| The etiology and mechanism of atherosclerosis (AS) has not been illustrated completely. Previous studies found that various risk factors contributed to the occurrence and development of AS. According to the response-to-injury hypothesis, AS is an chronic inflammatory disease in which inflammatory response to the injury of arterial intima induced by risk factors results in arterial atherosclerosis. Endothelial injury or dysfunction caused by various risk factors has been considered as the initial trigger of the occurrence of atherosclerosis. Therefore, clearing the mechanism of endothelial injury is vital to the prevention and the therapy of AS.Subendothelial deposition and accumulation of lipoprotein is an important pathological characteristics of AS. Epidemiological studies confirmed that low density lipoprotein cholesterol(LDL-C) was an important risk factors of AS, while oxidized low density lipoprotein (ox-LDL) was an product of LDL after oxidative modification. Ox-LDL is critical in the development of atherosclerosis. Endothelial injury caused by risk factors, particularly by ox-LDL, is the initial step of arterial AS. In addition, ox-LDL also causes the injury of endothelial progenitor cells, which suppresses the repair of injured endothelium and the restoration of endothelial function. However, the mechanism of endothelial cell injury and apoptosis induced by ox-LDL is not yet fully understood.Plenty of caveolin-1 is described to express on the cellular surface of many types of cells, such as endothelial cells, vascular smooth muscle cells and macrophages. Caveolin-1 mediates cell signal transduction and regulation widely. It is confirmed that caveolin-1 plays an important role in the progress of AS. Caveolin-1 modulates the permeability of endothelial cells, participates the uptake and transmembrane transport of LDL-C and ox-LDL by endothelial cells. Caveolin-1 also involved the proliferation and migration of endothelial cells, the expression of cytokines and the neoangiogenesis.Nuclear factor kappa B (NF-?B) plays vital roles in the modulation of oxidative stress, inflammation and cellular proliferation and apoptosis. The activation of NF-?B in various types cells located in atherosclerotic plaque was found to be elevated significantly. NF-?B mediates the pro-atherosclerosis effect of lipids, regulates the adhesion and migration of inflammatory cells. and is involved the apoptosis of endothelial cell after injury.In physiological status, high mobility group box-1 protein (HMGB1) mainly locates in cellular nucleus as a structural components of chromosome. HMGB1, a Late-Acting Pro-inflammatory factor, could be released to extracellular matrix by activated inflammatory cells and injured cells. Many risk factors of AS are able to cause the release of HMGB1 by endothelial cells, VSMCs, foam cells and macrophages. Inversely, the expression of HMGB1 receptors is rich on the surface of endothelial cells, VSMCs, foam cells and macrophages in atherosclerotic plaques. HMGB1 upregulates the expression of adhesion molecules, increase the release of cytokines, promotes the adhesion of monocytes and neutrophilic granulocyte, amplifies inflammatory response and accelerates the development of atherosclerosis. In addition, HMGB1 also promotes lipids accumulation and the infiltration of inflammatory cells by elevating the permeation of endothelial cells and decreasing the integrity of endothelium.It has been reported that both caveolin-1 and NF-?B played important roles on the regulation of HMGB 1 secretion in various types cells. NF-?B is a vital downstream signal molecule of caveolin-1, while caveolin-1 is involved the regulation of NF-?B activation. Caveolin-1/NF-KB pathway was described to mediate endothelial injury induced by ox-LDL. On the other hand, many receptors mediate the activation of HMGB1 on NF-?B, such as receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs). Given the relationship between caveolin-1, NF-?B and HMGB1, we considered caveolin-1, NF-?B and HMGB1 may cooperate in the ox- LDL-induced injury of endothelial cells. However, there is little report on this hypothetical synergistic effect in the injury of endothelial cells.Endothelial progenitor cells (EPCs), as an important cell population in the bone marrow and blood mononuclear cells (MNCs), play a critical role in angiogenesis, endothelial repair, vascular re-endothelialization, and prevention of neointima formation. The number and function of circulating EPCs are significantly decreased in patients with atherosclerosis, coronary artery disease. However, the exact mechanism for EPCs deficiency remains largely unknown. Ox-LDL also inhibits EPC proliferation and differentiation, and suppresses EPC function including inhibition of migration, adhesion, in vitro vasculogenesis, and ischemia-induced neovascularization in vivo. Previous studies has described that the crucial roles of ox-LDL on the endothelial cells, bone marrow stem cells, VSMCs, monocytes and macropages were mediated by ROS generated from ox-LDL stimulation. Our previous studies have confirmed that the venous injection of exogenous ox-LDL markedly elevated the generation of ROS in bone marrow and circulating blood in mice, which may explain the decreased population of circulating EPCs in patients with CAD, hyperlipidemia or AS.Probucol, a diphenolic compound, has anti-oxidant and anti-inflammatory properties. It was reported that probucol was able to decrease atherosclerosis and restenosis in coronary arteries. Probucol could preserve endothelial function by enhancing prostacyclin generation, reducing the level of endogenous nitric oxide (NO) synthase inhibitor, and inhibiting the expression of various adhesion molecules. Probucol could also promote proliferation and reduce apoptosis of endothelial cells that induced by oxidative injury. Probucol treatment could also protect EPCs from ox-HDL-induced apoptosis. For example, impaired ischemia-induced neovascularization by cigarette smoke could also be rescued by probucol via improving function of EPCs.The present study aimed to further illuminate the exact mechanism of endothelial injury by observing the roles of caveolin-1, NF-?B and HMGB1 in ox-LDL-induced injury of endothelial cells and macrophages migration induced by injured endothelial cells, and to further confirm whether the oxidative injury induced by ox-LDL plays a vital role in EPCs deficiency in patients with hyperlipidemia by investigating the protective effect of probucol on EPCs.The study consists of two sections.Part IThe roles of caveolin-1/NF-?B/HMGB1 signaling pathway in endothelial cells injury and macrophage migrationObjective:To further illuminate the exact mechanism of endothelial injury via observing the roles of caveolin-1, NF-?B and HMGB1 in ox-LDL-induced injury of endothelial cells and macrophages migration induced by injured endothelial cells.Methods:1. For observing ox-LDL-induced HUVECs injury, CRL-1730 cells were seeded into cultured in 25 cm2 culture flasks at 80% confluent monolayer and treated with different concentrations of ox-LDL for 24h to establish the model of endothelial injury. Endothelial cells were collected to extract cytoplasmic proteins, mitochondrial proteins, nuclear proteins and total cellular proteins following the respective protocol after incubation with ox-LDL for 24h. The expression and phosphorylation of caveolin-1 and NF-?B p65 in total cellular proteins and the subcellular distribution of Cytochrome C and HMGB1 were determined via Western Blot assay.2. For siRNA transfection, HUVECs were seeded into 6-well culture dishes at 50% confluent monolayer, and then transfected with prepared si-Cavl, si-p65 or si-HMGB1 for 12 h. After siRNAs transfection, ox-LDL was added to treat endothelial cells. Cells were collected to extract proteins followed respective protocols after incubation with ox-LDL for 24h. Western blot assay was performed to analysis the expression and phosphorylation of caveolin-1 and NF-?B p65 in total cellular proteins, cytoplasmic concentration of Cyt C, the expression of pro-caspase-3 and cleaved caspase-3 in total cellular proteins and HMGB1 expression in total cellular proteins and cytoplasmic proteins. We also collected the culture of different groups of HUVECs, and obtained the supernatant after centrifugation at 800g for 5min. The release of HMGB1 from HUVECs was detected by Western Blot.3. For observing the uptake ox-LDL using immunofluorescent assay, ox-LDL labeled with dil, rabbit anti-Human HMGB1 monoclonal antibody and FITC-labeled Goat Anti-Rabbit IgG were used to demonstrate the amount of ox-LDL and subcellular localization of HMGB1 in endothelial cells. The fluorescence intensity and location in HUVECs were observed by fluorescent microscopy.4. For macrophages migration assay, peripheral venous blood were obtained from healthy. Human peripheral blood monocytes (PBMC) were obtained using Ficoll density gradient centrifugation. Monocytes were seeded into 6-well culture dishes, and incubated with GM-CSF at a final concentration of 50ng/ml for 7d. The combination of DAPI and FITC-CD 14 were used to staining macrophages derived from monocytes for observing by immunofluorescent assay. CD14-FITC was used to determine monocyte-derived macrophages by flow cytometry. Macrophages derived from monocytes were seeded in the upper chambers, while the supernatants prepared previously was added into respective lower wells. After incubation for 48h, the filters were stained with 1% crystal violet. Cell migration was assessed by counting the number of migrated cells in 5 randomly selected microscopy fields per well.5. Data are expressed as means ± SD. Results were analyzed by one-way ANOVA and Student's t-test, using SPSS 17.0 software. Statistical significance was obtained when P values were less than 0.05.Result:1. Ox-LDL upregulated the expression and phosphorylation of caveolin-1, promoted Nf-KB p65 phosphorylation and Cyt C release from the mitochondria to the cytoplasm, and induced the cytoplasmic translocation of HMGB1 in a dose-dependent manner.2. Caveolin-1/NF-?B/HMGB1 signal pathway were significantly activated in ox-LDL treated HUVECs. Ox-LDL promoted the expression and cytoplasmic translation of HMGB1 in HUVECs by phosphorylating NF-?B p65 via upregulation on the expression and phosphorylation of caveolin-1.3. Caveolin-1/NF-?B/HMGB1 signal pathway played an vital role in the injury and apoptosis of HUVECs induced by ox-LDL. The knockdown of caveolin-1 significantly suppressed mitochondrial injury and endothelial apoptosis induced by ox-LDL. The knockdown of NF-?B markedly inhibited the injury of mitochondria, the activation of NF-?B and the apoptosis of endothelial cells after ox-LDL treatment. At the absent of ox-LDL stimulation, the suppression of si-HMGB1 on the expression of HMGB1 significantly enhanced mitochondrial injury. However, after stimulation with ox-LDL, si-HMGB1 markedly augmented mitochondrial injury, the activation of caspase-3 and the apoptosis of HUVECs.4. Caveolin-1/NF-KB/HMGB1 signal pathway was involved the uptake of ox-LDL by HUVECs. The blockage on caveolin-1 and NF-?B decreased ox-LDL uptake by HUVECs and HMGB1 expression in HUVECs, while si-HMGB1 markedly increased the uptake of ox-LDL by HUVECs.5. Caveolin-1/NF-KB/HMGB1 contributes to macrophage migration induced by endothelial injury, but HMGB1 may not play a crucial or final role in the migration of macrophages after endothelial injury.Conclusion: Caveolin-1/NF-?B/HMGB1 pathway was involved in the endothelial injury induced by ox-LDL and contributed to the ox-LDL uptake by endothelial cells. However, HMGB1 released by endothelial cells played an important but not a crucial role in the migration of macrophages induced by injured endothelial cells.Part ?Oxidative injury on endothelial progenitor cells induced by oxidized low-density lipoprotein and the protective effect of probucol via suppression of reactive oxygen species formation in vivoObjective: To further confirm the mechanism of the injury of endothelial progenitor cells induced by ox-LDL, to determine if probucol could protect EPCs from ox-LDL in vivo and to investigate the potential mechanisms.Methods:1. Ox-LDL preparation Peripheral blood was collected in heparinized tubes from healthy volunteers for the study. Plasma was obtained with centrifugation at 1500g for 20 min. Lipoproteins were isolated from the plasma by sequential ultracentrifugation using a Beckman TL-100 tabletop ultracentrifuge. The isolated lipoproteins were dialyzed against EDTA in 1x phosphate-buffered saline (PBS, pH 7.4) overnight and subsequently filter-sterilized. The protein concentration in the lipoproteins was determined using the Lowry's method. Ox-LDL was produced from the native LDL immediately after dialysis using copper sulphate, and was stopped by adding EDTA.2. The preparation of animal models Twenty-four wild type male C57 BL/6 mice (4-6 weeks old, Jackson Lab, ME, USA) were divided randomly into control group, oxLDL treatment group, and probucol group. Ox-LDL was injected into male C57BL/6 mice for 3 days with or without probucol pretreatment with PBS as control.3. Patients selection and probucol treatment Ten patients with hyperlipidemia and CAD were randomly divided into the placebo group and probucol group after informing consent. Five age- and sex-matched healthy volunteers informed consent were enrolled to be control. Patients of probucol group received oral treatment with probucol,500mg twice per day, while patients of placebo group received placebo treatment. Peripheral blood was collected from all patients and volunteers at baseline, after one week of treatment and one week after discontinuing probucol. blood samples were collected to determine the fasting lipid profile, serum ox-LDL level, blood glucose, CRP, SOD, TSH, kidney and liver functions, as well as the populations of circulating MNCs and EPCs. The patients'lipid profile was determined using an ARCHITECT ci16200 Integrated System and an electrochemiluminescent procedure. Patients' plasma ox-LDL was measured by ELISA.4. Intracellular and extracellular ROS detection After intravenous injection with PBS (control) or ox-LDL (50 ?g) with or without probucol treatment for 3 d, blood was harvested from the mice. Red blood cells (RBC) were eliminated using RBC lysis. The blood intracellular ROS formation was determined using flow cytometry assay. Plasma and BM fluid were collected for extracellular ROS detection using electron paramagnetic resonance (EPR).5. Murine MNCs, EPCs and human EPCs analysis After collection of BM and blood cells, MNCs were isolated. Cell surface markers CD34, Flk-1 and KDR were used to identify EPCs. The cells positive for CD34+/Flk-1+ were identified as EPCs for mice, and the combination of CD34 and KDR were used to determine EPCs for patients. After elimination of RBC, flow cytometry analysis was performed to identify EPCs using the LSRII system.6. Statistical analysis All the data were described as means ± standard deviation (SD), and statistically analyzed using unpaired Student t-est (two-sided) for two groups of data or two way ANOVA (analysis of variance) followed by post hoc conservative Bonferroni's test for three groups of data to minimize type I error as appropriate. Normal distribution of data was tested using the Shapiro-Wilk W-test, and equal variance was tested using the F-test. When the null hypothesis of normality and/or equal variance was rejected, the non-parametric Mann-Whitney U-test was used. The differences were considered statistically significant when a two-tailed p<0.05.Result:1. Ox-LDL induced EPCs injury via upregulating the generation of ROS.2. Probucol prevented ox-LDL-mediated reduction of BM mononuclear cells and circulating EPCs.3. Probucol attenuated both intracellular and extracellular ROS production.4. Probucol treatment decreased serum ox-LDL level and rescued circulating EPCs in hyperlipidemic patients.5. Probucol increased plasma SOD and decreased CRP levels in hyperlipidemic patientsConclusion: Oxidative stress induced by ox-LDL was further confirmed to play a vital role in EPCs injury in vivo. Probucol effectively reversed the effects of ox-LDL on circulating MNCs and EPCs in mouse via inhibition of BM and blood extracellular ROS and blood intracellular ROS production. For human study, probucol partially rescued the diminished EPCs level in hyperlipidemic patients in association with reduction of ox-LDL and CRP and increase in SOD level. |
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