Crocin inhibits neutrophil migration and activation to treat hypoxic pulmonary hypertension through targeting HCK.

Pulmonary artery (PA) smooth muscle cell (SMC) proliferation occurs with hypoxic pulmonary hypertension in vivo. However, proliferation of cultured PA SMC to hypoxia has not been demonstrated, and thus the mechanism by which these cells respond to hypoxia is unknown. Because protein kinase C (PKC) plays a role in intracellular transduction of proliferative signals, we asked whether PKC activation 1) causes proliferation of bovine PA SMC and 2) is important in PA SMC proliferative response to hypoxia. By measuring [3H]thymidine incorporation and cell counts, we found that quiescent PA SMC from four different cows proliferated with the PKC activator, phorbol 12-myristate 13-acetate (PMA), in a concentration-dependent manner. The proliferation was blocked with a PKC inhibitor, dihydrosphingosine, or by downregulating SMC PKC. We tested whether "priming" PA SMC by PKC activation was required for in vitro SMC proliferative response to hypoxia. Each SMC population was treated with PMA and then exposed for 24 h to 20, 10, 7, 3 or 0% O2. These cells proliferated with hypoxia reaching a peak response at 3% O2. The magnitude of the response to PMA and hypoxia was different for each cell population tested. No hypoxic proliferation occurred in control cells (no PMA). Dihydrosphingosine blocked the hypoxic response to the same extent that it inhibited the initial PMA conditioning stimulus. PKC-downregulated PA SMC did not proliferate to PMA or to subsequent hypoxia. The hypoxic response was not due to a reduction in O2 radical-mediated antiproliferative effect; rather, the PMA-primed cells seemed to "acquire" the ability to directly sense hypoxia and proliferate. In summary, PKC activation caused proliferation of PA SMC in vitro and allowed an additional proliferative response to hypoxia. Activation of PKC may be a requisite step for PA SMC to respond directly to hypoxia.

Piperlongumine attenuates vascular remodeling in hypoxic pulmonary hypertension by regulating autophagy

Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH.

Background N6-methyladenosine (m6A) is the most prevalent internal RNA modification in mammal mRNAs. Accumulating evidence has indicated the crucial role of m6A modification in cardiovascular diseases including cardiac hypertrophy, heart failure, ischemic heart disease, vascular calcification, restenosis, and aortic aneurysm. However, the role of m6A methylation in the occurrence and development of hypoxic pulmonary hypertension (HPH) remains largely unknown. Purpose The present study aims to explore the role of key transferase METTL3, in the development of HPH. Methods Hypoxic rat models and pulmonary artery smooth muscle cells (PASMCs) and were used to research the METTL3-mediated m6A in HPH in vivo and in vitro. CCK-8, EdU, PCNA, transwell and TUNEL assay were performed to evaluate the proliferation, migration and apoptosis rates of PASMCs. m6A RNA Methylation Quantification Kit and m6A-qPCR were utilized to measure the total m6A level and m6A-PTEN mRNA expression. RNA immunoprecipitation and RNA pull down were used to detect the interaction between METTL3 and PTEN mRNA. The half-life of mRNA was detected through actinomycin D assay. Results Both METTL3 mRNA and protein were found abnormally upregulated in pulmonary arteries of HPH rats and hypoxia induced PASMCs. Furthermore, downregulation of METTL3 attenuated PASMCs proliferation and migration exposed to hypoxia. In addition, m6A binding protein YTHDF2 was found significantly increased in HPH group in vivo and in vitro. Mechanistically, YTHDF2 recognized METTL3 mediated m6A-PTEN mRNA and promoted the degradation of PTEN. Decreased PTEN led to over-proliferation of PASMCs through activation of PI3K/Akt signaling pathway. Conclusion METTL3/YTHDF2/PTEN axis exerts a significant role in hypoxia induced PASMCs proliferation, providing a novel therapeutic target for HPH. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Natural Science Foundation of China Figure 1

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase suppresses glycolysis and proliferation of pulmonary artery smooth muscle cells in hypoxic pulmonary hypertension via inhibition of lactate dehydrogenase A.

The effect of activated κ-opioid receptor (κ-OR) on the role of calcium sensing receptor (CaSR) in preventing hypoxic pulmonary hypertension development

Rationale: Mitophagy plays an important role in pulmonary hypertension, a progressive disease characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). Long intergenic non-protein coding RNA 3047 (LINC03047), as a hypoxia-related lncRNA, is involved in the progression of various diseases. This study aims to elucidate the mechanism by which LINC03047 in hypoxic pulmonary hypertension (HPH). Methods: Dysregulated LINC03047 was identified through lncRNA sequencing. Reactive oxygen species assay, mitochondrial membrane potential assay, immunofluorescence staining, cell proliferation experiment, gain- and loss-of-function experiments were conducted to elucidate its role in mitophagy and proliferation of hypoxia-induced PASMCs and HPH progression. The upstream transcription regulation mechanism of LINC03047 was identified and verified by comprehensive methods, including reverse transcription-polymerase chain reaction, western blotting, luciferase assay, chromatin immunoprecipitation and rescue experiments. RNA pulldown, mass spectrometry, and RNA immunoprecipitation were employed to identify the potential interacting proteins of LINC03047, and further elucidate the regulatory mechanism between LINC03047 and its downstream targets. Furthermore, the HPH rat model and serum samples from PH patients were utilized to assess its in vivo impact and clinical relevance. Results: We observed that LINC03047 was significantly upregulated in hypoxia-induced PASMCs and promoted their mitophagy and proliferation.Mechanistically, signal transducer and activator of transcription 3 (STAT3) specifically interacted with the LINC03047 promoter and promoted the transcription of LINC03047. Furthermore, LINC03047 bound to heterogeneous nuclear ribonucleoprotein F (hnRNPF), altering the nuclear transport of hnRNPF, thereby upregulating the stability of connective tissue growth factor (CTGF) RNA. In vivo, targeting hnRNPF can ameliorate pulmonary vascular remodeling and HPH progression. The assessment of serum STAT3 and CTGF levels in PH patients indicated a strong positive correlation between the two biomarkers. Conclusions: The overexpression of LINC03047, driven by STAT3, facilitates PASMCs mitophagy by enhancing hnRNPF-mediated CTGF mRNA stability, thus promoting PASMCs proliferation and HPH progression. These findings highlight the critical role of LINC03047, providing new insights into the pathogenesis and potential treatment of HPH.

Hypoxic pulmonary hypertension (HPH) is a devastating and incurable disease characterized by pulmonary vascular remodeling, resulting in right heart failure and even death. Accumulated evidence has confirmed long coding RNAs (lncRNAs) are involved in hypoxia-induced pulmonary vascular remodeling in HPH. The exact mechanism of lncRNA in hypoxic pulmonary hypertension remains unclear. Microarray analysis was applied to investigate the profiles of lncRNA expression in pulmonary artery smooth muscle cells (PASMCs) cultured under hypoxia and normoxia condition. qRT-PCR was performed for the expression of lncRNAs, miRNA, and mRNAs, western blot analysis was employed for the detection of the expression of proteins. CCK-8 and transwell chamber assay were applied for the assessment of PASMC proliferation and migration, respectively. Besides, flow cytometry was performed for assessments of cell cycle progression. The binding between AC068039.4 and miR-26a-5p, miR-26a-5p, and TRPC6 3'UTR was detected by dual luciferase reporter assay. A total of 1,211 lncRNAs (698 up-regulated and 513 down-regulated) were differently expressed in hypoxia-induced PASMCs. Consistent with microarray analysis, quantitative PCR verified that AC068039.4 was obviously up-regulated in hypoxia-induced PASMCs. Knocking down AC068039.4 alleviated proliferation and migration of PASMCs and regulated cell cycle progression through inhibiting cells entering the G0/G1 cell cycle phase. Further experiment indicated AC068039.4 promoted hypoxic PASMCs proliferation via sponging miR-26-5p. In addition, transient receptor potential canonical 6 (TRPC6) was confirmed to be a target gene of miR-26a-5p. In conclusion, downregulation of lncRNA AC068039.4 inhibited pulmonary vascular remodeling through AC068039.4/miR-26a-5p/TRPC6 axis, providing new therapeutic insights for the treatment of HPH.

Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline

Research indicates that hypoxic pulmonary hypertension (HPH) potentially stimulates the sympathetic nervous system, which may increase norepinephrine (NE) release and cause excessive Ca2+ influx into pulmonary artery smooth muscle cells (PASMCs), leading to calcium overload and abnormal PASMC proliferation, factors closely associated with pulmonary vascular remodeling (PVR). This study investigates the potential mechanisms underlying echinacoside (ECH) treatment in HPH. In the in vitro experiment, NE-induced PASMCs were used to simulate HPH-induced PASMCs' calcium overload and abnormal proliferation. Postincubation with ECH, [Ca2+]cyt changes were detected using Fluo-4 AM. Flow cytometry was employed to ascertain ECH's inhibitory effect on PASMCs proliferation. For in vivo experiments, rats were exposed to a hypoxic and low-pressure oxygen environment to establish the HPH model. Post-ECH treatment, hematoxylin and eosin (HE) staining was conducted to assess PVR, and western blot analysis was used to examine protein expression in the lung tissues of the different groups. ECH was observed to inhibit [Ca2+]cyt increase in NE-induced PASMCs in a concentration-dependent manner, effectively reducing abnormal cell proliferation. It also reduced the expression of Transient receptor potential channel (TRPC) 1 (TRPC1), TRPC4, TRPC6, and calmodulin in PASMCs. In vivo studies demonstrated that ECH lowered the expression of these proteins in lung tissues of HPH rats, significantly decreased mean pulmonary artery pressure, and mitigated PVR.

BackgroundIn this study, we aimed to investigate whether and how lncRNA CASC2 was involved in hypoxia-induced pulmonary hypertension (PH)-related vascular remodeling.MethodsThe expression of lncRNAs or mRNAs was detected by qRT-PCR, and western blot analysis or immunochemistry was employed for detecting the protein expression. Cell number assay and EdU (5-ethynyl-2′-deoxyuridine) staining were performed to assess cell proliferation. Besides, flow cytometry and wound healing assay were employed for assessments of cell apoptosis and cell migration, respectively. Rat model of hypoxic PH was established and the hemodynamic measurements were performed. Hematoxylin and eosin (HE) and Masson′s trichrome staining were carried out for pulmonary artery morphometric analysis.ResultsThe expression of lncRNA CASC2 was decreased in hypoxia-induced rat pulmonary arterial tissues and pulmonary artery smooth muscle cells (PASMCs). Up-regulation of lncRNA CASC2 inhibited cell proliferation, migration yet enhanced apoptosis in vitro and in vivo in hypoxia-induced PH. Western blot analysis and immunochemistry showed that up-regulation of lncRNA CASC2 greatly decreased the expression of phenotype switch-related marker α-SMA in hypoxia-induced PH. Furthermore, it was indicated by the pulmonary artery morphometric analysis that lncRNA CASC2 suppressed vascular remodeling of hypoxia-induced rat pulmonary arterial tissues.ConclusionLncRNA CASC2 inhibited cell proliferation, migration and phenotypic switch of PASMCs to inhibit the vascular remodeling in hypoxia-induced PH.

A key characteristic of hypoxic pulmonary hypertension (HPH) is pulmonary vascular remodeling, involving abnormal proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Recent studies indicate that mesenchymal stem cell-derived exosomes (MSC-exo) exhibit therapeutic effects on HPH. MSC-exosomes were isolated from the conditioned medium of bone mesenchymal stem cells using ultracentrifugation, confirmed via Western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analyses (NTA). Platelet-derived growth factor BB (PDGFBB) induced pathological behavior in PASMCs, replicating the conditions observed in HPH. HPH rats were subjected to a low oxygen environment (10 ± 1% oxygen) for 8 h daily over 28 days. Parameters such as right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. MSC-exosomes suppressed PDGFBB-induced proliferation and migration of PASMCs. Additionally, MSC-exosomes protected rats from hypoxia-induced increases in RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling. The expression of epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ErbB2) was investigated in both HPH lung tissues and PDGFBB-induced PASMCs. Results indicated significant upregulation of EGFR/ErbB2 expression in HPH and PDGFBB-induced PASMCs, which was suppressed by MSC-exosomes. The study demonstrates that MSC-exosomes inhibit the development of HPH by suppressing excessive proliferation and migration of PASMCs through the inhibition of EGFR/ErbB2 heterodimerization.

miR-100 suppresses mTOR signaling in hypoxia-induced pulmonary hypertension in rats

Background: Hypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality, and so far patients have failed to benefit from therapeutics clinically available. Max interacting protein 1–0 (Mxi1-0) is one of the functional isoforms of Mxi1. Although it also binds to Max, Mxi1-0, unlike other Mxi1 isoforms, cannot antagonize the oncoprotein c-Myc because of its unique proline rich domain (PRD). While Mxi1-0 was reported to promote cell proliferation via largely uncharacterized mechanisms, it is unknown whether and how it plays a role in the pathogenesis of HPH.Methods: GEO database was used to screen for genes involved in HPH development, and the candidate players were validated through examination of gene expression in clinical HPH specimens. The effect of candidate gene knockdown or overexpression on cultured pulmonary arterial cells, e.g., pulmonary arterial smooth muscle cells (PASMCs), was then investigated. The signal pathway(s) underlying the regulatory role of the candidate gene in HPH pathogenesis was probed, and the outcome of targeting the aforementioned signaling was evaluated using an HPH rat model.Results: Mxi1 was significantly upregulated in the PASMCs of HPH patients. As the main effector isoform responding to hypoxia, Mxi1-0 functions in HPH to promote PASMCs proliferation. Mechanistically, Mxi1-0 improved the expression of the proto-oncogene c-Myc via activation of the MEK/ERK pathway. Consistently, both a MEK inhibitor, PD98059, and a c-Myc inhibitor, 10058F4, could counteract Mxi1-0-induced PASMCs proliferation. In addition, targeting the MEK/ERK signaling significantly suppressed the development of HPH in rats.Conclusion: Mxi1-0 potentiates HPH pathogenesis through MEK/ERK/c-Myc-mediated proliferation of PASMCs, suggesting its applicability in targeted treatment and prognostic assessment of clinical HPH.

Knockdown of PLOD2 inhibits pulmonary artery smooth muscle cell glycolysis under chronic intermittent hypoxia via PI3K/AKT signal.