| Atherosclerosis is one of the major diseases threatening the human health, at present accumulating evidence suggests that endothelial dysfunction is an early step in the atherosclerotic process and plays a pivotal role in the development and progression of atherosclerosis. And several studies have also found that endothelial progenitor cells (EPCs) play an important role in the development of atherosclerosis, such as promoting the regeneration of damaged endothelium. Walter et al demonstrated that circulating endothelial progenitor cells has the characteristics of homing to denuded parts of the artery after balloon injury, a body of evidence is available demonstrating that EPC levels are very strong vascular risk markers, even better than the Framingham risk score. Most of the clinical studies also confirmed that increased number and function of EPCs may have important therapeutic implications for the management of cardiovascular disease, such as coronary heart disease, heart failure etc. Traditional risk factors such as elderly, hypertension, hyperlipidemia, and diabetes are major causes of reducing the number and function of circulating EPCs Adiponectin is a30-kDa protein synthesized predominantly by white adipose tissue, which has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties. Enhancement of nitric oxide generation and attenuation of reactive oxygen species production in endothelial cells along with stimulated endothelial progenitor cells (EPCs) survival, proliferation and differentiation constitute some of adiponectin’s vasoprotective actions. Adiponectin exerts a pivotal role in vascular protection through activation of multiple intracellular signaling cascades and exists in the circulation in three isoforms:a low molecular weight trimer, a medium molecular weight hexamer, and a high molecular weight (HMW) multimer. The3isoforms might play different roles, but some arguments have suggested that the HMW isoform might be the major active protein. Both AdipoR1and AdipoR2, two receptors for adiponectin, are expressed in endothelial cells. The endothelium is a major target of adiponectin, where the adiponectin downregulates adhesion molecules, inhibits monocytes adhesion to endothelial cells and ultimately inhibits the migration and proliferation of vascular smooth muscle cells, which contribute to vascular damage.Endothelial progenitor cells from bone marrow or peripheral blood are mononuclear precursor cells that can differentiate into mature endothelial cells. During wound repair EPCs home to sites of vascular injury where these cells can restore vascular integrity, and play a significant role in maintaining vascular endothelial integrity, repairing the damaged endothelial cells, promoting new blood vessel formation and tissue repair, etc. EPCs are mainly isolated from peripheral, umbilical cord, and bone marrow. There are also a small number of EPCs in adipose tissue. The number of immature progenitors from bone marrow is500-fold higher compared to peripheral blood. Drug-induced EPCs can be increased to6%of the total number of white blood cells. EPCs can remarkably promote new blood vessel formation and repair damaged vessels, the mechanisms underlying this phenomenon are likely to be explained in two ways:(1) EPCs improve neovascularization of ischemic tissues by secretion of cytokines such as vascular endothelial growth factor (VEGF) and so on.(2) EPCs have been found to directly incorporate into injured vessels and that participate in angiogenesis and reconstruction by differentiation into endothelial cells.The atherosclerotic vascular remodelling and pathophysiology involve multiple cell types and a wide array of mediators and cascades. Of note, the PI3K/Akt signalling pathway impinges on several of them. Phosphoinositide3-kinases (PI3Ks) are protein and lipid kinases activated by G protein-coupled receptors and tyrosine kinase receptors. PI3K and Akt are important positive regulators of endothelial nitric oxide synthase (eNOS), which generates NO through the NADPH-dependent oxidation of L-arginine. The PI3K/Akt related pathways have a key role in regulating vascular tension, etc.With this background, this study sought to investigate whether the reduction in EPCs numbers is an independent risk for arteriosclerosis, the relationships between numbers of EPCs with cerebral atherosclerotic severity, and further investigate whether the activation of PI3K/Akt is involved in adiponectin promoting endothelial progenitor cell proliferation and migration.Chapter I Correlation of Different Subpopulations of Endothelial Progenitor Cells with Cerebral Arteriosclerosis SeverityObjective:Stroke remains a leading cause of death and long-term disability in adults’ worldwide, ischemic strokes (IS) account for approximately87%of these strokes. Cerebral atherosclerosis (CA) is one of the main causes of this type of obstruction and endothelial dysfunction also plays a critical role in its onset and progression. Endothelial dysfunction is characterized by a progressive loss of endothelial cell (ECs) and thought to be an important early event in the atherosclerotic disease process. It contributes to plaque initiation and progression. Mature ECs possess limited regenerative capacity. Thus, growing evidence suggests endothelial progenitor cells (EPCs), endothelial stem or precursor cells, may contribute to the restoration of the endothelial lining after injury and maintenance of endothelial function, which is a novel endogenous vascular repair system mediated by EPCs.Recent studies have indicated that EPCs are immature cells capable of homing to the injured endothelium, differentiating into mature endothelial cells and replacing the injured endothelial cells, thereby exerting a protective effect against atherosclerosis. The number of circulating EPCs is now considered a mirror of cardiovascular health. Recent studies suggest that reduced number of circulating EPCs was closely related to the degree of endothelial dysfunction in humans at various degrees of cardiovascular risk, smoking, diabetes, hyperlipidemia; and hypertension. Werner et al. also identified levels of EPCs correlate inversely with the severity of several atherosclerotic vascular diseases such as coronary artery disease and peripheral occlusive vascular disease. However, the relationship between the severity of the CA and EPCs levels is not known. With this background, this study sought to investigate the relationships between the number of EPCs and the CA severity. We also examined the degree to which EPCs count were associated with traditional cardiovascular risk factors (age, gender, smoking, hyperlipidemia, hypertension, diabetes, and family history). Finally, we assessed whether EPCs provided significant diagnostic value beyond traditional risk factors for estimating CA severity.Methods: 1. All subjects were enrolled from the consecutive inpatients who underwent cerebral angiography in the Department of Neurology at Jingling Hospital between December2011and December2013. They were thoroughly investigated for demographics and baseline clinical characteristics. All patients underwent conventional cardiac ultrasound and electrocardiogram examination. Neurological deficits were estimated using the National Institutes of Health Stroke Scale (NIHSS) score by two neurologists, who were blind of digital subtraction angiography (DSA) images and EPCs assay results. The Framingham risk score (FRS) was assessed at baseline for each subject’s cardiovascular risk factors, and the Ankle-brachial index (ABI) is used to confirm the diagnosis and determine the severity of peripheral arterial disease (PAD).2. Cerebral arteriosclerosis burden assessment:Locations of lesion were categorized as being in the intracranial, extracranial vessels or the combined extracranial and intracranial stenosis (IES). Intracranial lesions were defined as those at or above the precavernous or upper petrous carotid artery in the internal carotid artery. For the vertebral artery, the distinction was made at the point where the artery takes a turn and comes into the foramen magnum piercing the dura. Extracranial stenosis (ECS) was calculated using North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria; and intracranial stenosis (ICS) was measured by using Warfarin-Aspirin Symptomatic Intracranial Disease (WASID) criteria. The presence of arterial stenotic lesions was determined by the number of cerebral arteries showing steno-occlusion and the degree thereof. The extent of atherosclerotic burden (cerebral atherosclerotic burden, CAB) was graded according to the previous criteria (0indicating<50%stenosis,1indicating50%to99%stenosis, and2indicating occlusion). In the presence of multiple lesions within one arterial segment, data from the most significant stenosis were used. The total number of arterial segmental lesions ((≥50%stenosis) was also calculated (cerebral atherosclerotic number, CAN).3. Statistical analysis was performed using SPSS software, version16.0(SPSS Inc., Chicago, IL). Comparisons were performed using independent sample t tests, Kruskal-Wallis tests, Mann-Whitney U tests, or analysis of variance for normal distributed continuous variables, and the chi-square test or fisher’s exact test for categorical variables, as appropriate. Variables with a P value<0.2by univariate analysis were candidates for inclusion in multiple logistic regression analysis. A selection process was used to develop the final multivariable model, and adjusted odds ratios (OR) and95%confidence intervals (CI) were calculated as estimates of relative risk. Moreover, the relationship between number of arterial segmental lesions and EPCs levels was determined by Spearman rank test. Associations between EPCs level and arterial calcification or CA severity were assessed with logistic regression. A two-tailed P value<0.05was considered statistically significant.Results:1. Patients’CharacteristicsThe baseline characteristics of the197patients enrolled in this study are provided in Table1. Fifty-four patients (27.4%) had ECS and45patients (22.8%) had ICS. Mean age was60years, with29.4%women and12.2%no significant CA as a control group. The median values of were CD34+CD309+, CD133+CD309+and CD34+CD133+CD309+cells were0.172%(interquartile range,0.066%to0.370%),0.131%(interquartile range,0.041%to0.297%) and0.024%(interquartile range,0.005%to0.063%), respectively, in the total sample.2. Circulating EPCs Are Reduced in the Presence of CAIn binary logistic regression analysis, the only EPCs level was significantly conversely associated with the prevalence of CA after adjustment for age, smoking, hypertension, SBP, fibrinogen and FRS (CD45-/dimCD34+CD309+cells:B=-2.197, OR=0.111,95%CI:0.024to0.511, P=0.005; CD45-/dimCD133+CD309+cells: B=-2.172, OR=0.114,95%CI:0.023to0.556,p=0.007; CD45-/dim CD34+CD133+CD309+cells:B=-4.580, OR=0.010,95%CI:0.000to0.541, P=0.024). The result of multivariate logistic regression showed that only EPCs count was significantly negatively associated with the location of CA (extracranial or combined extra-and intra-cranial artery stenosis). However, EPCs were not significantly associated with intracranial atherosclerosis (P>0.05).3. Relationship of CA with EPCs, Calcification Score or Framingham Risk ScoreThe subjects were divided into three groups on the basis of their atherosclerosis score:low, intermediate and high group. In the ordinal regression analysis CA burden remained correlated conversely to EPCs and associated positively with fibrinogen and hypertension by controlling for confounders. We use Spearman’s rank correlation analysis to analyze the correlation between CA burden and EPCs levels, there was also a significant inverse relationship between CD133+CD309+cells count and atherosclerotic burden in78patients with ECS and control subjects (CAN and CAB: Spearman rho=-0.195and-0.197, P<0.05). Additionally, the atherosclerotic burden was inversely related to CD133+CD309+cells count in69patients with ICS (CAN: Spearman rho=-0.262, P=0.030; CAB:Spearman rho=-0.248, P=0.040). There are still significant differences between CA burden and EPCs levels after adjustment for potential confounders. However, the atherosclerotic burden was not related to FRS or the other two progenitor cell subsets.Conclusions:1. EPCs level was significantly conversely associated with the prevalence of CA after adjustment for age, smoking, hypertension, SBP, fibrinogen and FRS.2. There was a significant positive correlation between EPCs and atherosclerotic burden in patients with ECS. However, there was no statistically significant relationship of the atherosclerotic burden with EPCs in patients with ICS.Chapter II Isolation, culture and characterization of bone marrow derived EPCsObjective:EPCs, endothelial precursor cells, are mobilized early after vascular injury. They can home to sites of vascular injury and differentiate into mature endothelial cells, and avidly secrete angiogenic factors encouraging resident endothelial cell proliferation and migration. EPCs may play a critical role in re-establishing the endothelial integrity, repairing damaged endothelial cells, promoting angiogenesis in tissue repair. EPCs can be mainly isolated from peripheral blood or bone marrow, which is500-fold higher compared to peripheral blood. We try to establish a culture system to induce the differentiation of C57BL/6mouse bone marrow-derived mononuclear cells into EPCs.Methods:Bone marrow of tibia and fibula from C57BL/6mice (4-6week old, male) was washed out with PBS and eentrifugated with Histopaque1077at400g for30minutes. The bone marrow mononuclear cells (BMMNCs) were re-suspended in EGM-2SingleQuots and washed twice. The BMMNCs were then seeded in the cell culture flasks coated with human fibronectin and cultured with EGM-2SingleQuots at37℃5%CO2. Nonadherent cells were removed4days after seeding and the culture medium was replaced every other day thereafter. EPCs cultured up to3weeks were used for later cell characterization and transplantation.Cultured cells were observed and photographed by inverted microscope everyday. EPCs cultured up to3weeks were chosen for cell characterization. We identified the proportion of cultured cells which co-expressed CD34and VEGFR2by flow cytometer. Meanwhile, we also tested the abilities of of uptaking Dil-Ac-LDL and binding FITC-UEA-1of cultured cells.Results:After the first exchange of medium to remove adherent cells were growing rapidly. Then cell colonies began to appear on the5th day of culture. Colony morphology is small round cell aggregation center, surrounded by radial growth of cells. In cell culture, we also observed that cells can be present in linear and tubular growth, which suggested that the cells have the ability to grow new blood vessels. Since about2weeks of culture, the cells gradually changed to a cobblestone-like appearance.After3weeks culture, cells were verified as EPCs by flow cytometer. And the result exhibited that more than85%of the cultured cells co-expressed CD34and VEGFR2. Finally, by testing the abilities of uptaking Dil-Ac-LDL and binding FITC-UEA-1of cultured cells, we found most cultured EPCs were able to uptake DiI-Ac-LDL and bind FITC-UEA-1.Conclusions:1. EPCs were separated from bone marrow with density gradient centrifugation, wall sticking screening and amplified in vitro. Our method of culturing EPC from BMMNCs of C57BL/6mice is feasible.2. We observed a line-like and a tube-like appearance during the cell culture which indicating the vascular forming potential of cultured EPCs. The results of FACS and cell function tests all indicated that the cells we cultured were EPCs. This part of study provides the technological foundation for the later experiments. Chapter Ⅲ Effects of Adiponectin on proliferation and migration activities of endothelial progenitor cells and its possible mechanismObjective:Adiponectin is a30-kDa protein synthesized predominantly by white adipose tissue. Adiponectin exerts a pivotal role in vascular protection through activation of multiple intracellular signaling cascades. Enhancement of nitric oxide generation and attenuation of reactive oxygen species production in endothelial cells along with stimulated endothelial progenitor cells (EPCs) survival, proliferation and differentiation constitute some of adiponectin’s vasoprotective actions. Circulating adiponectin level is considered to be a new independent risk factor for coronary heart disease, hypoadiponectinemia will lead to vascular endothelial dysfunction. EPCs are the precursor cells that can proliferate and differentiate into endothelial cells and have some biological properties of endothelial cells and progenitor cells. Whether EPCs express adiponectin receptors, the effect of adiponectin on the proliferation and differentiation of EPCs and the possible mechanism are unclear.Adiponectin promotes endothelial progenitor cell number and function, enhances the ability to repair damaged endothelium and delays the occurrence and development of atherosclerotic lesions. With this background, we intend to use EPCs culture system successfully established in the second part, explore preliminarilly effects of Adiponectin on proliferation and migration activities of endothelial progenitor cells and its possible mechanism.Methods:1. We intend to study cell growth curves of EPCs. Bone marrow mononuclear cells (BMMNCs) were harvested from C57BL/6mice, and plated on fibronection-coated culture dishes in different concentrations. Cell growth curves of each group were drawn with CCK-8assay, which was used to determine the proliferation of the cultured EPCs in different inoculation concentration.2. To investigate the effects of adiponectin with different doses on proliferation and migration of EPCs. Obtain required amount of EPCs which were extracted from the bone marrow of C57BL/6mice, counted and seeded on the culture flasks. Namely, Control group and adiponectin-treated group (cell culture medium was supplemented with1and10ug/ml adiponectin respectively). EPCs’differentiation and migration were assessed with the CCK-8assay and Transwell respectively. The expression of Akt, p-Akt、p-eNOS、eNOS were measured by Western blot.3. Bone marrow mononuclear cells (BMMNCs) were harvested from C57BL/6mice and divided into6groups for cell culture. The cell groups were as follows: control group, adiponectin treatment groups (cell culture medium was supplemented with lug/ml adiponectin), NO donor group (sodium nitroprusside, SNP), SNP and adiponectin treatment group (cell culture medium was supplemented with SNP and lug/ml adiponectin), eNOS inhibitor group (cell culture medium was supplemented with L-NAME), L-NAME and adiponectin treatment group (cell culture medium was supplemented with L-NAME and1ug/ml adiponectin). The expression of nitric oxide in supernatant was measured by nitric oxide assay kit.4. Mouse BMMNCs were induced to differentiate into EPCs and divided into4groups for cell culture. The cell groups were as follows:control group, adiponectin treatment groups (cell culture medium was supplemented with lug/ml adiponectin), LY294002group (Akt inhibitor), LY294002and adiponectin treatment group (cell culture medium was supplemented with LY294002and lug/ml adiponectin). EPCs’ differentiation and migration were assessed with the CCK-8assay and Transwell respectively. The expression of Akt、p-Akt、p-eNOS、eNOS were measured by Western blot.5. Statistical analysis was performed using SPSS software, version16.0(SPSS Inc., Chicago, IL). Data are expressed as the mean value±standard deviation. Comparisons among more groups (such as levels of cell proliferation, migration, and NO, Akt, p-Akt, eNOS and p-eNOS, etc.) were performed by the analysis of variance. Statistical significance was accepted if the null hypothesis could be rejected at P≤0.05.Results:1. Monocyte proliferation curve in different inoculums concentrationsMonocytes cultured cells were seeded at a density of about5-11×105cells/cm2and reached its peak proliferation at2weeks. Thus, the concentration of mononuclear cells was used in the next experiment after2weeks of culture.2. Different doses of Adiponectin influence the proliferation and migration of EPCsAdiponectin inereased the number of EPCs with dose and time dependence. Adiponectin (lug/ml) signifieantly inereased proliferation and migration activities of EPCs after3days, Western blot analysis showed that there was statistically significant difference between adiponectin group and control group for Akt, p-Akt, p-eNOS and eNOS expression respectively (P<0.05).3. The eNOS-inhibitor could effectively inhibit the effect of adiponectin on EPCsAdiponectin increased the ability of migration and proliferation on cultured EPCs. And the properties revealed a dose-effect relationship and a positive relationship to p-eNOS expression which could be eliminated by eNOS inhibitor L-NAME. NO expression, the ability of migration and proliferation in adiponectin, SNP, SNP and adiponectin treatment group increased significantly compared with control group. Western blot analysis showed that there was statistically significant difference between adiponectin group and control group for p-Akt, eNOS and p-eNOS expression respectively. While cell proliferation and migration were inhibited and no significant differences of p-Akt, eNOS, p-eNOS and NO expressions existed among L-NAME, L-NAME and adiponectin treatment groups.4. Phosphatidylinositol3-kinase (PI3K) inhibitor LY294002could effectively inhibit the effect of adiponectin on EPCsAdiponectin exhibit pro-proliferative and anti-apoptotic properties on cultured EPCs, which could be eliminated by PI3K inhibitor LY294002.1ug/ml adiponectin treatment also significantly improved the number of cells, cell migration and the p-Akt, eNOS, p-eNOS expression levels. However, no significant differences of p-Akt expressions existed among the rest groups, the number of cells, cell migration and the p-Akt, eNOS, p-eNOS expression levels significantly decreased in LY294002group, and LY294002and adiponectin treatment group compared with control group (P<0.05).Conclusions:1. Adiponectin promote EPCs proliferation and migration activity in a time-and dose-dependent manner.2. Adiponectin improves EPCs proliferation and migration activity through PI3K/Akt signaling pathway. |
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