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Effects of panax notoginseng saponins on expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells injury induced by oxidized low density lipoprotein
Abstract
To study the protective effect of panax notoginseng saponins (PNS) on human umbilical vascular endothelial cells (HUVECs) injury induced by oxidized low-density lipoprotein (ox-LDL) for investigating the mechanism of PNS in treating arteriosclerosis obliterans (ASO).
Taking the cultured HUVECs as target cells, ox-LDL was used to establish a model of injured HUVEC and it was then intervened by PNS. The morphologic changes of HUVEC were observed under light microscope; activity of cells was determined by MTT method; the adhesive percentage between ox-LDL treated HUVEC and monocyte detennined by protein quantification and the protein expression of intercellular adhesion molecule-1 (ICAM-1) determined by flow cytometry.
At the time points of HUVEC being treated with ox-LDL (100 mg/L) for 12 h and 24 h, significant injury of HUVEC was shown, its activity reduced, the adhesion rate with monocytes elevated, and the protein expression of ICAM-l in HUVEC increased significantly (P < 0.05, P < 0.01). PNS showed significant effect in reversing all the above changes, as compared with the control group (without PNS treaded), respective significant difference was shown in all the four indexes (P < 0.05, P < 0.01).
PNS has a protective effect on endothelial cells injury induced by ox-LDL,which may be one of its mechanisms in treating ASO. The protective effect of PNS is probably by way of down-regulating the expression of ICAM-1 in endothelial cells and inhibiting the adherence of monocytes to endothelial cells.
... A previous study performed in ApoE-/-mice demonstrated that, PNS decreased the serum ox-LDL level and the ratio of plaque area to vessel area [27]. Another investigation showed that PNS protected HUVECs against ox-LDL-induced injury partially via down-regulation of ICAM-1 [28]. However, to our best of knowledge, only one literature has reported the anti-AS effect of NGR1 on an ApoE-/-mice model [13]. ...
Background/Aims: Radix notoginseng is a well-known traditional Chinese herbal medicine, has extensively pharmacological activities in cardiovascular system. Notoginsenoside R1 (NGR1) is one main active ingredient of Radix notoginseng. The purpose of this study was to evaluate the functional effects of NGR1 on atherosclerosis (AS). Methods: Human umbilical vascular endothelial cells (HUVECs) were subjected to oxidized low density lipoprotein (ox-LDL), before which cells were preconditioned with NGR1. Cell Counting Kit-8 (CCK-8) assay, flow cytometry, Transwell assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were carried out to assess the impacts of ox-LDL and NGR1 on HUVECs. Besides, the expression of microRNA-132 (miR-132), and the regulatory role of miR-132 in Matrix Gla Protein (MGP) expression were measured by qRT-PCR and Western blot. Results: NGR1 pre-conditioning prevented ox-LDL-induced apoptosis, migration and overproduction of Monocyte Chemoattractant Protein 1 (MCP-1) and Intercellular Adhesion Molecule 1 (ICAM-1). miR-132 was up-regulated in response to ox-LDL while was down-regulated by NGR1 pre-conditioning. The protective actions of NGR1 in ox-LDL-treated HUVECs were enhanced by miR-132 inhibitor, while were attenuated by miR-132 mimic. Besides, the up-regulated miR-132 could further decrease the expression of MGP, which acted as an anti-migratory and anti-adhesive factor. Furthermore, ox-LDL-induced the activation of c-Jun N-terminal Kinase (JNK) and Nuclear Factor Kappa B (NF-κB) pathways were partially attenuated by NGR1, and were fully eliminated by NGR1 treatment together with MGP overexpression. Conclusion: NGR1 prevents ox-LDL-induced apoptosis, migration and adhesion-related molecule release in HUVECs possibly via down-regulating miR-132, and subsequent up-regulating MGP.
... It is a well-known traditional Chinese herbal medicine as known to possess anti-inflammatory, anti-oxidative and anti-ischemia-reperfusion injury properties, and facilitates the treatment of cardiovascular disease (8,9). Previous studies have demonstrated that Panax notoginseng saponins improve ischemia/reperfusion-induced hepatic microcirculation disturbance (10), inhibit platelet aggregation (11,12) and adhesion molecule expression (13)(14)(15), and improve vascular endothelium function (16). The expression of lipopolysaccharide-induced vascular endothelial tumor necrosis factor-α is inhibited by NR1 by inhibiting degradation of inhibitor-κB (17). ...
Autologous fat particle transplantation has been widely used by surgeons. The present study evaluated the effect of Notoginsenoside R1 (NR1) treatment on rat autologous fat graft, along with the quality and retention rates. Male Sprague‑Dawley rats (n=60) received fat particle auto‑transplantation from the left abdominal cavity into lateral dorsum. A total of 14 days after surgery, NR1 in different doses (50, 100 and 200 mg/kg/day) was injected into rats, following which blood and fat graft samples were harvested at days 7, 14 and 28. Assessments were carried out by hematoxylin and eosin staining, western blotting, ELISA and immunohistochemistry (IHC). The survival rate of fat grafts was increased in three experimental groups, as detected by weight measurement. Histological scoring demonstrated that there were significant differences in tissue integrity between the 100 mg/kg/day group and the other 3 groups. hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, angiotensin and S100 levels in the 100 mg/kg/day NR1 group was increased compared with the other 2 treatment groups; however, all 3 treatment groups demonstrated increased expression of these proteins compared with the control group. Additionally, cluster of differentiation (CD)68 exhibited negative expression and CD31 showed weakly positive expression in all three experiments, as assessed by IHC. In conclusion, 100 mg/kg/day NR1 may potentially promote the retention rate and enhance the quality of autologous fat grafts via increasing vascularity in the recipient site. These results implicate NR1 as a therapeutic strategy for the improvement of outcome following fat graft surgery.
... To investigate the effects of PNS on antioxidant effect on the formation of atherosclerosis, one research [53] showed that PNS can lower the serum levels of lipid and oxidized low-density lipoprotein (oxLDL), ratio of plaque area to vessel area, and expression of CD40 and MMP-9 in the apolipoprotein Eknockout (apoE-KO) mice. Another in vivo study showed that PNS had an effect in reversing the injuries of human umbilical vascular endothelial cells (HUVEC) induced by oxidized low-density lipoprotein (ox-LDL), improving its activity, elevating the adhesion rate with monocytes, and increasing the protein expression of intercellular adhesion molecule-1 (ICAM-1) in HUVEC [54]. It suggested that the protective effect of PNS for treating arteriosclerosis obliterans (ASO) is probably by way of downregulating the expression of ICAM-1 in endothelial cells and inhibiting the adherence of monocytes to endothelial cells. ...
Panax notoginseng saponins (PNS) are one of the most important compounds derived from roots of the herb Panax notoginseng which are traditionally used as a hemostatic medicine to control internal and external bleeding in China for thousands of years. To date, at least twenty saponins were identified and some of them including notoginsenoside R1, ginsenoside Rb1, and ginsenoside Rg1 were researched frequently in the area of cardiovascular protection. However, the protective effects of PNS on cardiovascular diseases based on experimental studies and its underlying mechanisms have not been reviewed systematically. This paper reviewed the pharmacology of PNS and its monomers Rb1, Rg1, and R1 in the treatment for cardiovascular diseases.
... Although the exact target transcription factor for Rg3-mediated eNOS up-regulation is not clear, the ER mimetic effect of Rg3 or the PI3-kinase/Akt-dependent activation of an unknown transcription factor may be involved in the eNOS gene transactivation by Rg3. Although it has not been shown that Rg3's effect on oxidized LDLmediated endothelium dysfunction, ginsenoside Rb as well as Panax notoginseng saponins has protective effects against oxidized LDLinduced cell death and defect of endothelial functions (e.g., eNOS activity or monocyte adhesion) in endothelial cells (He et al., 2007; Qin et al., 2008). Moreover, exposure of endothelial cells to free fatty acids such as palmitic acid and linoleic acid cause decrease in eNOS phosphorylation and subsequently reduced NO production (Wang et al., 2006a,b). ...
Panax notoginseng saponins (PNS) have shown to be the biologically active constituents responsible for the therapeutic action of panax notoginseng. PNS could help to restrain the oxidative stress, however, whether biomechanical properties of the single cell involve in the protective effect exerted by PNS against oxidative stress injury remains unclear. In this work, we investigated the protective mechanism of PNS against oxidative stress based on the PeakForce Tapping technology firstly, focusing on the biomechanical properties of single human umbilical vascular endothelium cell (HUVEC). PNS display distinct inhibition on the reduction of the young's modulus of cells caused by oxidative stress damage. Combining with immunofluorescence assay, it indicates that improving the stability of cytoskeleton is a significant way for PNS to play a protective role in HUVEC cells during oxidative damage. This work provides a new idea for exploring the functional mechanism of traditional Chinese medicine at the single cell level, and reveals great potential of the atomic force microscope in studying the drug mechanism.
Objective
To investigate the mechanisms of Panax notoginseng saponins (PNS) in treating coronary heart disease (CHD) by integrating gene interaction network and functional enrichment analysis.
Methods
Text mining was used to get CHD and PNS associated genes. Gene–gene interaction networks of CHD and PNS were built by the GeneMANIA Cytoscape plugin. Advanced Network Merge Cytoscape plugin was used to analyze the two networks. Their functions were analyzed by gene functional enrichment analysis via DAVID Bioinformatics. Joint subnetwork of CHD network and PNS network was identified by network analysis.
Results
The 11 genes of the joint subnetwork were the direct targets of PNS in CHD network and enriched in cytokine-cytokine receptor interaction pathway. PNS could affect other 85 genes by the gene–gene interaction of joint subnetwork and these genes were enriched in other 7 pathways. The direct mechanisms of PNS in treating CHD by targeting cytokines to relieve the inflammation and the indirect mechanisms of PNS in treating CHD by affecting other 7 pathways through the interaction of joint subnetwork of PNS and CHD network. The genes in the 7 pathways could be potential targets for the immunologic adjuvant, anticoagulant, hypolipidemic, anti-platelet and anti-hypertrophic activities of PNS.
Conclusion
The key mechanisms of PNS in treating CHD could be anticoagulant and hypolipidemic which are indicated by analyzing biological functions of hubs in the merged network.
BACKGROUND: Modern pharmacological studies have confirmed that Buyang Huanwu Decoction could expand blood vessels, improve microcirculation, decrease oxidative stress, inflammation and platelet activation, so protect and improve the function of endothelial cells. But its certain mechanism is still unclear, especially for its impact on the restenosis of the coronary artery after percutaneous transluminal coronary angioplasty (PTCA). OBJECTIVE: To investigate the protective effects of Jiawei Buyang Huanwu Decoction on vascular stenosis and oxidative stress after balloon injury of rabbit iliac artery. METHODS: New Zealand rabbits, were randomized and divided into three groups, control group, model group and drug group respectively. Rabbits of control group were fed with common forage, but model group and drug group fed with high fat diet. Two weeks later, the iliac arteries were injured by balloon for model group and drug group. Meanwhile drug group were fed with Jiawei Buyang Huanwu Decoction after the operation, 2 mL/ kg per day. Control group underwent sham operation control. At the end of 4 weeks, serum samples were stored to assay the levels of cholesterol, activity of superoxide dismutase (SOD) and levels of malondialdehyde (MDA). Injured iliac artery was fixed by neutral formalin to observe the endothelial hyperplasia by light microscope, and the results were analyzed by picture analysis system. Differences of measurement were compared with one-way analysis of variance as well as repetitive measurements analysis of variance. RESULTS AND CONCLUSION: Iliac artery intima was thin, with intact structure, without arteriosclerosis in the control group. The vascular lumina were narrower, intima was thicker and there were more enormous arteriosclerosis plaques in model group rabbits. The plaque thickness was reduced and stenosis was mild in drug group. Total cholesterol, triglyceridemia, low - density lipoprotein of cholesterol (LDL-C) and MDA levels were significantly lower in the drug group compared with the model group, whereas high-density lipoprotein of cholesterol (HDL-C) and serum SOD levels were significantly greater than the model group (P < 0.05). The results showed that Jiawei Buyang Huanwu Decoction has significant preventive effect on intimal hyperplasia and the development of artherosclerosis in rabbits with iliac artery injury, and the mechanism of which may be related to modifying lipid metabolism and cleaning oxygen free radical anti-oxidative stress.
We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.
Endothelial dysfunction is a feature of atherosclerosis and is associated with CHD (coronary heart disease) risk factors. This study aimed to determine the relationship between the degree of endothelial dysfunction and calculated cardiovascular risk. Endothelial function, as determined by the ACh/NP (acetycholine/sodium nitroprusside response) ratio on brachial plethysmography, was compared with cardiovascular risk as calculated from the Framingham, PROCAM (Prospective Cardiovascular Munster) and MRFIT (Multiple Risk Factor Intervention Trial) algorithms in 246 (187 male) patients, including 44 (22%) with established CHD. Endothelial dysfunction correlated with the total number of risk factors (r2=0.22; P=0.002) and was related to LDL (low-density lipoprotein)-cholesterol in men and triacylglycerols (triglycerides) in women. The ACh/NP ratio correlated with the occurrence of diabetes, CHD and the LDL-cholesterol concentration (r2=0.58; P<0.001). Endothelial dysfunction was associated with presence of CHD on receiver-operating characteristic plot analysis (area=0.706+/-0.04; P=0.001). There was no correlation between ACh/NP ratio and CHD risk calculated with the Framingham algorithm in men, although both ACh and NP response correlated separately with risk in women. The endothelial ACh/NP ratio correlated with absolute risk in the PROCAM algorithm (r2=0.41; P<0.005). Intermediate results were obtained with MRFIT. Individual risk factors make different contributions to endothelial dysfunction compared with their role in risk calculators. The stronger relationship of endothelial dysfunction with PROCAM risk reflects the contribution of male sex, LDL-cholesterol and triacylglycerols to risk calculated by this algorithm.
Background: In traditional Chinese medicine, both deficiency as primary and excess as secondary and deficiency of qi and blood stasis are common symptoms in dialectical logy of atherosclerosis (AS). Therefore, qi-benefiting drugs are the main component of qi-benefiting and blood-activating intervention. However, the best dose relationship between qi-benefiting and blood-activating drugs needs to be further studied. Objective: To observe the effect of qi-benefiting and blood-activating intervention on the expression of aortic vascular cell adhesion molecule-1mRNA (VCAM-1mRNA) in AS models and analyze dose-effect relationship between astragalus and sanchi. Design: Randomized control animal study. Setting: Shanxi Medical University. Materials: The experiment was carried out in the Shanxi Medical University in April 2005. A total of 60 healthy male Wistar rats were selected in this study. The main reagents were quercetin (Shaanxi Huike Plant Co., Ltd., batch number: 20041112), saponins of panax notoginseng (PNS, Kunming Yagechen Pharmaceutical Co., Ltd., batch number: 20050118) and ligustrazine (Yuxin Guoji Longyuan Pharmaceutical Co., Ltd., batch number: 20041204). Methods: Model establishment: Wistar rats were administrated AS feeds (including 10% yolk powder, 5% lard, 0.5% bile salt and 85% basic feed) for 3 months. Grouping and administration: At three days after suitability feeding, 8 rats were randomly selected, regarded as the normal control group and given general feeds, and other 52 rats were fed with AS feeds. Three months later, 4 rats were randomly selected for the measurements of lipid and aortic tissue. And then, the models were established successfully. In addition, 48 rats were randomly divided into 6 groups. 1 Astragalus treatment group: Rats were perfused with 0.1 g/(kg · d) quercetin. 2 Sanchi treatment group: Rats were perfused with 0.1 g/(kg · d) PNS. 3 2:1 of astragalus/sanchi treatment group: Rats were perfused with 0.1 g/(kg · d) quercetin and 0.05 g/(kg · d) PNS. 4 3:1 of astragalus/sanchi treatment group: Rats were perfused with 0.15 g/(kg · d) quercetin and 0.05 g/(kg · d) PNS. 5 Ligustrazine treatment group: Rats were perfused with 0.2 g/(kg · d) ligustrazine (Yuxin Guoji Longyuan Pharmaceutical Co., Ltd.). 6 Normal control group: Rats were fed with general feeds. 7 Model group: Rats were fed with general feeds after successful model estalishement. Thirty days after administration, relative expression of VCAM-1mRNA in aorta was measured with reverse transcription polymerase chain reaction (RT-PCR) technique; moreover, 2 mL venous blood was collected from tail to measure the levels of serum total cholesterol (TC), triacylglycerol (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). Main outcome measures: 1 Relative expression of VCAM-1mRNA in aorta; 2 level of lipid. Results: Among 60 male Wistar rats, three rats in the treatment group died because of perfusion and two rats in the model group died due to accident; therefore, a total of 51 rats were involved in the final analysis. 1 High-lipid diet could promote the formation of AS models. Level of lipid was higher in the model group than that in the normal control group (P < 0.05), and leves of serum TC, TG and LDL-C were lower in the interventions group than those in the model group (P < 0.05). 2 Expression of VCAM-1mRNA was not found in the normal control group. Expression of VCAM-1mRNA was lower in the intervention groups than that in the model group. Expression of VCAM-1mRNA (0.42± 0.02) was the lowest in 3:1 of astragalus/sanchi treatment group, and there were significant differences as compared with other intervention groups (P< 0.05). Conclusions: Astragalus and sanchi, a main component of qi-benefiting and blood-activating herbs, can down-regulate the level of lipid and resist AS; meanwhile, the combination of them is superior to the single application; in addition, with the increasing deal of qi-benefiting drugs, the function against AS is strengthened.
Aim: To observe the effects of astragalus polysaccharides (APS) on the proliferation of human cardiac microvascular endothelial cells (HCMEC) and adhesion between HCMEC injured by reperfusion and polymorphonuclear neutrophils (PMN). Methods: The experiment was performed at the Key Laboratory of Chinese Internal Medicine of Ministry of Education, Beijing University of Chinese Medicine from July to December 2006. The fourth and fifth generation HCMECs cultured in vitro were selected, and their morphous and growth was observed under inverted phase contrast microscope; the proliferation of HCMECs was detected by means of MTT after HCMEC was treated with APS of different concentrations for 24 hours and separately treated with 100, 50, and 25 μg/mL APS for 4, 6, and 8 hours. Ischemia/reperfusion (I/R) injury model was established by oxygen-glucose deprivation for 2 hours and recovery 6 hours method. The effect of APS on PMN adhesion to HCMEC after reperfusion injury was measured by rose Bengal staining. Expression of intracellular adhesion molecule-1 (ICAM-1) was determined by immunocytochemistry and image quantitative analysis system. Results: 1 The HCMEC showed a typical cobblestone appearance after recovered for 48 to 72 hours and its morphological, biological and physiological characters were stable during the 15 population doubling periods. 2 The proliferation of HCMEC was lower (P < 0.05 or 0.01) when APS was more than 5 g/L, and higher (P < 0.05 or 0.01) when APS was between 1.25 g/L-18.78 mg/L. There was no obvious change in proliferation when treated with 2.5 g/L APS. 3 Compared with control group, there was no evident change in the proliferation of HCMEC when APS was treated separately with 100, 50, and 25 mg/L for 4, 6, and 8 hours. 4 50 mg/L APS could decrease the adhesion of PMN to HCMEC apparently (P < 0.01) and 100 mg/L of APS could reduce the expression of ICAM-1 in HCMEC significantly (P < 0.05). Conclusion: 1 APS can promote the proliferation of HCMEC during a certain concentration. However, this effect requires a long period of time. 2 APS displays a protective effect on HCMEC injured by I/R. Its mechanism might be related with promoting the proliferation of HCMEC, inhibiting the expression of ICAM-1 in HCMEC and declining the adhesion of PMN to HCMEC.
Aim: To observe effect of Huoxue Injection on the expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion between monocyte and endothelial cells in cultured human umbilical vein endothelial cells (HUVEC) injury induced by the oxidized low-density lipoprotein (ox-LDL), and explore the mechanism of Chinese medicine compound preparation on preventing and treating atherosclerosis. Methods: The experiment was performed in Key Laboratory of Chinese Internal Medicine of Ministry of Education, Dongzhimen Hospital, Beijing University of Chinese Medicine from March to December 2004. 1 Huoxue Injection with 2 000 g/L was made up of Salvia miltiorrhiza Bge, Ligusticurn chuanxiong Hort, Carthamus tinctorius L and Paeonia lactiflora Pal at the proportion of 2:11:1. Infant umbilical cords of healthy parturients were supplied by Department of Gynaecology and Obstetrics, China-Japan Friendship Hospital, with consent of puerperant and their families. The experiment met ethical standard of Helsinki declaration. 2 Using the cultured HUVEC as target cells, the ox-LDL was added to the endothelial cell culture to prepare the injury model of human endothelial cell. 3 The adhesive percentage between HUVEC treated with ox-LDL and monocytes was determined by protein quantification. HUVEC were incubated in M199 culture fluid (control group) and culture fluid containing different concentrations (10,20,40 ml/L) of Huoxue Injection and ox-LDL (experimental group) for 12 and 24 hours and in M199 culture fluid containing monocytes. Expression of mRNA and protein of VCAM-1 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry respectively. HUVEC were divided into control group, ox-LDL group and ox-LDL and Huoxue Injection group (end concentrations were 10,20,40 ml/L respectively, for 12 and 24 hours respectively). Results: 1 The adhesion rate of human monocyte to HUVEC with ox-LDL for 12 and 24 hours significantly increased, which was obviously higher than that in the control group (P < 0.01). Different dosages of Huoxue Injection could significantly inhibit the adhesion between monocyte and endothelial cells. Significant difference was found compared with the ox-LDL group (P < 0.01). The effects were enhanced with increasing of the Huoxue Injection dosage. 2 The ox-LDL could significantly enhance the expressions of mRNA and protein of VCAM-1. Difference dosages of Huoxue Injection could significantly inhibit the expression of mRNA and protein of VCAM-1 in HUVEC (P < 0.05, P < 0.01), and significant difference was found compared with the ox-LDL group (P < 0.05, P < 0.01). The effects were strengthened with increasing the dosage of Huoxue Injection. Conclusion: Ox-LDL could stimulate the expression of VCAM-1. Huoxue Injection has an inhibitory effect on the adhesion between monocyte and endothelial cells, probably by way of down-regulating the expression of mRNA and protein of VCAM-1 in endothelial cells, which can bring about the protective effect on endothelial cell injury induced by ox-LDL and decrease the progression of atherosclerosis.
Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.
Probucol is a potent inhibitor of atherosclerosis in animal models. However, the mechanism of its antiatherogenic effect is not known. To investigate the effects of probucol on gene expression of VCAM-1, MCP-1, and M-CSF in vivo during the early stages of atherogenesis, we determined gene expression in 12 control WHHL rabbits and 12 WHHL rabbits fed 1% probucol from age 3 weeks. Three animals from each group were killed at 6, 9, 12, and 18 weeks of age. Two intimal/medial segments of the thoracic aorta, each comprising the orifices of a pair of intercostal arteries, were analyzed by semiquantitative RT-PCR using GAPDH as an internal standard. A third segment located between these two segments was studied by immunocytochemistry. A basal level of VCAM-1 gene expression was observed in lesion-free aortas of both treated and untreated WHHL rabbits (and in normal NZW aortas). Immunocytochemistry showed some VCAM-1 protein in normal arteries and confirmed that VCAM-1 protein expression generally correlated with gene expression. In the untreated WHHL rabbits, a marked upregulation of VCAM-1 expression was observed at 18 weeks. To correlate gene expression with intimal monocyte/macrophages in each animal, the macrophage area was determined by morphometry of immunostained sections. In addition, a scoring system of lesions was used. VCAM-1 expression showed a highly significant correlation with the extent of intimal macrophage presence (P < .001). A lesser degree of correlation between gene expression and macrophage accumulation was also seen for MCP-1. In contrast, M-CSF expression remained constant over the entire study period and showed no correlation with the intimal macrophage accumulation. Probucol treatment completely prevented lesion formation in all animals up to 18 weeks of age. Probucol reduced the level of basal VCAM-1 expression and prevented its upregulation. MCP-1 expression was not affected by probucol treatment, whereas M-CSF expression was significantly lowered by probucol. Our results support the idea that VCAM-1 plays an important role in early atherogenesis and suggest that the antiatherogenic effect of probucol may in part be due to a downregulation of VCAM-1. Reduction of the basal level of M-CSF gene expression by probucol treatment may also contribute to its ability to inhibit atherogenesis.
Oxidatively modified low density lipoprotein (OxLDL) is thought to be involved in the early development of atherosclerotic lesions. The appearance of lipid-laden foam cells is known to be one of the typical features of atherosclerotic lesions, and accumulating evidence has demonstrated that foam cells are formed after taking up OxLDL by macrophages in vitro. However, the modified structures, distribution, and metabolism of OxLDL present in vivo are poorly understood. Recently, our studies, together with others, have demonstrated that OxLDL is actually present in circulating human plasma. Furthermore, we have provided evidence that foam cells accumulate modified apoB fragments derived from OxLDL in the cells. This article reviews recent progress in this field, including the intracellular metabolism of OxLDL in foam cells and the relevance of OxLDL as an in vivo ligand for macrophages.
Atherosclerosis is a chronic inflammatory process of the arterial wall associated with systemic and local immune responses to various antigens, oxidized low-density lipoprotein (oxLDL) being the most significant. Both IgM and IgG antibodies to oxLDL are produced during atherosclerosis. Some studies have shown that elevated levels of antibody to oxLDL correlate with the degree of atherosclerosis. Other studies reported that immunization of experimental animals with oxLDL induces high levels of antibodies to oxLDL, with decreased atherosclerosis, suggesting that the immune response to oxLDL may be antiatherogenic. The accelerated development of atherosclerosis has been observed in patients with systemic autoimmune diseases. In patients with antiphospholipid syndrome (APS), beta2-glycoprotein I (beta2GPI) is a major antigenic target for anticardiolipin antibodies (aCLs). We recently reported that oxLDL interacts with beta2GPI via oxLDL-derived specific ligands, such as 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) to form complexes. In vitro, anti-beta2GPI autoantibodies bind to oxLDL/beta2GPI complexes that are actively taken up by macrophages via Fcgamma receptors. Circulating oxLDL/beta2GPI complexes were detected in patients with systemic lupus erythematosus (SLE) and APS, at higher levels than in healthy individuals. Autoantibodies against these complexes were also present; however, IgG anti-oxLig-1/beta2GPI antibody levels in SLE patients with APS were significantly higher than those in SLE patients without APS and those in healthy individuals.
The oxidative modifications of low-density lipoprotein (LDL) are crucial for the atherosclerosis process. The aim of this study was to determine if the minimally modified LDL, obtained after the ingestion of three different diets, produce differential effects on the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression in human umbilical endothelial cells (HUVECs).
Twenty healthy young males were exposed to three dietary periods. Each period lasted four weeks. During the first period, all subjects consumed a saturated fat (SFA) enriched diet (38% fat, 20% SFA). The second and third dietary periods were administered following a randomized crossover design: a low fat high carbohydrates diet (CHO diet) and a Mediterranean diet. LDL particles, isolated during each dietary period, were oxidized by exposure to UV light and incubated for 48 h with HUVEC. Thereafter, 100 U/mL of TNF-alpha was added and incubation continued for 6 h. Cellular ELISA determined adhesion molecules expression. Lag time, propagation rate and total amounts of formed conjugated dienes were calculated in LDL incubated with 10mumol/L Cu(2+). When compared to the SFA diet, LDL isolated from the Mediterranean and CHO diets induced a lower expression of VCAM-1 and E-selectin in HUVECS (P<0.007). There were no differences between both lipid lowering diets. However, lag time of LDL from the Mediterranean diet was higher than with the CHO diet (P<0.042). This parameter was inversely correlated with E-selectin expression (r=-0.497; P<0.04).
Our results suggest that both the Mediterranean and CHO diets may decrease the pro-inflammatory environment induced by modified LDL in endothelial cells.
To study the effect of Wenmaitong (WMT) and its disassembled formulas on the adhesion of monocytes to endothelial cells induced by hyperlipidemic serum to explore the mechanism of WMT on early arteriosclerosis obliterans (ASO).
Serums containing whole WMT and its disassembled formulas, including the formula consisted of warming Jing and boosting qi part (Wenjin Yiqi, WY) and that of promoting blood circulation part (Huoxue Tongmai, HT), as well as the serum contained high concentration of lipids were prepared conventionally, respectively. The adhesion of monocytes cell strain THP-1 to human umbilical vascular endothelial cells (HUVEC) was determined by rose bengal stain method, and ELISA was used to detect expressions of intercellular adhesion molecule (ICAM-1), vascular cellular adhesion molecule (VCAM-1) and P-selectin on HUVEC surface.
WMT could inhibit THP-1 to HUVEC adhesion induced by hyperlipidemic serum, and down-regulate the expression of ICAM-1, VCAM-1, P-selectin on HUVEC surface, the two disassembled formulas could down-regulate different adhesion molecules.
One mechanism of WMT on ASO may be its inhibition on arteriosclerosis by way of down-regulating the expression of vascular endothelial cells adhesion molecules to decrease the adhesion of monocyte to VEC, therefore to inhibit the monocytes migrating into vascular intima to develop foam cells.
Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis, most likely mediating an anti-inflammatory action. In order to understand the mechanisms involved in this protection, we evaluated the effects of DHEA on several molecules involved in the inflammatory response. Reactive oxygen species (ROS), expression of adhesion molecules, activation of the NF-kappaB/IkappaB-alpha pathway and of the AP-1 transcription factor were evaluated in human umbilical vein endothelial cells (HUVECs) treated with oxidized low density lipoproteins (oxLDL) and DHEA. We also determined if DHEA affected LDL oxidation in vitro. 100 microM DHEA-treatment inhibited the oxLDL-induced expression of ICAM-1, VCAM-1, PECAM-1, ROS production, and U937 cells adhesion to HUVECs. DHEA also delayed the kinetics of LDL oxidation in vitro. While DHEA did not affect the translocation of NF-kappaB neither the degradation IkappaB-alpha, it led to an increased translocation of AP-1. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process in endothelial cells activated with oxLDL, therefore its potential anti-inflammatory properties should be evaluated for the treatment of chronic inflammatory diseases such as atherosclerosis.
Endothelial dysfunction, common to diabetes and cardiovascular diseases, is an early step in the development of atherosclerosis and diabetic angiopathies. Deficiencies of taurine have been related to diabetes and cardiovascular diseases.
We investigated whether taurine provides protective action against endothelial dysfunction induced by hyperglycemia and/or oxidized low density lipoproteins (oxLDL).
Quiescent human umbilical cord venous endothelial cells were exposed for 20 h to high glucose (35 mM) and/or oxLDL (60 microg/ml) alone and in presence of taurine (0.5-2.5 mg/ml). Apoptosis, caspase-3 activity, soluble(s) and cell surface expressions of vascular cellular (VCAM-1) and intercellular (ICAM-1) adhesion molecules were determined. Results are given as a percentage of the low glucose medium control. Apoptosis, VCAM-1 and ICAM-1 expressions were related to cell number.
Hyperglycemia increased apoptosis to 162.5 +/- 19.2%, caspase-3 activity to 153.2 +/- 10.3%, cell-surface expression of VCAM-1 to 125.1 +/- 5.8%, the expression of ICAM-1 to 123.7 +/- 2.8% and sICAM-1 to 146.5 +/- 7.9%. Taurine (0.5-2.5 mg/ml) restored apoptosis, caspase-3 activity and expressions of VCAM-1 and ICAM-1. OxLDL (60 microg/ml) increased apoptosis to 114.8 +/- 3.1%; taurine (2.5 mg/ml) reduced this apoptosis to 40.5 +/- 4.1%. The combination of hyperglycemia and oxLDL increased apoptosis to 211.7 +/- 11.6%. This increase was normalized by taurine (2.5 mg/ml) to 97.9 +/- 12.8%.
Taurine protects HUVECs from endothelial dysfunction induced by hyperglycemia through down-regulation of apoptosis and adhesion molecules. Counteracting the combination of oxLDL and hyperglycemia requires pharmacological concentrations of taurine.