RESUMO
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.
Assuntos
Aorta Abdominal , Aneurisma da Aorta Abdominal , Masculino , Camundongos , Humanos , Animais , Suínos , Aorta Abdominal/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Elastina/efeitos adversos , Elastina/metabolismo , Papaína/efeitos adversos , Papaína/metabolismo , Camundongos Endogâmicos C57BL , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Modelos Animais de Doenças , Elastase Pancreática/efeitos adversos , Elastase Pancreática/metabolismoRESUMO
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 cellsï¼these 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.
Assuntos
Daunorrubicina , Miócitos Cardíacos , Canal de Cátion TRPC6 , Animais , Ratos , Apoptose , Cardiotoxicidade/metabolismo , Morte Celular , Daunorrubicina/toxicidade , Dinaminas/metabolismo , Sistema de Sinalização das MAP Quinases , Dinâmica Mitocondrial , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6/metabolismoRESUMO
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.
Assuntos
Hipertensão Pulmonar , Monocrotalina , Animais , Cálcio , Células Endoteliais , Hipertensão Pulmonar/induzido quimicamente , Magnésio/farmacologia , Camundongos , Monocrotalina/efeitos adversos , Artéria Pulmonar , Ratos , Ratos Sprague-Dawley , VasoconstriçãoRESUMO
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.
Assuntos
Transtornos Cognitivos , Disfunção Cognitiva , Idoso , Cognição , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/psicologia , Disfunção Cognitiva/terapia , Função Executiva , Humanos , Qualidade de VidaRESUMO
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.
Assuntos
Hipertensão Pulmonar , Canais de Cátion TRPV , Animais , Células Endoteliais , Hipóxia/complicações , Pulmão , Permeabilidade , Ratos , Canais de Cátion TRPV/genéticaRESUMO
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.
Assuntos
Endotélio Vascular/metabolismo , Microscopia Confocal , Miografia , Óxido Nítrico/metabolismo , Artéria Pulmonar/metabolismo , Vasodilatação , Acetilcolina/farmacologia , Animais , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos Endogâmicos ICR , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
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.
Assuntos
Cálcio/metabolismo , Ginsenosídeos/farmacologia , Hipertensão Arterial Pulmonar/prevenção & controle , Animais , Modelos Animais de Doenças , Masculino , Monocrotalina , Panax/química , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacosRESUMO
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.
Assuntos
Genômica , Hipertensão Arterial Pulmonar/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Hipóxia/complicações , Masculino , Mapas de Interação de Proteínas , Hipertensão Arterial Pulmonar/etiologia , Hipertensão Arterial Pulmonar/patologia , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Aorta/metabolismo , Cálcio/metabolismo , Caveolina 1/metabolismo , Hipertensão/metabolismo , Animais , Masculino , Artérias Mesentéricas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Apoptose , Calcineurina/metabolismo , Proliferação de Células , Hipertensão Pulmonar/patologia , Fatores de Transcrição NFATC/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Apoptose/efeitos dos fármacos , Calcineurina/química , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclosporina/farmacologia , Modelos Animais de Doenças , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/metabolismo , Masculino , Monocrotalina/toxicidade , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Artéria Pulmonar/citologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
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.
Assuntos
Transdiferenciação Celular , Galectina 3/metabolismo , Remodelação Vascular , Animais , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Galectina 3/antagonistas & inibidores , Galectina 3/genética , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/metabolismo , Masculino , Proteína MyoD/antagonistas & inibidores , Proteína MyoD/genética , Proteína MyoD/metabolismo , Artéria Pulmonar/citologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Receptor Notch1/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Fator de Resposta Sérica/antagonistas & inibidores , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Remodelação Vascular/efeitos dos fármacosRESUMO
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.
Assuntos
Cálcio/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Canais de Cátion TRPM/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Endotelina-1/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão Pulmonar/metabolismo , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Pirimidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPC/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
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.
Assuntos
Endotelina-1/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/tratamento farmacológico , Magnésio/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Óxido Nítrico/metabolismo , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
This study was aimed to establish an optimized method to observe the synchronous changes of vascular tension and intracellular Ca2+ signal in the third-order branches of mesenteric arteries (sMA, diameter: 100-300 µm). The vascular tension and intracellular Ca2+ signal changes in response to potassium chloride (KCl), endothelin-1 (ET-1) and Gd3+ were detected using confocal wire myograph system and confocal laser scanning microscopy imaging technique, respectively. The experimental results were analyzed to explore the optimal experimental conditions. The results showed that KCl caused contraction in sMA significantly, and the intracellular Ca2+ level of vascular smooth muscle cells (VSMCs) was also increased under 20× and 40× objective lens. Compared with those under the 40× objective lens, the Ca2+ signal change was larger and the fluorescence value was more stable under the 20× objective lens, whereas the Ca2+ signal change was not obvious under the 10× objective lens. ET-1 (1-10 nmol/L) caused concentration dependent contraction in sMA significantly, and the intracellular Ca2+ signal of VSMCs was also enhanced in a concentration dependent manner. Additionally, Gd3+ significantly reduced the contraction of sMA and the intracellular Ca2+ signal of VSMCs caused by ET-1. The results suggest that the intracellular Ca2+ signal of VSMCs changes with vascular contraction or relaxation caused by the agonists or antagonists of Ca2+ channels. We successfully recorded both changes synchronously using confocal wire myograph system and confocal laser scanning microscopy imaging technique at the same time. Based on the analysis of the experimental results, we concluded that 20× objective lens provides the best experimental condition. Compared to combination of vascular tone detection method and real-time cellular fluorescence imaging technique, the present synchronous method is convenient and helpful to reduce experimental error.
Assuntos
Sinalização do Cálcio/fisiologia , Artérias Mesentéricas/fisiologia , Animais , Endotelina-1/farmacologia , Masculino , Microscopia Confocal , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
This study was designed to observe the differences between main pulmonary arteries and the third-order branches of pulmonary arteries in the contractile response to phenylephrine (Phen), endothelin-1 (ET-1) and potassium chloride (KCl). The vascular tension changes of main and the third-order branches of pulmonary arteries induced by KCl, ET-1 and Phen were recorded by traditional vascular tone detection methods and microvascular ring technique, respectively. The results showed that Phen could cause a significant contraction in main pulmonary arteries, but did not induce apparent contraction in the third-order branches of pulmonary arteries. Compared with main pulmonary arteries, ET-1 contracted the third-order branches of pulmonary arteries with reduced maximal response value and PD2 value. In comparison with the main pulmonary arteries, contraction caused by KCl was enhanced in the third-order branches of pulmonary arteries. The results suggest that the vascular reactivity of main and the third-order branches of pulmonary arteries is different and it is important to study the vascular function of small branches of pulmonary arteries. This study could provide an important experimental basis for the further study on vascular function of small branches of pulmonary arteries and the functional changes in pulmonary hypertension.
Assuntos
Endotelina-1/farmacologia , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Vasoconstrição , Animais , Masculino , RatosRESUMO
BACKGROUND/AIMS: Alterations in intracellular Ca2+ concentration ([Ca2+]i) underlie the pathogenesis of various cardiovascular diseases. Caveolin-1 (Cav-1) is the primary functional protein associated with caveolae, which are invaginations in the plasma membrane, and is a regulator of [Ca2+]i signaling. Caveolae and Cav-1 increase the activity of store-operated Ca2+ channels (SOCC) in rat pulmonary arterial smooth muscle cells (PASMCs), and these enhancing effects were more pronounced in rats with pulmonary hypertension (PH). Classical transient receptor potential (TRPC) proteins are highly expressed in vascular smooth muscle cells, and these proteins form functional receptor-operated Ca2+ channels (ROCC) and SOCC in PASMCs. Previous studies suggested that functional and structural changes in aortas might occur during the pathological process of PH. Our data demonstrated that Cav-1 and TRPC were also abundant in the aorta smooth muscle cells (AoSMCs) of PH rats. However, previous PH research primarily focused on Ca2+ channels in pulmonary arteries, but not functional changes in Ca2+ channels in aortas. The contribution of Cav-1 of AoSMCs to alterations of Ca2+ signaling in aortic functions during the pathological process of PH has not been fully characterized. Therefore, this study investigated alterations in Cav-1 expression and the relationship of these changes to Ca2+ channels in AoSMCs of PH rats. METHODS: The present study examined physiological caveolae and Cav-1 expression and characterized the function of altered Cav-1 expression in rat aortas with PH. RESULTS: The appearance of caveolae with Cav-1 expression increased significantly in the aortas of rats with PH, but TRPC1 and TRPC6 expression was not altered. In vitro experiments demonstrated that caveolae contributed to phenylephrine, endothelin-1, and 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced aortic vasoreactivity, but KCl and cyclopiazonic acid had no effect, which suggests the vital ability of Cav-1 to regulate ROCC activity. The introduction of Cav-1 scaffolding domain peptide enhanced OAG-induced ROCC function in primary AoSMCs. CONCLUSION: Cav-1 is specifically associated with ROCC in aortas and plays a vital role in altering vasoreactivity, which affects cardiovascular diseases pathology. Caveolae and Cav-1 up-regulation may affect the function of ROCC in rat models of PH.
Assuntos
Aorta/metabolismo , Cálcio/metabolismo , Caveolina 1/metabolismo , Hipertensão Pulmonar/metabolismo , Animais , Aorta/fisiologia , Aorta/ultraestrutura , Western Blotting , Cavéolas/metabolismo , Caveolina 1/genética , Células Cultivadas , Expressão Gênica , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Masculino , Microscopia Eletrônica de Transmissão , Miócitos de Músculo Liso/metabolismo , Fenilefrina/farmacologia , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
BACKGROUND: Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, enhanced vasoreactivity and vascular remodeling, which leads to right heart failure and death. Despite several treatments are available, many forms of PH are still incurable. Ginsenoside Rb1, a principle active ingredient of Panax ginseng, exhibits multiple pharmacological effects on cardiovascular system, and suppresses monocrotaline (MCT)-induced right heart hypertrophy. However, its effect on the pulmonary vascular functions related to PH is unknown. METHODS: We examined the vasorelaxing effects of ginsenoside Rb1 on endothelin-1 (ET-1) induced contraction of pulmonary arteries (PAs) and store-operated Ca(2+) entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs) from chronic hypoxia (CH) and MCT-induced PH. RESULTS: Ginsenoside Rb1 elicited concentration-dependent relaxation of ET-1-induced PA contraction. The vasorelaxing effect was unaffected by nifedipine, but abolished by the SOCE blocker Gd(3+). Ginsenoside Rb1 suppressed cyclopiazonic acid (CPA)-induced PA contraction, and CPA-activated cation entry and Ca(2+) transient in PASMCs. ET-1 and CPA-induced contraction, and CPA-activated cation entry and Ca(2+) transients were enhanced in PA and PASMCs of CH and MCT-treated rats; the enhanced responses were abolished by ginsenoside Rb1. CONCLUSION: Ginsenoside Rb1 attenuates ET-1-induced contractile response via inhibition of SOCE, and it can effectively antagonize the enhanced pulmonary vasoreactivity in PH.
Assuntos
Cálcio/metabolismo , Ginsenosídeos/farmacologia , Contração Muscular/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Animais , Canais de Cálcio/metabolismo , Hipóxia Celular , Células Cultivadas , Modelos Animais de Doenças , Endotelina-1/metabolismo , Gadolínio/toxicidade , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Indóis/farmacologia , Masculino , Monocrotalina/toxicidade , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nifedipino/farmacologia , Panax/química , Panax/metabolismo , Artéria Pulmonar/citologia , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/metabolismoRESUMO
BACKGROUND: Daunorubicin (DNR)-induced cardiotoxicity, which is closely associated with cardiomyocyte apoptosis, limits the drug's clinical application. The activation of the extracellular regulated protein kinases (ERKs) pathway is responsible for the pro-apoptosis effect of DNR Sodium ferulate (SF) has recently been found to attenuate both DNR-induced cardiotoxicity and mitochondrial apoptosis in juvenile rats. Nonetheless, the precise mechanism underlying SF-induced cardio-protection remains unclear. METHODS: The DNR-injured H9c2 cell model was prepared by incubating the cells in 1 µM DNR for 24 h. Amounts of 15.6, 31.3 or 62.5 µM SF were simultaneously added to the cells. The effect of SF on the cytotoxic and apoptotic parameters of the cells was studied by monitoring apoptosis regulation via the ERKs pathway. RESULTS: SF attenuated DNR-induced cell death (particularly apoptotic death), cTnI and ß-tubulin degradation, and cellular morphological changes. SF reduced mitochondrial membrane potential depolarization, cytochrome c leakage, and caspase-9 and caspase-3 activation. SF also decreased ERK1/2, phospho-ERK1/2, p53 and Bax expression and increased Bcl-2 expression. These effects were similar to the results observed when using the pharmacological ERKs phosphorylation inhibitor, AZD6244. CONCLUSION: We determined that SF protects H9c2 cells from DNR-induced apoptosis through a mechanism that involves the interruption of the ERKs signaling pathway.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Daunorrubicina/efeitos adversos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Caspase 9/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , Concentração Inibidora 50 , Células K562 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ratos , Troponina I/metabolismo , Tubulina (Proteína)/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Daunorubicin (DNR) is a widely used chemotherapeutic agent; however, its clinical use is limited because of its cardiotoxicity. This study was aimed to investigate the protective effect of sodium ferulate (SF), an effective component from traditional Chinese herbs, against DNR-induced cardiotoxicity in juvenile rats. DNR was administered intraperitoneally to rats at the dosage of 2.5 mg·kg(-1)·wk(-1) for 5 consecutive weeks (cumulative dose of 12.5 mg/kg) or in combination with intraperitoneal injection of SF at 50 mg·kg(-1)·d(-1) over a period of 30 days. The animals were killed 6 days after the last injection of DNR. SF significantly ameliorated the DNR-induced cardiac dysfunction, structural damage of the myocardium, and release of lactate dehydrogenase and creatine kinase. Treatment with SF also reversed DNR-induced oxidative stress as evidenced by a decrease in malondialdehyde levels with a concomitant increase in myocardical superoxide dismutase activities. Furthermore, SF afforded significant cardioprotection against DNR-induced apoptosis in vivo and effectively suppressed the complex mitochondrion-dependent apoptotic signaling triggered by DNR. This study indicates that SF may improve cardiac function by inhibition of oxidative stress and apoptosis, thus providing a beneficial effect on the prevention of DNR-induced cardiotoxicity.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Antibióticos Antineoplásicos/antagonistas & inibidores , Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Ácidos Cumáricos/uso terapêutico , Daunorrubicina/antagonistas & inibidores , Daunorrubicina/toxicidade , Cardiopatias/induzido quimicamente , Cardiopatias/prevenção & controle , Mitocôndrias/efeitos dos fármacos , Animais , Cardiopatias/patologia , Hemodinâmica , Masculino , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The study was designed to explore the alteration of intracellular calcium concentration ([Ca²âº]i), induced by transient receptor potential melastatin 8 (TRPM8) channel-specific agonist menthol, in pulmonary arterial smooth muscle cells (PASMCs) between control and pulmonary hypertensive (PH) rats. PH rat models were established by means of chronic hypoxia (CH) and monocrotaline (MCT) injection, respectively. PASMCs from control and PH rats were cultured. The change of [Ca²âº]i in PASMCs induced by menthol, and the effect of TRPM8 channel-specific antagonist BCTC on the change of [Ca²âº]i, were observed. Cellular localization of TRPM8 was examined by using immunohistochemistry. Results showed that menthol increased [Ca²âº]i in the control PASMCs both in Ca²âº -normal and Ca²âº - free Tyrode's solutions, and at the same time BCTC could inhibit these two kinds of elevations. Compared with the control group, elevations of [Ca²âº]i were decreased notably in CH- and MCT-pretreated PASMCs superfused with 2 mmol/L Ca²âº - or 0 Ca²âº -Tyrode's solutions. Immunohistochemical localization experiments showed that the whole PASMCs were dyed brown except for the nucleus. This study verified that TRPM8 exists both in membrane and sarcoplasmic reticulum of PASMCs. In addition, CH- and MCT-pretreatment could independently down-regulate the Ca²âº influx and Ca²âº release mediated by TRPM8 channel.