung vascular ECs. Hypoxia also increased cell proliferation and disrupted intercellular junctions, leading to enhanced endothelial permeability. Reduced miR-153 expression was observed in both hypoxia- and TGF-β1-induced EndMT, as well as in ECs isolated from PH patients exhibiting an EndMT phenotype. Similar to hypoxia, TGF-β1 promoted EC permeability. Loss of miR-153 enhanced SNAI1-mediated EndMT, endothelial survival, and permeability under normoxic conditions, whereas miR-153 overexpression attenuated EndMT induced by hypoxia or TGF-β1. However, miR-153 restoration did not completely recover endothelial barrier integrity disrupted by these stimuli. In conclusion, miR-153 serves as a critical regulator of EndMT, maintaining endothelial identity and barrier function. Therapeutic delivery of miR-153 may therefore represent a novel strategy to inhibit EndMT and attenuate pulmonary vascular remodeling in PH.

The association between lung microbiome and pulmonary hypertension (PH) remain unknown. This study aims to define the airway mycobiome signature and its potential correlation with clinical parameters of PH. Overall, 244 patients with PH and 120 healthy controls (CON) were recruited from three independent centers. The PH group was divided into subgroups not using antibiotics or corticosteroids (non-ANT/CORT), and those using ANT, CORT, or ANT + CORT within 1 month, and clinical classification (Groups 1, 3, and 4), World Health Organization functional class (I–IV), and disease severity based on mean pulmonary artery pressure or pulmonary vascular resistance levels for in-depth comparison. Distinct airway mycobiome profiles were observed in PH, CON, and PH subgroups. Linear discriminant analysis effect size analysis showed increased Purpureocillium, Issatchenkia, and Cyberlindnera and decreased Peroneutypa, Simplicillium, and Metarhizium in patients with PH (non-ANT/CORT, ANT, CORT, and ANT + CORT) than in CON. Receiver operating characteristic analysis indicated a strong prediction of the two fungal genera sets in distinguishing PH and its subgroups from CON. The two major fungal phyla, Ascomycota and Basidiomycota, correlated differently with major clinical factors. Increased connections among the top fungal phyla or genera were observed in the PH than in the CON group. Dominant enrichment (Purpureocillium, Issatchenkia, and Cyberlindnera) and diminishment (Peroneutypa, Simplicillium, and Metarhizium) of fungal genera consistently and strongly predicted PH without being influenced by different PH subgroups. This study provides the first description of the unique airway mycobiome signature in PH and among different PH subgroups.

Rationale: Clinical observations have suggested an association between alkylating agent-based chemotherapy and pulmonary arterial hypertension (PAH). The Fanconi anemia (FA) pathway, the principal mechanism for resolving alkylating agent-induced DNA damage, has been implicated in this process. Objectives: To establish the interplay among the FA pathway, DNA damage, and PAH. Methods: A knockout-first mouse model for FA complementation group L (Fanclkf/kf) and an adenovirus-associated virus 9-mediated Fancl overexpression (AAV-Fancl) model were used. Lung specimens, pulmonary arterial endothelial cells from patients with PAH, and primarily cultured pulmonary microvascular endothelial cells (PMVECs) from wild-type and Fanclkf/kf mice were analyzed. Measurements and Main Results: Data analysis on lung single-cell RNA-sequencing datasets revealed significant downregulation of FANCL in endothelial cells from patients with idiopathic PAH, a finding consistently validated in both clinical samples (lung specimens and pulmonary arterial endothelial cells) and the monocrotaline-induced PAH rat model. Notably, Fanclkf/kf mice developed spontaneous PAH and showed heightened susceptibility to alkylating agent (mitomycin C)-induced PAH, characterized by severe DNA damage and apoptosis in PMVECs. These pathological phenotypes were rescued through Fancl gene supplementation via AAV-Fancl or pharmacological intervention with the DNA damage protector amifostine. Mechanistically, transcriptomic profiling combined with functional validation demonstrated a suppressed bone morphogenetic protein signaling coupled with hyperactivated transforming growth factor-β pathways in PMVECs from Fanclkf/kf mice. Importantly, this imbalance was fully restored in PMVECs from AAV-Fancl-treated mice. Conclusions: Deficient Fancl plays a key role to promote PAH, and targeted rescue of Fancl could be a novel effective strategy for the treatment of PAH.

BACKGROUND:Pulmonary hypertension (PH), particularly secondary to hypoxic lung diseases like chronic obstructive pulmonary disease (COPD), lacks effective targeted therapies. Emerging evidence suggests that microbiota imbalances contribute to PH progression, raising the possibility of microbiome-targeted interventions. This study explores the role of antibiotics in modulating microbiota and ameliorating PH. METHODS:A retrospective cohort analysis was conducted using the Medical Information Mart for Intensive Care (MIMIC) database to assess changes in mean pulmonary artery pressure (mPAP) after antibiotic treatment. Subsequently, clinical data of 220 PH patients (including group 1, 3, and 4 PH) from single clinical center were analyzed, with 16S rRNA sequencing performed on pharyngeal and fecal samples to evaluate microbiota composition. A hypoxia-induced PH rat model was used to investigate the effects of antibiotic treatment on hemodynamics, pulmonary vascular remodeling, and gut microbiota. RESULTS:Antibiotic use was associated with reduced mPAP in PH patients, particularly in hypoxic associated PH. Microbiota diversity decreased with antibiotic treatment, but probiotic species like Lactobacillus were enriched. In hypoxia-induced PH rats, antibiotics attenuated right ventricular systolic pressure (RVSP), reduced pulmonary vascular thickening, and preserved gut villi integrity. Lactobacillus and Anaerostipes correlated negatively with PH severity, suggesting a protective role. CONCLUSION:Antibiotic-driven microbiota modulation may alleviate PH progression by targeting dysbiosis and reducing inflammation. These findings support further investigation into optimized antibiotic regimens as a therapeutic strategy for PH, particularly in hypoxic lung disease-associated cases.

RATIONALE: Endothelial-to-mesenchymal transition (EndMT) is a cellular process in which endothelial cells (ECs) transition into mesenchymal cells, a mechanism recently linked to pulmonary vascular remodeling in pulmonary hypertension (PH). However, the regulatory mechanisms driving EndMT in PH remain largely undefined. MicroRNA-153 (miR-153), a small non-coding RNA, regulates gene expression by targeting the 3'-untranslated region (3'-UTR) of mRNA, affecting mRNA translation and stability. This study investigates miR-153's role in modulating EndMT in PH. METHODS: Human lung vascular endothelial cells (LVECs) were isolated from healthy individuals and patients with idiopathic pulmonary arterial hypertension (IPAH). In vitro, normal LVECs were exposed to hypoxia (3% O₂) for 72 hours or treated with TGF-β1 (10 ng/mL) for 7 days to induce EndMT. Levels of miR-153 and EndMT markers were quantified by qPCR, Western blotting, or immunostaining. Proliferation and apoptosis of ECs were assessed using EdU and TUNEL assays, respectively. LVECs were transfected with miR-153 inhibitor or mimic, then exposed to normoxic or hypoxic conditions, respectively for 72 hours. In vivo, mice were subjected to either hypoxia (10% O₂ for 6 weeks, Hx-PH) or Sugen5416/hypoxia (20 mg/kg i.p. plus 10% O₂ for 6 weeks, SuHx-PH) to induce PH. The PH phenotype in mice was evaluated by hemodynamic measurements and histological analysis of H&E-stained lung tissues. RESULTS:In silico analysis identified miR-153 as a direct regulator of human SNAI1 and SNAI2 genes via binding to their 3'-UTR. Downregulation of miR-153 in IPAH LVECs was correlated with elevated SNAI1/2 expression, increased EndMT, and higher proliferation compared to normal LVECs. Hypoxia or TGF-β1 treatment reduced miR-153 expression, increased EndMT transcription factors (SNAI1/2), mesenchymal markers (SM-22, vimentin), and reduced EC markers (CD31, VE-cadherin). In normoxic LVECs, miR-153 inhibition significantly upregulated SNAI1/2, increased EndMT and EC proliferation, and reduced apoptosis. Conversely, miR-153 mimic abolished hypoxia- and TGF-β1-induced EndMT. Overexpression of miR-153 attenuated hypoxia-induced EC survival. In PH mouse models, reduced miR-153 levels were associated with activated SNAI1/2, EndMT, and pulmonary vascular remodeling. CONCLUSIONS: Downregulation of miR-153 drives phenotypic transition of LVECs to proliferative mesenchymal cells via SNAI-mediated EndMT, contributing to pulmonary vascular remodeling and PH. Therefore, miR-153 could be a potential therapeutic target for PH treatment.

Chronic kidney disease (CKD) is a significant risk factor for pulmonary hypertension (PH), a complication that adversely affects patient prognosis. However, the mechanisms underlying this association remain poorly understood. A major obstacle to progress in this field is the lack of a reliable animal model replicating CKD-PH. This study aimed to establish a stable rat model of CKD-PH. We employed a combined approach, inducing CKD through a 5/6 nephrectomy and concurrently exposing the rats to a high-salt diet. The model's hemodynamics were evaluated dynamically, alongside a comprehensive assessment of pathological changes in multiple organs. Lung tissues and serum samples were collected from the CKD-PH rats to analyze the expression of angiotensin-converting enzyme 2 (ACE2), evaluate the activity of key vascular components within the renin–angiotensin–aldosterone system (RAAS), and characterize alterations in the serum metabolic profile. At 14 weeks post-surgery, the CKD-PH rats displayed significant changes in hemodynamic parameters indicative of pulmonary arterial hypertension. Additionally, right ventricular hypertrophy was observed. Notably, no evidence of pulmonary vascular remodeling was found. Further analysis revealed RAAS dysregulation and downregulated ACE2 expression within the pulmonary vascular endothelium of CKD-PH rats. Moreover, the serum metabolic profile of these animals differed markedly from the sham surgery group. Our findings suggest that the development of pulmonary arterial hypertension in CKD-PH rats is likely a consequence of a combined effect: RAAS dysregulation, decreased ACE2 expression in pulmonary vascular endothelial cells, and metabolic disturbances.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats ( Uchl1 −/− ), and conditional Uchl1 knockout mice ( Tie2Cre-Uchl1 fl/fl ) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1 −/− animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice ( Gadd45α −/− ) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Pulmonary arterial hypertension (PAH) is a rare and fatal vascular disease with heterogeneous clinical manifestations. To date, molecular determinants underlying the development of PAH and related outcomes remain poorly understood. Herein, we identify pulmonary primary oxysterol and bile acid synthesis (PPOBAS) as a previously unrecognized pathway central to PAH pathophysiology. Mass spectrometry analysis of 2,756 individuals across five independent studies revealed 51 distinct circulating metabolites that predicted PAH-related mortality and were enriched within the PPOBAS pathway. Across independent single-center PAH studies, PPOBAS pathway metabolites were also associated with multiple cardiopulmonary measures of PAH-specific pathophysiology. Furthermore, PPOBAS metabolites were found to be increased in human and rodent PAH lung tissue and specifically produced by pulmonary endothelial cells, consistent with pulmonary origin. Finally, a poly-metabolite risk score comprising 13 PPOBAS molecules was found to not only predict PAH-related mortality but also outperform current clinical risk scores. This work identifies PPOBAS as specifically altered within PAH and establishes needed prognostic biomarkers for guiding therapy in PAH.

Delta-like ligand 4 (DLL-4) inhibitor drugs are an emerging cancer treatment. In clinical trials for solid organ malignancies, intravenous administration of monoclonal antibodies that inhibit DLL-4 is associated with development of pulmonary hypertension, in the absence of left ventricular dysfunction. Analysis of 13 clinical trials showed that pulmonary hypertension is a complication of DLL-4 inhibition.

A novel class of antidiabetic drugs, called gliflozins, inhibit sodium-glucose cotransporter 2 (SGLT2i) to lower blood glucose levels in patients with Type 2 diabetes (T2DM). Gliflozins reduce mortality and heart failure (HF) hospitalizations in HF patients with or without T2DM, but the molecular mechanisms are unclear. We hypothesize that modulating effects on cardiac calcium homeostasis are involved in SGLT2i-induced benefits during HF, and that the SGLT2i ertugliflozin (ERTU) will alter cytosolic calcium concentration ([Ca2+]cyt) in contracting cardiomyocytes after adrenergic stimulation. Cardiomyocytes were isolated from 0–2-day old C57BL/6J mice, treated for 72 hours with either ERTU (100 nM, and 1 μM), the NHE-1 inhibitor cariporide (CARI, 10 μM), ERTU and CARI, or vehicle (Ctrl). After 72 hours, cells were either A) loaded with Fura-2, baseline cytosolic calcium levels ([Ca2+]cyt) recorded, cells stimulated with phenylephrine (PE, 100 μM), and [Ca2+]cyt recorded for additional 30 minutes, B) lysed and protein isolated for analysis by immunoblotting, or C) fixed for immunohistochemical assessment. Results: 100 nM and 1 uM ERTU reduced [Ca2+]cyt peaks after PE, but 100 nM ERTU to a lesser degree. In contrast, 72-hour-long NHE1 inhibition with CARI increased [Ca2+]cyt at baseline when compared to vehicle and ERTU, and further enhanced [Ca2+]cyt peaks after PE. Immunoblotting revealed increased protein levels of S16 phosphorylated phospholamban, decreased total phospholamban, and upregulated sarcomeric α-actinin post-ERTU. Immunohistochemical assessment of sarcomeric α-actinin localization revealed improved sarcomeric Z-line organization after ERTU administration. Sustained exposure to the SGLT2i ERTU reduced, but the NHE-1 inhibitor CARI increased [Ca2+]cyt in contracting cardiac myocytes. These data indicate that ERTU’s beneficial effect on [Ca2+]cyt homeostasis is independent of NHE1 inhibition. Since disturbed calcium handling and calcium overload are hallmarks of HF, reduced [Ca2+]cyt levels after sympathetic stimulation may contribute to the cardioprotective effect of SGLT2i. Further studies are needed to unravel the molecular mechanisms behind ERTU’s influence on cardiac calcium homeostasis. This study was funded by Merck & Co., Inc. (Kenilworth, NJ). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.