A physiological membrane-receptor agonist typically stimulates oscillations, of varying frequencies, in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). Whether and how [Ca²⁺]_i oscillation frequency regulates agonist-stimulated downstream events, such as gene expression, in non-excitable cells remain unknown. By precisely manipulating [Ca²⁺]_i oscillation frequency in histamine-stimulated vascular endothelial cells (ECs), we demonstrate that the gene expression of vascular cell adhesion molecule 1 (VCAM1) critically depends on [Ca²⁺]_i oscillation frequency in the presence, as well as the absence, of histamine stimulation. However, histamine stimulation enhanced the efficiency of [Ca²⁺]_i-oscillation-frequency-regulated VCAM1 gene expression, versus [Ca²⁺]_i oscillations alone in the absence of histamine stimulation. Furthermore, a [Ca²⁺]_i oscillation frequency previously observed to be the mean frequency in histamine-stimulated ECs was found to optimize VCAM1 mRNA expression. All the above effects were abolished or attenuated by blocking histamine-stimulated generation of intracellular reactive oxygen species (ROS), another intracellular signaling pathway, and were restored by supplementary application of a low level of H₂O₂. Endogenous NF-κB activity is similarly regulated by [Ca²⁺]_i oscillation frequency, as well as its co-operation with ROS during histamine stimulation. This study shows that [Ca²⁺]_i oscillation frequency cooperates with ROS to efficiently regulate agonist-stimulated gene expression, and provides a novel and general strategy for studying [Ca²⁺]_i signal kinetics in agonist-stimulated downstream events.

Mitochondrial transplantation attenuates hypoxic pulmonary hypertension

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.

BACKGROUND: Hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) and consequent pulmonary vascular remodeling are the crucial pathological features of pulmonary hypertension (PH). Protein methylation has been shown to be critically involved in PASMC proliferation and PH, but the underlying mechanism remains largely unknown. METHODS: PH animal models were generated by treating mice/rats with chronic hypoxia for 4 weeks. SMYD2-vTg mice (vascular smooth muscle cell-specific suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 (deformed epidural auto-regulatory factor-1) domain-containing protein 2 transgenic) or wild-type rats and mice treated with LLY-507 (3-cyano-5-{2-[4-[2-(3-methylindol-1-yl)ethyl]piperazin-1-yl]-phenyl}-N-[(3-pyrrolidin-1-yl)propyl]benzamide) were used to investigate the function of SMYD2 (suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2) on PH development in vivo. Primary cultured rat PASMCs with SMYD2 knockdown or overexpression were used to explore the effects of SMYD2 on proliferation and to decipher the underlying mechanism. RESULTS: We demonstrated that the expression of the lysine methyltransferase SMYD2 was upregulated in the smooth muscle cells of pulmonary arteries from patients with PH and hypoxia-exposed rats/mice and in the cytoplasm of hypoxia-induced rat PASMCs. More importantly, targeted inhibition of SMYD2 by LLY-507 significantly attenuated hypoxia-induced pulmonary vascular remodeling and PH development in both male and female rats in vivo and reduced rat PASMC hyperproliferation in vitro. In contrast, SMYD2-vTg mice exhibited more severe PH phenotypes and related pathological changes than nontransgenic mice after 4 weeks of chronic hypoxia treatment. Furthermore, SMYD2 overexpression promoted, while SMYD2 knockdown suppressed, the proliferation of rat PASMCs by affecting the cell cycle checkpoint between S and G2 phases. Mechanistically, we revealed that SMYD2 directly interacted with and monomethylated PPARγ (peroxisome proliferator-activated receptor gamma) to inhibit the nuclear translocation and transcriptional activity of PPARγ, which further promoted mitophagy to facilitate PASMC proliferation and PH development. Furthermore, rosiglitazone, a PPARγ agonist, largely abolished the detrimental effects of SMYD2 overexpression on PASMC proliferation and PH. CONCLUSIONS: Our results demonstrated that SMYD2 monomethylates nonhistone PPARγ and inhibits its nuclear translocation and activation to accelerate PASMC proliferation and PH by triggering mitophagy, indicating that targeting SMYD2 or activating PPARγ are potential strategies for the prevention of PH.

Pulmonary artery hypertension (PAH) is a rare and fatal disorder that involves extensive remodeling of the pulmonary arteries mediated by hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Aberrant platelet-derived growth factor (PDGF) activity can lead to hyperproliferation of PASMCs; however, little is known about the role of long noncoding RNA (lncRNA) in this process. Using RNA sequencing, we identified 725 lncRNAs in rat PASMCs, 95 of which were expressed differentially in response to PDGF-BB treatment. Depletion of four lncRNAs affected the proliferation of rat PASMCs as measured by 5-ethynyl-2′-deoxyuridine incorporation assay. Among these, one lncRNA, named LnRPT (lncRNA regulated by PDGF and transforming growth factor β), was found to be the most potent in promoting the proliferation of PASMCs when knocked down. In contrast, proliferation of PASMCs was repressed when LnRPT was overexpressed. Mechanistically, LnRPT inhibited the expression of two genes involved in the Notch signaling pathway (notch3 and jag1) as well as the cell-cycle–regulating gene ccna2. In addition, downregulation of LnRPT induced by PDGF-BB was abrogated when phosphatidylinositol 3′-kinase activity was inhibited with pictilisib. Downregulation of LnRPT was also observed in the pulmonary arteries of rats with monocrotaline-induced PAH. This study provides novel insights into the effects of PDGF-BB on lncRNA expression in PASMCs, and identifies one lncRNA, LnRPT, that plays a role in PAH development as a regulator of PASMC proliferation by mediating the Notch signaling pathway and cell cycle.

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 ⁺ Tgfb1 ^fl/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.