Pulmonary Hypertension Induces Serotonin Hyperreactivity and Metabolic Reprogramming in Coronary Arteries via NOX1/4-TRPM2 Signaling Pathway.

BACKGROUND: Clinical evidence revealed abnormal prevalence of coronary artery (CA) disease in patients with pulmonary hypertension (PH). The mechanistic connection between PH and CA disease is unclear. Serotonin (5-hydroxytryptamine), reactive oxygen species, and Ca2+ signaling have been implicated in both PH and CA disease. Our recent study indicates that NOXs (NADPH [nicotinamide adenine dinucleotide phosphate] oxidases) and TRPM2 (transient receptor potential cation channel subfamily M member 2) are key components of their interplay. We hypothesize that activation of the NOX-TRPM2 pathway facilitates the remodeling of CA in PH. METHODS: Left and right CAs from chronic hypoxia and monocrotaline-induced PH rats were collected to study vascular reactivity, gene expression, metabolism, and mitochondrial function. Inhibitors or specific siRNA were used to examine the pathological functions of NOX1/4-TRPM2 in CA smooth muscle cells. RESULTS: Significant CA remodeling and 5-hydroxytryptamine hyperreactivity in the right CA were observed in PH rats. NOX1/4-mediated reactive oxygen species production coupled with TRPM2-mediated Ca2+ influx contributed to 5-hydroxytryptamine hyperresponsiveness. CA smooth muscle cells from chronic hypoxia-PH rats exhibited increased proliferation, migration, apoptosis, and metabolic reprogramming in an NOX1/4-TRPM2-dependent manner. Furthermore, the NOX1/4-TRPM2 pathway participated in mitochondrial dysfunction, involving mitochondrial DNA damage, reactive oxygen species production, elevated mitochondrial membrane potential, mitochondrial Ca2+ accumulation, and mitochondrial fission. In vivo knockdown of NOX1/4 alleviated PH and suppressed CA remodeling in chronic hypoxia rats. CONCLUSIONS: PH triggers an increase in 5-hydroxytryptamine reactivity in the right CA and provokes metabolic reprogramming and mitochondrial disruption in CA smooth muscle cells via NOX1/4-TRPM2 activation. This signaling pathway may play an important role in CA remodeling and CA disease in PH.

TRPC6 promotes daunorubicin-induced mitochondrial fission and cell death in rat cardiomyocytes with the involvement of ERK1/2-DRP1 activation.

Daunorubicin (DNR-) induced cardiotoxicity seriously restricts its clinical application. Transient receptor potential cation channel subfamily C member 6 (TRPC6) is involved in multiple cardiovascular physiological and pathophysiological processes. However, the role of TRPC6 anthracycline-induced cardiotoxicity (AIC) remains unclear. Mitochondrial fragmentation greatly promotes AIC. TRPC6-mediated ERK1/2 activation has been shown to favor mitochondrial fission in dentate granule cells. The aim of the present study was to elucidate the effects of TRPC6 on daunorubicin- induced cardiotoxicity and identify the mechanisms associated with mitochondrial dynamics. The sparkling results showed that TRPC6 was upregulated in models in vitro and in vivo. TRPC6 knockdown protected cardiomyocytes from DNR-induced cell apoptosis and death. DNR largely facilitated mitochondrial fission, boosted mitochondrial membrane potential collapse and damaged debilitated mitochondrial respiratory function in H9c2 cells，these effects were accompanied by TRPC6 upregulation. siTRPC6 effectively inhibited these mitochondrial adverse aspects showing a positive unexposed effect on mitochondrial morphology and function. Concomitantly, ERK1/2-DRP1 which is related to mitochondrial fission was significantly activated with amplified phosphorylated forms in DNR-treated H9c2 cells. siTRPC6 effectively suppressed ERK1/2-DPR1 over activation, hinting at a potential correlation between TRPC6 and ERK1/2-DRP1 by which mitochondrial dynamics are possibly modulated in AIC. TRPC6 knockdown also raised the Bcl-2/Bax ratio, which may help to block mitochondrial fragmentation-related functional impairment and apoptotic signaling. These findings suggested an essential role of TRPC6 in AIC by intensifying mitochondrial fission and cell death via ERK1/2-DPR1, which could be a potential therapeutic target for AIC.

Mouse Abdominal Aortic Aneurysm Model Induced by Periarterial Incubation of Papain.

For decades, numerous experimental animal models have been developed to examine the pathophysiologic mechanisms and potential treatments for abdominal aortic aneurysms (AAAs) in diverse species with varying chemical or surgical approaches. This study aimed to create an AAA mouse model by the periarterial incubation with papain, which can mimic human AAA with advantages such as simplicity, convenience, and high efficiency. Eighty C57BL/6J male mice were randomly assigned to 1 of the 4 groups: papain (1.0 or 2.0 mg), porcine pancreatic elastase, and phosphate-buffered solution. The aortic segment was wrapped for 20 minutes, and the diameter was measured using ultrasound preoperatively and postoperative days 7 and 14. Then, the mice were killed for histomorphometric and immunohistochemical analyses. According to ultrasound measurements and histomorphometric analyses, on postoperative day 7, 65% of mice in the 1.0-mg papain group and 60% of mice in the 2.0-mg papain group developed AAA. In both papain groups, 100% of mice developed AAA, and 65% of mice in the porcine pancreatic elastase group developed AAA on postoperative day 14. Furthermore, hematoxylin/eosin, elastin van Gieson, and Masson staining of tissues from the papain group revealed thickened media and intimal hyperplasia, collagen sediments, and elastin destruction, indicating that AAA histochemical alteration was similar to that of humans. In addition, the immunohistochemical analysis was conducted to detect infiltrated inflammatory cells, such as macrophages and leukocytes, in the aortic wall and hyperplasic adventitia. The expression of matrix metalloproteinase 2 and 9 was significantly upregulated in papain and human AAA tissues. Periarterial incubation with 1.0 mg of papain for 20 minutes can successfully create an experimental AAA model in mice for 14 days, which can be used to explore the mechanism and treatment of human AAA.

Attenuating effect of magnesium on pulmonary arterial calcification in rodent models of pulmonary hypertension.

OBJECTIVE: Vascular calcification has been considered as a potential therapeutic target in pulmonary hypertension. Mg2+ has a protective role against calcification. This study aimed to investigate whether Mg2+ could alleviate pulmonary hypertension by reducing medial calcification of pulmonary arteries. METHODS: Monocrotaline (MCT)-induced and chronic hypoxia-induced pulmonary hypertension rats were given an oral administration of 10% MgSO4 (10 ml/kg per day). Additionally, we administered Mg2+ in calcified pulmonary artery smooth muscle cells (PASMCs) after incubating with ß-glycerophosphate (ß-GP, 10 mmol/l). RESULTS: In vivo, MCT-induced and chronic hypoxia-induced pulmonary hypertension indexes, including right ventricular systolic pressure, right ventricular mass index, and arterial wall thickness, as well as Alizarin Red S (ARS) staining-visualized calcium deposition, high calcium levels, and osteochondrogenic differentiation in pulmonary arteries, were mitigated by dietary Mg2+ intake. In vitro, ß-GP-induced calcium-rich deposits stained by ARS, calcium content, as well as the detrimental effects of calcification to proliferation, migration, and resistance to apoptosis of PASMCs were alleviated by high Mg2+ but exacerbated by low Mg2+. Expression levels of mRNA and protein of ß-GP-induced osteochondrogenic markers, RUNX Family Transcription Factor 2, and Msh Homeobox 2 were decreased by high Mg2+ but increased by low Mg2+; however, Mg2+ did not affect ß-GP-induced expression of SRY-Box Transcription Factor 9. Moreover, mRNA expression and protein levels of ß-GP-reduced calcification inhibitor, Matrix GLA protein was increased by high Mg2+ but decreased by low Mg2+. CONCLUSION: Mg2+ supplement is a powerful strategy to treat pulmonary hypertension by mitigating pulmonary arterial calcification as the calcification triggered physiological and pathological changes to PASMCs.

Reduced CircSMOC1 Level Promotes Metabolic Reprogramming via PTBP1 (Polypyrimidine Tract-Binding Protein) and miR-329-3p in Pulmonary Arterial Hypertension Rats.

BACKGROUND: Pulmonary arterial hypertension maintains rapid cell proliferation and vascular remodeling through metabolic reprogramming. Recent studies suggested that circRNAs play important role in pulmonary vascular remodeling and pulmonary arterial smooth muscle cells proliferation. However, the relationship between circRNA, cell proliferation, and metabolic reprogramming in pulmonary arterial hypertension has not been investigated. METHODS: RNA-seq and qRT-PCR reveal the differential expression profile of circRNA in pulmonary arteries of pulmonary arterial hypertension rat models. Transfection was used to examine the effects of circSMOC1 on pulmonary artery smooth muscle cells, and the roles of circSMOC1 in vivo were investigated by adenoassociated virus. Mass spectrometry, RNA pull-down, RNA immunoprecipitation, and dual-luciferase reporter assay were performed to investigate the signaling pathway of circSMOC1 regulating the metabolic reprogramming. RESULTS: CircSMOC1 was significantly downregulated in pulmonary arteries of pulmonary arterial hypertension rats. CircSMOC1 knockdown promoted proliferation and migration and enhanced aerobic glycolysis of pulmonary artery smooth muscle cells. CircSMOC1 overexpression in vivo alleviates pulmonary vascular remodeling, right ventricular pressure, and right heart hypertrophy. In the nucleus, circSMOC1 directly binds to PTBP1 (polypyrimidine tract-binding protein), competitively inhibits the specific splicing of PKM (pyruvate kinase M) premRNA, resulting in the upregulation of PKM2 (pyruvate kinase M2), the key enzyme of aerobic glycolysis, to enhance glycolysis. In the cytoplasm, circSMOC1 acted as a miR-329-3p sponge, and its reduction in pulmonary arterial hypertension suppressed PDHB (pyruvate dehydrogenase E1 subunit beta) expression, leading to the impairment of mitochondrial oxidative phosphorylation. CONCLUSIONS: circSMOC1 is crucially involved in the metabolic reprogramming of pulmonary artery smooth muscle cells through PTBP1 and miR-329-3p to regulate pulmonary vascular remodeling in pulmonary arterial hypertension.

Effects of an art-based intervention in older adults with mild cognitive impairment: a randomised controlled trial.

BACKGROUND: Art-based interventions may delay cognitive decline and improve health-related outcomes in older adults with mild cognitive impairment (MCI). OBJECTIVE: To examine the effects of the Creative Expressive Arts-based Storytelling (CrEAS) program compared to active and waitlist controls on neurocognitive and other health-related outcomes in older people with MCI. DESIGN: Three-arm parallel-group, randomised controlled design. PARTICIPANTS: One-hundred and thirty-five adults with MCI (mean age: 70.93 ± 6.91 years). METHODS: Participants were randomly assigned to intervention (CrEAS, n = 45), active control (n = 45) or waitlist control (n = 45) groups. Interventions were applied once per week for 24 weeks. The primary outcome was global cognitive function; secondary outcomes were specific cognition domains (memory, executive function, language and attention) and other health-related outcomes (anxiety, depression and quality of life [QoL]). All variables were measured at baseline (T0), 24-week follow-up (T1) and 48-week follow-up (T2). RESULTS: Participants in the CrEAS group showed significantly higher global cognitive function (adjusted mean difference [MD] = -0.905, 95% confidence interval [CI] -1.748 to -0.062; P = 0.038) and QoL (adjusted MD = -4.150, 95% CI -6.447 to -1.853; P = 0.001) and lower depression symptoms (adjusted MD = 2.902, 95% CI 0.699-5.104; P = 0.011) post-intervention at the 24-week follow-up compared with the active control group. At 48-week follow-up, only the Auditory Verbal Learning Test Immediate recall score was significantly improved compared with the active control group (adjusted MD = -2.941, 95% CI -5.262 to -0.620; P = 0.014). CONCLUSIONS: Older adults with MCI who participated in the CrEAS program improved their neuropsychological outcomes and QoL and reduced their rate of cognitive deterioration.

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

NEW FINDINGS: What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. ABSTRACT: Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

[TRPV4 channel mediates the increase of pulmonary microvascular endothelial permeability in rats with chronic hypoxic pulmonary hypertension].

The purpose of the present study was to investigate the effect of transient receptor potential vanilloid 4 (TRPV4) channel on the permeability of pulmonary microvascular endothelial cells (PMVECs) in rats with chronic hypoxia-induced pulmonary hypertension (CHPH), so as to clarify the mechanism of vascular endothelial dysfunction during the occurrence of pulmonary hypertension (PH). CHPH rat model was established by exposure to chronic hypoxia (CH) for 21 days. Primary PMVECs were cultured by adherent tissue blocks at the edge of the lung. The permeability coefficient of primary cultured PMVECs was detected by fluorescein isothiocyanate (FITC)-dextran. The structure of tight junction (TJ) was observed by transmission electron microscope. The expression of TRPV4 and TJ-related proteins, such as, Occludin, Claudin-5, ZO-1 were examined by real-time fluorescence quantitative PCR and Western blotting. The intracellular calcium concentration ([Ca2+]i) in PMVECs and its effect on PMVECs permeability were observed after the intervention of TRPV4 specific agonist GSK1016790A (GSK, 10 nmol/L) and specific inhibitor HC-067047 (HC, 1 µmol/L, 0.5 µmol/L). The results showed that the CHPH model was successfully established in rats treated with CH for 21 days. In CHPH rats, the structure of TJ was destroyed, the function of PMVECs barrier was decreased, the intercellular permeability was increased, the expression of TJ-related proteins were significantly decreased and the expression of TRPV4 was significantly increased (P < 0.01). The amplitude of [Ca2+]i in PMVECs of CHPH rats was significantly increased after activation of TRPV4. The inhibition ratio of HC on [Ca2+]i in PMVECs of CHPH rats was significantly higher than that in normal PMVECs. TRPV4 specific inhibitor HC reversed the increase of PMVECs permeability and increased the expression of three TJ-related proteins in CHPH rats (P < 0.01, P < 0.05). These results suggest that TRPV4 channel can induce endothelial dysfunction by increasing the [Ca2+]i, resulting in the destruction of TJ structure and the decrease of TJ-related proteins expression on PMVECs in CHPH rats.

TRPC3 promotes tumorigenesis of gastric cancer via the CNB2/GSK3ß/NFATc2 signaling pathway.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3ß and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3ß-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression.

Serotonin and chronic hypoxic pulmonary hypertension activate a NADPH oxidase 4 and TRPM2 dependent pathway for pulmonary arterial smooth muscle cell proliferation and migration.

5-Hydroxytryptamine (5-HT)-dependent signaling mediated through its transporters and receptors plays important roles in chronic hypoxic pulmonary hypertension (CHPH), which is associated with aberrant reactive oxygen species (ROS) production. NADPH oxidase 4 (NOX4) is one of the major sources of ROS in pulmonary vasculature, and has been implicated in the development of PH. NOX4 generates H2O2, which can activate the transient receptor potential melastatin 2 (TRPM2) channels, providing Ca2+ signals for cell proliferation and migration. However, the connection between 5-HT, NOX4, ROS and TRPM2 in the context of PH has not been established. Here we examined the level of 5-HT and expression of NOX4 and TRPM2, and their roles in pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration. NOX4 and TRPM2 were upregulated in pulmonary arteries of CHPH rats, which were associated with elevated levels of 5-HT and ROS, and enhanced proliferation and migration in PASMCs. The increase in ROS, and the enhanced proliferation and migration of PASMCs from CHPH rats were mimicked by treating normoxic PASMCs with 5-HT. 5-HT; and CH-induced ROS production were reversed by catalase, the NOX1/NOX4 inhibitor GKT137831, and Nox4 siRNA. 5-HT and H2O2 elicited Ca2+ responses were significantly augmented in CHPH PASMCs; and the augmented Ca2+ responses were obliterated by the 2-Aminoethoxydiphenyl borate (2-APB) and Trpm2-specific siRNA. Moreover, 5-HT and CH-induced proliferation and migration were suppressed by Nox4 or Trpm2 siRNA; and simultaneous transfection of both siRNA did not cause further inhibition. These results suggest that the 5-HT and CH-induced PASMC proliferation and migration were mediated, at least in part, by TRPM2 via activation of NOX4-dependent ROS production; and revealed a novel NOX4-ROS-TRPM2 signaling pathway for the pathogenesis of CHPH.

Magnesium Supplementation Attenuates Pulmonary Hypertension via Regulation of Magnesium Transporters.

Pulmonary hypertension (PH) is characterized by profound vascular remodeling and altered Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Magnesium ion (Mg2+), a natural Ca2+ antagonist and a cofactor for numerous enzymes, is crucial for regulating diverse cellular functions, but its roles in PH remains unclear. Here, we examined the roles of Mg2+ and its transporters in PH development. Chronic hypoxia and monocrotaline induced significant PH in adult male rats. It was associated with a reduction of [Mg2+]i in PASMCs, a significant increase in gene expressions of Cnnm2, Hip14, Hip14l, Magt1, Mmgt1, Mrs2, Nipa1, Nipa2, Slc41a1, Slc41a2 and Trpm7; upregulation of SLC41A1, SLC41A2, CNNM2, and TRPM7 proteins; and downregulation of SLC41A3 mRNA and protein. Mg2+ supplement attenuated pulmonary arterial pressure, right heart hypertrophy, and medial wall thickening of pulmonary arteries, and reversed the changes in the expression of Mg2+ transporters. Incubation of PASMCs with a high concentration of Mg2+ markedly inhibited PASMC proliferation and migration, and increased apoptosis, whereas a low level of Mg2+ produced the opposite effects. siRNA targeting Slc41a1/2, Cnnm2, and Trpm7 attenuated PASMC proliferation and migration, but promoted apoptosis; and Slc41a3 overexpression also caused similar effects. Moreover, siRNA targeting Slc41a1 or high [Mg2+] incubation inhibited hypoxia-induced upregulation and nuclear translocation of NFATc3 in PASMCs. The results, for the first time, provide the supportive evidence that Mg2+ transporters participate in the development of PH by modulating PASMC proliferation, migration, and apoptosis; and Mg2+ supplementation attenuates PH through regulation of Mg2+ transporters involving the NFATc3 signaling pathway.

Keratin 1 attenuates hypoxic pulmonary artery hypertension by suppressing pulmonary artery media smooth muscle expansion.

AIM: Abnormally activated vascular smooth muscle cells are key factors in pulmonary artery remodelling (PAR) and pulmonary artery hypertension (PAH). Keratin 1 is involved in inflammatory diseases; however, its role in PAH is unknown. We speculated that keratin 1 could regulate PASMCs and prevent PAH. METHODS: Rats were exposed to hypoxia (10% O2 ) or MCT (50 mg/kg, intraperitoneal injection) or treated with AAV6 virus. PAR was measured through HE and Masson staining. PASMC activities were measured using MTS assay, EdU and Western blot analyses after cell knockdown with siRNAs or overexpression with Krt1 vectors. RESULTS: 1. Hypoxic PAR was associated with a decrease in keratin 1, especially in PASMCs. 2. Keratin 1 knockdown led to cell proliferation, migration and contraction to synthetic transformation, while keratin 1 overexpression attenuated hypoxia-induced changes in PASMCs. 3. Decreased keratin 1 induced TLR7 upregulation and mediated increases in the inflammatory factors S100a8 and S100a9. 4. Keratin 1 overexpression reduced the inflammatory factor expression induced by TLR7 activation. 5. Further studies demonstrated that keratin 1 expression was negatively correlated with pulmonary vascular pressure following prolonged hypoxia. 6. Pre-treatment with keratin 1 decreased pulmonary artery pressure and the right heart hypertrophy index and alleviated PAR in two model rats. 7. Keratin 1 exhibited a hypermethylation status in hypoxic pulmonary arteries in the sequencing. Hypoxia-induced decrease in keratin 1 expression was associated with Dnmt1 upregulation induced by YY1 downregulation in PASMCs. CONCLUSION: This study suggests that keratin 1 regulates PASMC expansion and has a preventive effect on PAH.

Preventive treatment with ginsenoside Rb1 ameliorates monocrotaline-induced pulmonary arterial hypertension in rats and involves store-operated calcium entry inhibition.

CONTEXT: Ginsenoside Rb1, the main active ingredient of ginseng, exhibits ex vivo depression of store-operated calcium entry (SOCE) and related vasoconstriction in pulmonary arteries derived from pulmonary hypertension (PH) rats. However, the in vivo effects of ginsenoside Rb1 on PH remain unclear. OBJECTIVE: This study explored the possibility of using ginsenoside Rb1 as an in vivo preventive medication for type I PH, i.e., pulmonary arterial hypertension (PAH), and potential mechanisms involving SOCE. MATERIALS AND METHODS: Male Sprague-Dawley rats (170-180 g) were randomly divided into Control, MCT, and MCT + Rb1 groups (n = 20). Control rats received only saline injection. Rats in the MCT + Rb1 and MCT groups were intraperitoneally administered single doses of 50 mg/kg monocrotaline (MCT) combined with 30 mg/kg/day ginsenoside Rb1 or equivalent volumes of saline for 21 consecutive days. Subsequently, comprehensive parameters related to SOCE, vascular tone, histological changes and hemodynamics were measured. RESULTS: Ginsenoside Rb1 reduced MCT-induced STIM1, TRPC1, and TRPC4 expression by 35.00, 31.96, and 32.24%, respectively, at the protein level. SOCE-related calcium entry and pulmonary artery contraction decreased by 162.6 nM and 71.72%. The mean pulmonary artery pressure, right ventricle systolic pressure, and right ventricular mass index decreased by 19.5 mmHg, 21.6 mmHg, and 39.50%. The wall thickness/radius ratios decreased by 14.67 and 17.65%, and the lumen area/total area ratios increased by 18.55 and 15.60% in intrapulmonary vessels with 51-100 and 101-150 µm o.d. CONCLUSION: Ginsenoside Rb1, a promising candidate for PH prevention, inhibited SOCE and related pulmonary vasoconstriction, and relieved MCT-induced PAH in rats.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy.

OBJECTIVES: To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. METHODS: In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100-300 µM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate Calbiochem (2.5 µM), and then precontracted with phenylephrine (Phen, 10-6 M), and subsequently dilated in acetylcholine (ACh, 10-6 M - 10-4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10-4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10-6 M) followed by ACh (10-5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. RESULTS: ACh (10-6 M - 10-4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10-4 M) significantly attenuated the vasodilatation and the intracellular NO release. CONCLUSIONS: This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

Revealing the pathogenic changes of PAH based on multiomics characteristics.

BACKGROUND: Pulmonary artery hypertension (PAH), which is characterized by an increase in pulmonary circulation blood pressure, is a fatal disease, and its pathogenesis remains unclear. METHODS: In this study, RNA sequencing (RNA-seq), tandem mass tags (TMT) and reduced representation bisulfite sequencing (RRBS) were performed to detect the levels of mRNA, protein, and DNA methylation in pulmonary arteries (PAs), respectively. To screen the possible pathways and proteins related to PAH, pathway enrichment analysis and protein-protein interaction (PPI) network analysis were performed. For selected genes, differential expression levels were confirmed at both the transcriptional and translational levels by real-time PCR and Western blot analyses, respectively. RESULTS: A total of 362 differentially expressed genes (|Fold-change| > 1.5 and p < 0.05), 811 differentially expressed proteins (|Fold-change| > 1.2 and p < 0.05) and 76,562 differentially methylated regions (1000 bp slide windows, 500 bp overlap, p < 0.05, and |Fold-change| > 1.2) were identified when the PAH group (n = 15) was compared with the control group (n = 15). Through an integrated analysis of the characteristics of the three omic analyses, a multiomics table was constructed. Additionally, pathway enrichment analysis showed that the differentially expressed proteins were significantly enriched in five Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways and ten Gene Ontology (GO) terms for the PAH group compared with the control group. Moreover, protein-protein interaction (PPI) networks were constructed to identify hub genes. Finally, according to the genes identified in the PPI and the protein expression fold-change, nine key genes and their associated proteins were verified by real-time PCR and Western blot analyses, including Col4a1, Itga5, Col2a1, Gstt1, Gstm3, Thbd, Mgst2, Kng1 and Fgg. CONCLUSIONS: This study conducted multiomic characteristic profiling to identify genes that contribute to the hypoxia-induced PAH model, identifying new avenues for basic PAH research.

Increased caveolin-1 expression enhances the receptor-operated Ca2+ entry in the aorta of two-kidney, one-clip hypertensive rats.

NEW FINDINGS: What is the central question of this study? The aim was to examine and compare the contributions of caveolin-1 to the contractile responses mediated by L-type voltage-dependent calcium channels, store-operated Ca2+ channels and receptor-operated Ca2+ channels in two different types of arteries from two-kidney, one-clip hypertensive rats. What is the main finding and its importance? We demonstrated that the density of caveolae and caveolin-1 expression were significantly upregulated in the aorta of two-kidney, one-clip hypertensive rats, but not in the third-order branches of mesenteric arteries. We highlight that caveolin-1 plays an important role in aortic constriction by enhancing receptor-operated Ca2+ entry in the hypertensive rat model. ABSTRACT: Calcium and its multiple regulatory mechanisms are crucial for the development of hypertension. Among these regulatory mechanisms, store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mediate agonist-induced calcium influx, contributing to vascular contraction. The SOCE and ROCE are regulated by a variety of mechanisms involving caveolin-1 (Cav1), which has been found to be strongly associated with hypertension in gene polymorphism. In the present study, we investigated the role of Cav1 during the enhanced activity of calcium channels in hypertensive arteries. We demonstrated that the expression level of Cav1 was significantly increased in the aorta of two-kidney, one-clip (2K1C) hypertensive rats. The disruption of caveolae by methyl-ß-cyclodextrin did not cause a marked difference in agonist-induced vasoconstriction in the third-order branches of the mesenteric arteries but strongly suppressed the aortic contractile response to endothelin-1 in the 2K1C group, which was not found in the control group. The increase in Cav1 by introduction of Cav1 scaffolding domain enhancing peptide promoted the 1-oleoyl-2-acetyl-glycerol-induced ROCE in hypertensive aortic smooth muscle cells but did not enhance the cyclopiazonic acid-induced SOCE. In the resistance arteries, similar changes were not observed, and no statistical changes of Cav1 expression were evident in the third-order branches of the mesenteric arteries. Our results indicate that increased Cav1 expression might promote the altered [Ca2+ ]i -induced aortic vasoreactivity by enhancing ROCE and be involved in the pathogenesis of hypertension.

Galectin-3- Mediated Transdifferentiation of Pulmonary Artery Endothelial Cells Contributes to Hypoxic Pulmonary Vascular Remodeling.

BACKGROUND/AIMS: Vascular muscularity is a key event in vessel remodeling during pulmonary artery hypertension (PAH). Endothelial-mesenchymal transdifferentiation (EndMT) has been increasingly reported to play a role in disease occurrence. Galectin-3, a carbohydrate-binding protein regulates cell proliferation, differentiation, migration and neovascularization. However, whether galectin-3 controls endothelial cell transdifferentiation during the development of PAH is unknown. METHODS: Rats were exposed to normoxic or hypoxic conditions (fraction of inspired O2 0.10) for 21 d to establish PAH models. Hemodynamic changes were evaluated through surgery of the right jugular vein and ultrasound biomicroscopy inviVue. And vessel pathological alterations were detected by H&E staining. Galectin-3 (Gal-3)-induced pulmonary artery endothelium cell (PAEC) dynamic alterations were measured by MTT assays, Cell immunofluorescence, Flow cytometry, Real-time PCR and Western blot. RESULTS: Our study demonstrated that Gal-3 was expressed in hypoxic pulmonary vascular adventitia and intima. The increased Gal-3 expression was responsible for hypoxic vessel remodeling and PAH development in vivo. Gal-3 was found to inhibit cell proliferation and apoptosis in cultured endothelial cells. Meanwhile endothelial cell morphology was altered and exhibited smooth muscle-like cell features as demonstrated by the expression of α-SMA after Gal-3 treatment. Gal-3 activated Jagged1/Notch1 pathways and induced MyoD and SRF. When MyoD or SRF were silenced with siRNAs, Gal-3-initiated transdifferentiation in endothelial cells was blocked as indicated by a lack of α-SMA. CONCLUSION: These results suggest that Gal-3 induces PAECs to acquire an α-SMA phenotype via a transdifferentiation process which depends on the activation of Jagged1/Notch1 pathways that mediate MyoD and SRF expression.

Calcineurin/NFAT Signaling Modulates Pulmonary Artery Smooth Muscle Cell Proliferation, Migration and Apoptosis in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats.

BACKGROUND/AIMS: Pulmonary arterial hypertension (PAH) is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, which is driven by excessive proliferation and migration and apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs). The calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway is the most important downstream signaling pathway of store-operated Ca2+ entry (SOCE), which is increased in PAH. CaN/NFAT has been reported to contribute to abnormal proliferation in chronic hypoxia (CH)-induced PAH. However, the effect of CaN/NFAT signaling on PASMC proliferation, migration and apoptosis in monocrotaline (MCT)-induced PAH remains unclear. METHODS: PAH rats were established by a single intraperitoneal injection of MCT for 21 days. PASMCs were isolated and cultured in normal and MCT-induced PAH Sprague-Dawley rat. PASMCs were treated with CsA targeting CaN and siRNA targeting NFATc2-4 gene respectively by liposome. We investigated the expression of calcineurin/NFAT signaling by immunofluorescence, qRT-PCR and Western blotting methods. Cell proliferation was monitored using MTS reagent or by assessing proliferating cell nuclear antigen (PCNA) expression. Cell apoptosis was evaluated with an Annexin V - FITC/propidium iodide (PI) apoptosis kit by flow cytometry. PASMC migration was assessed with a Transwell chamber. RESULTS: MCT successfully induced PAH and pulmonary vascular remodeling in rats. CaN phosphatase activity and nuclear translocation of NFATc2-4 were increased in PASMCs derived from MCT-treated rats. In addition, CaNBß/NFATc2-4 expression was amplified at the mRNA and protein levels. PASMC proliferation and migration were markedly inhibited in a dosedependent manner by cyclosporin A (CsA). Furthermore, siRNA targeting NFATc2 and NFATc4 attenuated the excessive proliferation and migration and apoptosis resistance in PASMCs derived from both CON and MCT-treated rats, while NFATc3 knockdown specifically affected MCT-PASMCs. CONCLUSION: Our results demonstrate that CaN/NFAT signaling is activated and involved in the modulation of PASMC proliferation, migration and apoptosis in MCT-induced PAH.

Transient Receptor Potential Melastatin-8 Activation Induces Relaxation of Pulmonary Artery by Inhibition of Store-Operated Calcium Entry in Normoxic and Chronic Hypoxic Pulmonary Hypertensive Rats.

Pulmonary hypertension (PH) is characterized by enhanced vasoconstriction and vascular remodeling, which are attributable to the alteration of Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). It is well established that store-operated Ca2+ entry (SOCE) is augmented in PASMCs during PH and that it plays a crucial role in PH development. Our previous studies showed that the melastatin-related transient receptor potential 8 (TRPM8) is down-regulated in PASMCs of PH animal models, and activation of TRPM8 causes relaxation of pulmonary arteries (PAs). However, the mechanism of TRPM8-induced PA relaxation is unclear. Here we examined the interaction of TRPM8 and SOCE in PAs and PASMCs of normoxic and chronic hypoxic pulmonary hypertensive (CHPH) rats, a model of human group 3 PH. We found that TRPM8 was down-regulated and TRPM8-mediated cation entry was reduced in CHPH-PASMCs. Activation of TRPM8 with icilin caused concentration-dependent relaxation of cyclopiazonic acid (CPA) and endothelin-1 contracted endothelium-denuded PAs, and the effect was abolished by the SOCE antagonist Gd3+ Application of icilin to PASMCs suppressed CPA-induced Mn2+ quenching and Ca2+ entry, which was reversed by the TRPM8 antagonist N-(3-aminopropyl)-2-([(3-methylphenyl)methyl])-oxy-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB). Moreover, the inhibitory effects of icilin on SOCE in PA and PASMCs of CHPH rats were significantly augmented due to enhanced SOCE activity in PH. Our results, therefore, demonstrated a novel mechanism of TRPM8-mediated inhibition of SOCE in pulmonary vasculature. Because SOCE is important for vascular remodeling and enhanced vasoconstriction, down-regulation of TRPM8 in PASMCs of CHPH rats may minimize its inhibitory influence to allow unimpeded SOCE activity for PH development.

Magnesium attenuates endothelin-1-induced vasoreactivity and enhances vasodilatation in mouse pulmonary arteries: Modulation by chronic hypoxic pulmonary hypertension.

NEW FINDINGS: What is the central question of this study? The central goal of this study was to elucidate the role of magnesium in the regulation of pulmonary vascular reactivity in relationship to hypoxic pulmonary hypertension. What is the main finding and its importance? We found that magnesium is essential for normal vasoreactivity of the pulmonary artery. Increasing the magnesium concentration attenuates vasoconstriction and improves vasodilatation via release of nitric oxide. Pulmonary hypertension is associated with endothelial dysfunction resulting in the suppression of magnesium modulation of vasodilatation. These results provide evidence that magnesium is important for the modulation of pulmonary vascular function. ABSTRACT: Pulmonary hypertension (PH) is characterized by enhanced vasoreactivity and sustained pulmonary vasoconstriction, arising from aberrant Ca2+ homeostasis in pulmonary arterial (PA) smooth muscle cells. In addition to Ca2+ , magnesium, the most abundant intracellular divalent cation, also plays crucial roles in many cellular processes that regulate cardiovascular function. Recent findings suggest that magnesium regulates vascular functions by altering the vascular responses to vasodilator and vasoactive agonists and affects endothelial function by modulating endothelium-dependent vasodilatation in hypertension. Administration of magnesium also decreased pulmonary arterial pressure and improved cardiac output in animal models of PH. However, the role of magnesium in the regulation of pulmonary vascular function related to PH has not been studied. In this study, we examined the effects of magnesium on endothelin-1 (ET-1)-induced vasoconstriction, ACh-induced vasodilatation and the generation of NO in PAs of normoxic mice and chronic hypoxia (CH)-treated mice. Our data showed that removal of extracellular magnesium suppressed vasoreactivity of PAs to both ET-1 and ACh. A high concentration of magnesium (4.8 mm) inhibited ET-1-induced vasoconstriction in endothelium-intact or endothelium-disrupted PAs of normoxic and CH-treated mice, and enhanced the ACh-induced production of NO in PAs of normoxic mice. Moreover, magnesium enhanced ACh-induced vasodilatation in PAs of normoxic mice, and the enhancement was completely abolished after exposure to CH. Hence, in this study we demonstrated that increasing the magnesium concentration can attenuate the ET-1-induced contractile response and improve vasodilatation via release of NO from the endothelium. We also demonstrated that chronic exposure to hypoxia can cause endothelial dysfunction resulting in suppression of the magnesium-dependent modulation of vasodilatation.