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Objective:To investigate the effect of interleukin-33 (IL-33) on lipopolysaccharide (LPS)-induced permeability of rat cardiac microvascular endothelial cells (RCMECs).Methods:RCMECs were cultured in vitro to be divided into control group, LPS group, IL-33 group and LPS+IL-33 group. The effect of IL-33 on the proliferation of RCMECs was detected by cell counting reagent (CCK8). Fluorescein isothiocyanate (FITC)-dextran assay was used to evaluate the permeability of RCMECs. The expression of vascular endothelial calmodulin, ras homologous gene family (Rho) member A (RhoA) and phosphorylated Rho-associated coiled-coil-containing protein kinase (p-ROCK2) proteins were tested by western blot. High-throughput sequencing and gene ontology (GO) were performed for gene expression in LPS and LPS+IL-33 groups.Results:No significant effect of IL-33 at 10-50 ng/ml on the proliferation of RCMECs was observed ( P>0.05). Compared with the control group, the permeability of RCMECs (permeability coefficient ratio 1.404±0.029 vs. 1.000±0.200, P<0.05) was significantly increased in LPS group and the expression of vascular endothelial calmodulin (relative gray value 0.429 5±0.012 9 vs. 0.594 9±0.014 2, P<0.05) was down-regulated, while the permeability of monolayers (permeability coefficient ratio, 0.948±0.013, P<0.01) was decreased in LPS+IL-33 group and the expression of vascular endothelial calmodulin (relative grayscale value 0.549 1±0.012 0, P<0.005) was up-regulated compared with the LPS group. High-throughput sequencing data revealed that the differential genes downregulated in the LPS and LPS+IL-33 groups were associated with cytoskeleton and Rho signaling pathway. Compared with the control group, RhoA (relative gray value 0.211 4±0.009 9 vs. 0.135 0±0.007 6, P<0.000 1) and p-ROCK (relative gray value 0.656 3±0.013 2 vs. 0.503 6±0.036 2, P<0.000 1) protein expression was upregulated in the LPS group. When compared with LPS group, RhoA (relative gray value 0.157 7±0.010 7, P=0.000 2), p-ROCK (relative gray value 0.427 7±0.003 8, P<0.000 1) protein expression was decreased in LPS+IL-33 group. Conclusion:IL-33 may improve LPS-induced hyperpermeability of RCMECs by inhibiting RhoA and p-ROCK protein expression in Rho/Rho-associated coiled-coil-containing protein kinase signaling pathway.
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Objective To observe the protective effect of bisoprolol against hypoxia/reoxygenation injury of cardiac microvascular endothelial cells and explore the mechanism. Methods Left ventricular of cardiac microvascular endothelial cells (CMECs) were isolated from 8-week-old male C57BL/6N mice. CMECs were randomized into four groups: control group, vehicle group, hypoxia/reoxygenation group (H/R group), hypoxia/reoxygenation + bisoprolol group. The level of cell proliferation, apoptosis, superoxide anion, Cleaved caspase-3 and Nox2 expression were measured in each group. Results Compared with control group, H/R group had lower cell proliferation, higher apoptotic level, more superoxide anion level and the expression of Cleaved caspase-3 and Nox2 (P < 0.05). Furthermore, bisoprolol reversed hypoxia/reoxygenation-induced the decreased cell proliferation, the increased apoptosis, superoxide anion level, Cleaved caspase-3 and Nox2 expression (P < 0.05). Conclusion Bisoprolol can protect CMECs against hypoxia/reoxygenation injury by reducing the expression of Nox 2 that decreases oxidative stress.
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Objective To investigate the effect of high glycolipid on mouse cardiac microvascular endothelial cells (MCMECs),clarify the role of Tom70 (translocase of the outer mitochondrial membrane 70,Tom70) in it,and explore the related mechanism.Methods MCMECs cultured with normal glucose medium were divided into normal glucose group (NG,5.5mmol/L),high glucose group (HG,25mmol/L) and HG combined with high fat group (HG+HF,glucose concentration 25mmol/L,500μmol/L,16h).Then,the expression of Tom70 in MCMECs was knocked down by siRNA,and the HG+HF group was further divided into vehicle group,Scramble siRNA group and Tom70-siRNA group.To further confirm the specific mechanism of Tom70 in MCMEC injury induced by high glycolipid,Tom70-siRNA group was subgrouped into N-acetylcysteine (NAC)-free group and NAG-containing group.Accordingly,the apoptosis levels were measured by flow cytometer,the generation of nitric oxide (NO) was detected by ELISA kit,the expressions of Tom70 were determined by immunofluorescence and qRT-PCR,and the levels of reactive oxygen species (ROS) by DHE staining and ELISA kit.Results The apoptosis level increased after exposure to HG,and the generation of NO decreased (P＜0.05),while merging HF could aggravate these injuries (P＜0.05).Moreover,HG inhibited the expressions of Tom70 and promoted the production of ROS in MCMECs (P＜0.05).Compared with HG alone,and combination with HF significantly inhibited the expression of Tom70,and significantly increased the production of ROS (P＜0.05).Most importantly,compared with the vehicle group and Scramble siRNA group,the intracellular ROS content and apoptosis rate increased in the Tom70-siRNA group,while generation of NO was significantly decreased (P＜0.01).In contrast,these damage effects mentioned above were partially reversed by the application of antioxidants NAC (P＜0.05).Conclusions High fat can further aggravate the damage on diabetic MCMECs and Tom70 could exert its effect against cardiac microvascular endothelial injury induced by diabetes via inhibiting oxidative stress.
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Aim To investigate the effects of classic calcium antagonists verapamil(Ver),nifedipine(Nif),diltiazem(Dil)and the novel calcium antagonist N-n-butyl haloperidol iodide(F2)which was synthesized by our lab by regulating Ca2+-independent phospholipase A2(iPLA2)on hypoxia/reoxygenation(H/R)injury of cardiac microvascular endothelial cells(CMECs)and the mechanisms.Methods The CMECs were isolated from SD neonatal rats.The H/R model was established,then cells were treated with different concentrations of calcium antagonists and F2.The content of LDH in the cell supernatant was measured by colorimetric method.The levels of IL-6 and AA in cell supernatant were measured by ELISA;and late-stage apoptosis was measured by TUNEL.The mRNA and protein expression levels of iPLA2 in CMECs were examined by real time-PCR and Western blot analysis.Results Calcium antagonists except Dil decreased the generation of LDH,IL-6 and AA in a dose-dependent manner(P<0.05),and reduced the apoptosis(P<0.05).F2 and Ver decreased the mRNA and protein expression of iPLA2 in a dose-dependent manner,while there were no such effects for Nif and Dil.Conclusions Calcium antagonists except Dil have protective effects against H/R injury.F2 and Ver protect CMECs against H/R injury partly through iPLA2.
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[Abstract ] Objective Cardiac shock wave therapy (CSWT) can promote arteriogenesis in ischemic myocardia , but the mo-lecular mechanism remains unclear .The study aimed to explore the effect of CSWT on arteriogenesis in human cardiac microvascular endothelial cells ( HCMEC ) and the role of focal adhesion kinase (FAK) and Calcium-activated potassium channels (KCa) in the sig-nal conduction pathway of CSWT arteriogenesis . Methods HC-MEC cells cultured in vitro were randomly divided into control group , CSWT group , CSWT +T ( FAK inhibitor PF-573228 ) group and CSWT+F( SCa inhibitor iberiotoxin ) group.Each group received one CSWT(0.09 mJ/mm2, 200Times) 48 h after added stimulant.24 hours'conventional culture later , tests were made on the levels of endothelial nitric oxide synthase ( eNOS ) and vascular endothelial growth factor ( VEGF) mRNA as well as the changes of related protein expression . Results ①QPCR test showed that eNOS , VEGF mRNA expressions increased in CSWT group compared with control group (4.61 ±0.19 vs 3.99 ±0.17, P<0.05), while compared with CSWT group, eNOS, VEGF mRNA expressions in CSWT +T group were decreased (0.62 ±0.10 vs 0.40 ±0.02, P<0.05), eNOS, VEGF mRNA expressions in CSWT +F group were also decreased (0.53 ±0.02 vs 0.64 ±0.02, P<0.05), all the differ-ences were of statistical significance .②Western blot showed that eNOS , VEGF protein expressions increased in CSWT group compared with control group(0.63 ±0.02 vs 0.43 ±0.02, P<0.05), while compared with CSWT group , eNOS, VEGF protein expressions in CSWT+T group were decreased (0.36 ±0.01 vs 0.29 ±0.02, P<0.05), eNOS, VEGF protein expressions in CSWT +F group were also decreased (0.37 ±0.02 vs 0.30 ±0.02, P<0.05), all the differences were of statistical significance . Conclusion CSWT can improve eNOS , VEGF mRNA and protein expressions in HCMEC cells and FAK and KCa may participate in the signal conduction pathway of CSWT arteriogenesis .
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Objective To observe the effect of the advanced glycation end products(AGEs)on the iNOS expression in cultured cardiac microvascular endothelial cells.Methods Cultured the cardiac microvascular endothelial cells in vitro.After AGEs of the different dose(50～200 mg/L)and different times(0～24 h)played the role on the cells,we determined the NO generation and iNOS protein expression of all groups.Results NO generation and iNOS protein expression increased with the AGEs-dose increasing and treatment time.The results were significantly different.Conclusion These results demonstrate that AGEs may induce iNOS to produce toxic NO in cardiac microvascular endothelial cells,then the diabetic cardiomyopathy occures.
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Objective To investigate the effects of low frequency pulsed magnetic fields (LF-PMFs) on proliferation, the cell cycle, apoptosis, migration, cytoskeleton formation and nitric oxide (NO) secretion in cardiac microvascular endothelial cells (CMECs). Methods LF-PMFs with rectangular and triangular waveforms at 15 Hz were tested. The CMECs were randomly divided into a control group, 0.6 mT group, 1.2 mT group, 1.8 mT group and 2.4 mT group. Except for the control group, the CMECs were exposed to LF-PMFs 4 h/day for 5 days. Results After 5 days of intervention with rectangular wave LF-PMFs the proliferation of CMECs accelerated and NO secretion was enhanced significantly. The percentages of cells at ( S+G2 ) phase increased significantly, whereas apoptosis rates were significantly lower than in the control group. The migration of CMECs was facilitated, stress fibers increased and cytoskeleton components were reorganized. After 5 days of intervention with triangular wave LF-PMFs proliferation of CMECs accelerated and NO secretion was significantly enhanced. The percentages of cells at(S+G2)phase and migration increased, while apoptosis was inhibited. Cytoskeleton components were reorganized after exposure to 0.6 mT and 2.4 mT triangular waveform LF-PMFs. No significant change was detected with 1.2 mT and 1.8 mT triangular waveform LF-PMFs with regard to these variables. Conclusions The effects of LF-PMFs on proliferation, the cell cycle, apoptosis, migration, cytoskeleton formation and NO secretion function of CMECs were infiuenced by the waveform in addition to it's intensity and frequency.