Four PKC-related proteases involved in atherosclerosis

Atherosclerosis is a multiple-pathogeny disease that consists of many complicated processes, including oxidative stress, endothelium dysfunction, abnormal cell proliferation, apoptosis and extracellular matrix disorder. The pivotal proteases for these courses are NAD(P)H oxidase, eNOS, MAPKs and MMPs, respectively. As an upstream kinase, protein kinase C (PKC) may involve in the development of atherosclerosis through affecting these enzymes.     

Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.     

Effects of short-chain acyl-CoA dehydrogenase on hypertensive vascular remodeling

AIM: To investigate the changes of short-chain acyl-CoA dehydrogenase (SCAD) in hypertensive vascular remodeling and to explore the relationship between SCAD and vascular remodeling in hypertension.METHODS: The spontaneously hypertensive rats (SHR; 24 weeks old) and Wistar rats (24 weeks old) were used as experimental control groups. The SHR and Wistar rats of 16 weeks old were trained by swimming as experimental groups. The systolic pressure was measured periodically. The thickness of vascular wall and the diameter of the vascular lumen were measured. The contents of ROS and ATP, the enzyme activity of SCAD, and the expression of SCAD at mRNA and protein levels in the aorta were determined. The free fatty acid in the serum and aorta was also measured.RESULTS: Compared with Wistar group, the diameter of vascular lumen decreased in SHR group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR group were increased significantly (P<0.05). Compared with SHR group, the diameter of vascular lumen increased in SHR+swim group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR+swim group were decreased significantly. Compared with control group, the expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, and the content of ATP were decreased in SHR group. However, the free fatty acid in the serum and aorta, and the content of ROS in the aorta were increased in SHR group. The expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, the content of ATP were increased in Wistar+swim group and SHR+swim group. However, the free fatty acid in serum and aorta, and the content of ROS in the aorta were decreased in Wistar+swim group and SHR+swim group.CONCLUSION: Decrease in SCAD expression may be associated with hypertensive vascular remodeling. Swimming training can reverse hypertensive vascular remodeling by increasing the expression of SCAD in the aorta.     

CHIP participates in high glucose-induced vascular endothelial cell injury

AIM To investigate the effects of carboxy terminus of heat shock protein 70-interacting protein (CHIP) on high glucose (HG)-induced vascular endothelial cell injury. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with 5.5 mmol/L glucose (normal glucose， NG) or 25.5 mmol/L glucose (HG) for 24 h. Down-regulation of CHIP expression by RNA interference was conducted. Before the experiment, mannitol was used to eliminate the interference of osmotic pressure. Subsequently, the cells was divided into 4 groups: NG+siRNA NC group, NG+siRNA CHIP group, HG+siRNA NC group, and HG+siRNA CHIP group. Additionally, MTT assay and TUNEL staining were used to detect the viability and apoptosis. The level of endothelin-1 (ET-1) was measured by ELISA, and the level of reactive oxygen species (ROS) was detected by fluorescence probe dihydroethidium. The level of nitric oxide (NO), and the activity of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in the cells were detected by their respective kits. The mRNA expression of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) was detected by RT-qPCR. The protein levels of CHIP, NADPH oxidase (NOX) 2, NOX4, p38, p65, p-p38, p-p65, Bax and Bcl-2 were determined by Western blot. RESULTS Compared with NG+siRNA NC group, the cell viability was decreased, the apoptosis rate, the mRNA expression of IL-8 and MCP-1, and the level of ROS were increased (P<0.05), the activity of SOD was decreased (P<0.05), while the levels of ET-1, NO and iNOS and the protein levels of p-p38, p-p65 and Bax were increased in HG+siRNA NC group (P<0.05). Compared with HG+siRNA NC group, the inflammatory response, the oxidative stress, the apoptosis rate, and the protein levels of p-p38, p-p65 and Bax were significantly increased in HG+siRNA CHIP group (P<0.05). CONCLUSION Down-regulation of CHIP expression aggravates HG-induced vascular endothelial cell injury.     

Effect of hydrogen molecule on apoptosis-related proteins and Nrf2 signaling pathway in glomerular mesangial cells cultured with high glucose

AIM: To investigate the role of hydrogen molecule on apoptosis-related proteins in glomerular mesangial cells cultured with high glucose and to explore its possible mechanism. METHODS: Mouse glomerular mesangial cells cultured in vitro were divided into 4 groups:normal control group (C group, 5.5 mmol/L glucose), mannitol group (G group, 5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (H group, 25 mmol/L glucose), high glucose+hydrogen-rich water group (HH group, 25 mmol/L glucose+hydrogen-rich water), and cultured for 48 h. The protein levels of Bax, Bcl-2, cleaved caspase-3, nuclear factor E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1) were determined by Western blot, and the mRNA expression of HO-1 and NQO-1 was determined by RT-PCR. The level of intracellular reactive oxygen species (ROS) was detected by dihydroethidium method, and the activity of superoxide dismutase (SOD) was measured by WST-8 assay. RESULTS: Compared with C group, the protein levels of Bax and cleaved caspase-3 were up-regulated, and Bcl-2 was down-regulated in H group (P <0.05). No significantly difference of the protein levels mentioned above between C and HH group was observed. Compared with H group, the protein levels of Bax and cleaved caspase-3 were down-regulated, and Bcl-2 was up-regulated in HH group (P <0.05). The level of intracellular ROS was higher and the activity of SOD was lower in H group than those in C group (P<0.05). However, there was no difference of the SOD activity between C group and HH group. The level of intracellular ROS decreased and the activity of SOD increased in HH group as compared with H group (P<0.05). Compared with C group, clearly reduced protein expression of Nrf2, HO-1 and NQO-1, and decreased mRNA expression of HO-1 and NQO-1 in H group were observed (P<0.05). Compared with H group, the protein levels of Nrf2, HO-1 and NQO-1 as well as the mRNA levels of HO-1 and NQO-1 were obviously increased in HH group (P<0.05).CONCLUSION: Hydrogen molecule inhibits the expression of pro-apoptotic proteins and induces the expression of anti-apoptotic proteins in glomerular mesangial cells cultured with high glucose. The mechanism may be related to activation of Nrf2 signaling pathway.     

Effects of sinapic acid on proliferation and apoptosis of rat vascular smooth muscle cells induced by high glucose

AIM: To investigate the effects of sinapic acid(SA) on the proliferation and apoptosis of rat vascular smooth muscle cells(VSMCs) induced by high glucose(HG). METHODS: Cultured A7r5 cells were randomly divided and treated as indicated. The cell viability was determined by MTT assay. DNA synthesis was measured by BrdU assay. Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis. The levels of reactive oxygen species(ROS) were detected by ELISA. The protein levels of cyclin D1, P21, P27, phosphorylated protein kinase C(p-PKC), p-P38 and β-actin were evaluated by Western blot. RESULTS: Compared with control group, the viability of A7r5 cells was significantly enhanced, the DNA synthesis was increased, the cell cycle progression was promoted, the levels of ROS were elevated, the cell apoptotic rate was reduced, the protein expression of P21 and P27 was decreased, and the protein levels of cyclin D1, p-PKC and p-P38 were increased in HG group(all P<0.05). These effects were reversed by SA(0.1, 1 and 10 μmol/L) treatment in a dose-dependent manner(all P<0.05). Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability(P<0.05).CONCLUSION: SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.     

Norepinephrine attenuates injury of vascular endothelial cells induced by LPS

AIM: To investigate the effects of norepinephrine (NE) on vascular endothelial cell damage induced by lipopolysaccharides (LPS). METHODS: Human umbilical vein endothelial cells (HUVEC-12) were cultured with LPS at 100 mg/L to establish the cell damage model. Real-time PCR and Western blot were used to determine the expressions of VE-cadherin at mRNA and protein levels. The levels of TNF-α, IL-1β, IL-2 and IL-10 in culture supernatant were measured by ELISA. The reactive oxygen species (ROS) production in the endothelial cells was detected by ROS assay kit. RESULTS: LPS decreased both mRNA and protein levels of VE-cadherin accompanied by increased levels of TNF-α, IL-1β, IL-2 and intracellular ROS, and decreased level of IL-10 in the endothelial cells. NE reversed the expression of VE-cadherin at mRNA and protein levels under the condition of LPS treatment in a dose-dependent manner, and also alleviated the intracellular oxidative stress. CONCLUSION: NE reverses the endothelial damage induced by LPS, which may be related to the up-regulation of VE-cadherin level and the decreases in oxidative stress and inflamatory mediators.     

核黄素处理促进‘巨峰’葡萄提早成熟的研究

基于核黄素在光照下易分解产生活性氧的特性,设其浓度梯度为0(对照)、0.1、0.5、1.0mmol·L~(-1),分别于‘巨峰’葡萄花后40 d喷施,研究外源活性氧对葡萄果实发育的影响。结果表明:不同浓度的核黄素处理均可促进‘巨峰’葡萄果实提前成熟,以0.5 mmol·L~(-1)效果最好,使果实提前成熟16 d。0.5 mmol·L~(-1)核黄素处理后,显著提升了果实发育前期可溶性糖及转色期花青苷的含量,果实发育前期过氧化氢(H_2O_2)含量,同时也提高了果胶酶(PG)、纤维素酶及脂氧合酶(LOX)的活性,虽然NADPH氧化酶(NOX)活性有所提升,但超氧化物歧化酶(SOD)活性及超氧阴离子产生速率均下降,推测前期升高的H_2O_2可能来源于LOX等其他途径,而H_2O_2含量的上升并没有引起细胞损伤(MDA含量无差异),适宜浓度的外源活性氧能够启动与葡萄果实快速发育有关活性氧产生途径,从而促进果实的发育。     

Production and cytotoxicity of the reactive oxygen species induced by diallyl trisulfide in human myeloid leukemia HL-60 cells

AIM: To explore the production and cytotoxicity of the reactive oxygen species (ROS) induced by diallyl trisulfide (DATS) in HL-60 cells.METHODS: HL-60 cells were either treated with various doses of DATS alone, or DATS combination with apocynin, a specific NADPH oxidase inhibitor, or with antioxidant N-acetyl-L-cysteine (NAC) for 0, 1, 3, 6, 12 and 24 h, respectively. The intracellular ROS level was measured by flow cytometry. The activity of NADPH oxidase was evaluated by NBT reduction experiment. The content of both malondialdehyde (MDA) and the protein carbonyl were analyzed by spectrophotometer. RESULTS: The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL-60 cells (P<0.05), which was dose-and time-dependent. The fluorescence intensities of ROS reached at maximum when HL-60 cells were incubated with 150 μmol/L DATS for 3 h. The NBT reduction experiment showed that DATS activated NADPH oxidase, the highest activity was observed when the cells were exposed to 150 μmol/L DATS for 3 h. DATS induced MDA and protein carbonyl production in HL-60 cells. Furthermore, both MDA and protein carbonyl reached the highest level in the cells exposed to 150 μmol/L DATS for 3 h. Apocynin and NAC attenuated the production of MDA and protein carbonyl, suggesting that ROS induced by DATS was involved in the toxicity to the cells. CONCLUSION: DATS induces ROS production through activating NADPH oxidase in HL-60 cells. ROS increases the oxidation of membrane lipid and protein in HL-60 cells.     

Erianin induces apoptosis of human lung cancer A549 cells via ROS/p38 MAPK pathway

AIM:To investigate the effect of erianin on the viability and apoptosis of human lung cancer A549 cells and its possible mechanism. METHODS:A549 cells and BEAS-2B cells were cultured in vitro and treated with erianin at 10, 20, 40, 80 and 160 nmol/L for 48 h. The cell viability was measured by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. The activity of superoxide dismutase (SOD) was detected by WST-8 method, and the content of malondialdehyde (MDA) was detected by barbituric acid method. The protein levels of nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), p38 MAPK, p-p38 MAPK, caspase-3 and cleaved caspase-3 were determined by Western blot.RESULTS:Erianin remarkably reduced the viability of A549 cells in a dose-dependent manner (P<0.05) with IC₅₀ at 52.64 nmol/L. Erianin also induced apoptosis (P<0.05), increased ROS level and MDA content (P<0.05), diminished SOD activity (P<0.05), and down-regulated the protein levels of Nrf2, NQO1 and HO-1 (P<0.05), in a dose-dependent manner. Meanwhile, erianin up-regulated the levels of p-p38 MAPK and cleaved caspase-3 (P<0.05), and these effects were inhibited by N-acetyl-L-cysteine and SB203580 (P<0.05).CONCLUSION:Erianin may induce apoptosis of human lung cancer A549 cells most likely via inhibiting SOD activity and down-regulating the protein levels of Nrf2, NQO1 and HO-1, thus resulting in an increase in ROS and activation of p38 MAPK.     

Role of nitric oxide in proliferation and secretion effect of vascular endothelial cells induced by vascular endothelial growth factor

AIM: To investigate the role of nitric oxide in proliferation and secretion of vascular endothelial cells induced by vascular endothelial growth factorr (VEGF). METHODS: The in vitro cultured vascular endothelial cells of rabbit aorta were divided into control group, VEGF-treated group and VEGF+L-NAME treated group, the absorbance (A) value of vascular endothelial cells, endothelin-1(ET-1) and von Willebrand factor (vWF) in the supernatant were examined by WST-1 assay, radioimmunoassay and ELISA. RESULTS: The A value in VEGF and VEGF+L-NAME treated group were higher than that in control group (P＜0.01). A value in VEGF group was higher than that in VEGF+L-NAME group the ET-1 and vWF were markedly decreased in VEGF group compared with the control and VEGF+L-NAME treated group (P＜0.05, P＜0.01). These results indicated that VEGF promoted the proliferation and inhibited the secretion of ET-1 and vWF in vascular endothelial cells, and L-NAME inhibited the effect of VEGF. CONCLUSION:Nitric oxide is an important mediator in the process of stimulating proliferation and regulating secretion of vascular endothelial cells by VEGF.     

Cholesterol induces endothelial cells injury by increasing production of reactive oxygen species and activating NF-κB

AIM: To study the increased level of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and their correlation with the injury caused by cholesterol on HUVECs, and to clarify the original source of intracellular ROS. METHODS: The cells of HUVECs-12 were cultured in F12 medium with 10% FBS and divided into normal control group (without any treatment), solvent group (treated with 0.25% dehydrated alcohol), cholesterol group (treated with 50 mg/L cholesterol) and N-acetyl-L-cysteine(NAC) group(pretreated with 10 mmol/L NAC for 1 h and then treated with 50 mg/L cholesterol for 48 h). The intracellular ROS levels were determined by flow cytometry (FCM) with DCFH-DA as fluorescent probe. Nuclear translocation of NF-κB subunit p65 was detected by immunocytochemistry staining. LDH activity and concentration of nitric oxide in the supernatant of the cell culture were also determined. The concentration of MCP-1 protein in cultured supernatant was measured by ELISA. The intracellular levels of ROS and the changes after adding 4 kinds of enzyme inhibitors (NADPH oxidase inhibitor diphenyl iodide, mitochondrial respiratory chain enzyme complex inhibitor rotenone, NOS inhibitor L-NAME and xanthine oxidase inhibitor oxypurinol) were observed. RESULTS: (1)Compared to the normal control cells, 50 mg/L cholesterol increased intracellular ROS (P<0.01) and activated the nuclear translocation of NF-κB p65. A significant increases in LDH activity and the MCP-l protein were also observed. The NO level decreased in the cells. (2)Compared to the cholesterol control cells, diphenyl iodide decreased intracellular ROS significantly (P<0.01).Retenone also inhibited the generation of ROS partially (P<0.05). The other inhibitors almost did not affect the level of ROS caused by cholesterol (P>0.05). CONCLUSION: Free cholesterol increases ROS generation in endothelial cells, activates intracellular NF-κB, thus leading to endothelial cell injury. NADPH oxidase was the main source of ROS generation in HUVECs cultured with free cholesterol.     

Effect of shikonin on high glucose-induced apoptosis and oxidative stress in vascular endothelial cells

AIM:To investigate the effect of shikonin on the apoptosis and oxidative stress induced by high concentration of glucose in vascular endothelial cells. METHODS:Rat thoracic aortic endothelial cells were randomly divided into 5 groups:normal control group (with glucose at concentration of 5.5 mmol/L in cell culture medium), high glucose group (with glucose at concentration of 33 mmol/L in cell culture medium), high glucose+low shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 0.1 μmol/L in cell culture medium), high glucose+medium shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 1 μmol/L in cell culture medium), and high glucose+high shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 10 μmol/L in cell culture medium). After treatments, the cell viability was measured by CCK-8 assay and cell apoptotic rate was analyzed by flow cytometry. In addition, the status of oxidative stress was evaluated by determining the levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of Nrf2/HO-1 signaling pathway was determined by Western blot. RESULTS:Compared with high glucose group, shikonin reversed high glucose-induced decrease in cell viability and increase in apoptosis in a concentration-dependent manner. High concentration of glucose induced high levels of MDA and ROS, while decreased the levels of SOD and GSH-Px. However, after treatment with shikonin, the contents of MDA and ROS were decreased, while the activities of SOD and GSH-Px were increased as compared with high glucose group. Furthermore, the high concentration of glucose up-regulated the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear). Compared with high glucose group, the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear) were partly decreased after treatment with shikonin. CONCLUSION:Shikonin alleviates high glucose-induced vascular endothelial cell apoptosis. Its mechanism may be related to activation of Nrf2/HO-1 signaling pathway and down-regulation of oxidative stress in vascular endothelial cells.     

Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human umbilical vein endothelial cells

AIM:To explore the effect of atorvastatin on high glucose-induced oxidative stress and underlying mechanisms in human endothelial cells. METHODS:Human umbilical vein endothelial cells(HUVECs) were cultured in medium 199 containing normal concentration of glucose(5.5 mmol/L). For high glucose treatment, glucose solution was added to the final concentration of 25 mmol/L. Reactive oxygen species(ROS) were detected by flow cytometry and confocal microscopy. The activity of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase was measured by lucigenin assay. Phosphorylated protein kinase C(PKC) and the expression levels of NADPH oxidase subunits Nox4 and Nox2/gp91^phox were determined by quantitative real-time PCR and immunoblotting. RESULTS:High glucose increased ROS production, NADPH oxidase activity and the expression of Nox4 and Nox2/gp91^phox subunits. Treatment of endothelial cells with atorvastatin resulted in significant inhibition(in a concentration-dependent manner) of high glucose-induced ROS production, NADPH oxidase activation and the expression of Nox4 and Nox2/gp91^phox subunits. PKC inhibitor showed a similar effect to that of atorvastatin on high glucose-induced oxidative stress. Furthermore, atorvastatin rapidly inhibited high glucose-induced activation of protein kinase C, an upstream activator of NADPH oxidase. CONCLUSION:PKC is involved in high glucose-induced oxidative stress in HUVECs. Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human endothelial cells.     

Metallothionein inhibits rat vascular fibroblasts activation induced by homocysteine

AIM:To study the effects of exogenous metallothionein (MT) and ZnCl₂-induced MT production on biological action of homocysteine(HCY)in vascular fibroblasts.METHODS:[³H]-TdR, [³H]-Pro incorporation and LDH leakage were measured, the cellular viabilities were calculated by trypan blue exclusion test and the intracellular contents of MT were assayed by [¹⁰⁹Cd]-hemoglobin saturation method in cultured rat vascular fibroblasts.RESULTS:Proliferation, collagen production of vascular fibroblasts in HCY-treated group were significantly increased compared with control group in a concentration-depedant manner. HCY (500 μmol/L) increased LDH leakage and decreased the cellular viabilities (P＜0.05 or P＜0.01). [³H]-TdR incorporation, [³H]-Pro incorporation, collagen secretion and LDH leakage were all decreased in MT (1×10^-5 mol/L, 1×10^-4mol/L) plus HCY(500 μmol/L) incubated group, compared with HCY alone group, respectively (P＜0.05 or P＜0.01). MT content in ZnCl₂ pretreatment group was increased compared with control group. Proliferation, collagen production and LDH leakage in HCY group pretreated with ZnCl₂ were decreased whereas the cellular viabilities were increased compared with HCY alone group.CONCLUSIONS:The results shows that HCY induces proliferation and collagen production of vascular fibroblasts. Both exogenous MT and endogenous MT induced by ZnCl₂ inhibite the above-mentioned effects of HCY on vascular fibroblasts. MT inhibites vascular fibroblast activation induced by HCY, which may be related to its vascular protection.     

Changes of heme oxygenase-carbon monoxide system-in vascular calcification in rats

AIM:To investigate the change of heme oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in vascular calcification, to clarify the cellular and molecular mechanimsm in vascular calcification.METHODS:Vascular calcification model was established in rats by using vitamin D₃ and nicotine. The relative content of HO-1 mRNA, immunochemistry (IH) for HO-1, HO activity, HbCO formation and content of cGMP in aorta were measured.RESULTS:Compared to those of control rats, the HO-1 mRNA level in vessels of rats in VDN group(vascular calcification group) were decreased by 34.9% (P＜0.05);expression of HO-1 protein were decreased too, there were trace positive staining of HO-1 in the endothelium, and no obvious immunoreactivity in the medial layer;HO-1 activity was decreased by 60.6% (P＜0.01), CO concentration was decreased by 53.9% (P＜0.01) and cGMP content was decreased by 77.1% (P＜0.01) in vessels of rats in VDN group.CONCLUSION:There were obvious down regulation in HO-CO-cGMP pathway in calcified vessels.     

Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting caspase-12

AIM: To investigate the effect of D4F, an apolipoprotein A-I mimetic peptide, on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium (DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respectively. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in the cells and the activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined. The protein level of caspase-12 was examined by Western blot analysis. RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM (an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner. Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced oxidative stress, as expressed by the decreased generation of ROS and MDA (P <0.01), the increased activity of SOD and the decreased activity of NADPH oxidase (P <0.05). Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner (P <0.05). Furthermore, D4F also inhibited the caspase-12 activation induced by TM (P <0.05). CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.