SDF-1 Involves In Mobilization And Recruitment Of Endothelial Progenitor Cells After Artery Injury In Mice

1. Background and Objective:Revascularization procedures such as percutaneous balloon angioplasty, stent implantation, or atherectomy are widely used in the treatment of coronary artery disease but are often prone to failure because of restenosis, thrombosis, and vasospasm. Initial endothelial denudation is a major contributing factor to these consequences, in which the availability of vascular protective molecules such as nitric oxide (NO) and prostacyclin as well as antioxidant systems such as heme oxygenase-1(HO-1) are decreased, and the production of growth-promoting substances are increased, which ultimately lead to the formation of neointima. Reendothelialization at sites of spontaneous or iatrogenic endothelial denudation has classically been thought to be the result of the migration and proliferation of endothelial cells from the viable endothelium adjacent to the injury site. Neighboring endothelial cells, however, it may not constitute the exclusive source of endothelial cells for vascular repair. Recently, a series of investigations has suggested that endothelial progenitor cells (EPCs) derived from the bone marrow are present in peripheral blood and that these cells can be recruited to denuded areas and incorporated into nascent endothelium.EPCs may be defined as adherent cells derived from peripheral blood- or bone marrow-derived mononuclear cells demonstrating ac-LDL uptake and isolectin-binding capacity. The number of circulating EPCs inversely correlates with the number of cardiovascular risk factors and is reduced in cardiovascular disease. Moreover, several studies have demonstrated that patients with coronary heart disease or severe heart failure may suffer from impaired function of peripheral circulating EPCs. EPCs mobilized or transfused systematically can home to the sites of endothelial denudation, and accelerates reendothelialization of injured arteries and effectively impairs smooth muscle cell proliferation and neointima formation. However, the means of EPCs homing to the sites of endothelial denudation is not clear yet.First clinical trials have been performed investigating the effects of EPCs transplantation into cardiac ischemic areas after myocardial infarction, in patients with peripheral atherovascular disease, and on endothelialization of artificial heart valves. Next to EPCs transplantation, the pharmacological mobilization and functional modification of EPCs may also play a major role in future therapies.Chemokine stromal cell derived factor-1 (SDF-1, also known as CXCL12) is constitutively produced by bone marrow stromal cells and by other cells including CD34+ cells. It was initially characterized as a pre-B cell-stimulating factor and believed to be involved in retention of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in bone marrow. SDF-1 has been demonstrated to increase EPCs number through enhancement of (BM) c-kit+ stem cell adhesion onto extracellular matrix components by integrin receptors and protect EPCs from serum starvation-induced apoptosis. Increasing the level of SDF-1 in perepheral blood has been shown to mobilize bone marrow-derived EPCs into peripheral blood. Up-regulated SDF-1 expression in ischemic tissues or increasing SDF-1 expression in ischemic tissues through several established methods could also mobilize bone marrow-derived EPCs into peripheral blood and mediate them home to the site of neovascularization in ischemic tissues. Recently, studies had shown that the expression of SDF-1αwas up-regulated in injured carotid arteries of apoE-/- mice, which resulted in a marked mobilization of circulating Sca-1+lineage- progenitor cells (PBPCs) in the peripheral blood and mediated these cells home to the site of neointimal lesions, where they can adopt an SMC-like phenotype. It is not clear whether the up-regulation of SDF-1αcould also induce a mobilization of EPCs and mediate them home to site of injured arteries.In the present study, we attempted to demonstrate that SDF-1 is a biological function modificator of EPCs. For this purpose, we evaluated the effects of SDF-1 on the number, proliferation, migration, adhesiveness and apoptosis of murine bone marrow-derived EPCs. To clarify the role of local SDF-1αin repair of injured artery, we investigated the effect of up-regulated SDF-1αexpression on mobilization of EPCs in peripheral blood with fluorescence-activated cell sorter analysis after wire-induced arterial injury in mice. Furthermore, spleen-derived EPCs co-cultured with AMD3100 (a highly selective antagonist of SDF-1 that binds to its receptor, CXCR4) were injected into mice after carotid injury to evaluate the effect of local SDF-1αexpression on homing of EPCs to the site of endothelial denudation.2. Methods:In part one, mononuclear cells (MNCs) in murine bone marrow were isolated by density gradient centrifugation and bone marrow-derived EPCs were cultured according to previously described techniques. Murine bone marrow-derived EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under an inverted fluorescent microscope and further documented by demonstrating the expression of Sca-1 and VEGFR-2 by flow cytometer. To investigate the potential effects of SDF-1 on EPCs biological functions, bone marrow mononuclear cells or bone marrow-derived EPCs were stimulated with different concentrations of SDF-1 (1 ng/ml,10 ng/ml or 100 ng/ml) for different times. EPCs number, proliferation, migration, adhesion and apoptosis were evaluated by counting the double positive cells, MTT assay, modified Boyden chamber assay, counting the adherent cells and TUNEL staining, respectively.In part two, we established an injury model of the mouse carotid artery with a non- microscopical surgery method. The specimens of carotid arteries 30 minutes after the denuding procedure were examined using scanning electron microscopy, and the intimal lesion 2 weeks after injury was measured with pathological method. SDF-1αwas detected by RT-PCR and Western blot in carotid arteries of mice at different time points (day 0, day 1, day 3, day 7,day14) after wire-induced injury. Peripheral blood murine samples and bone marrow were obtained from mice at different time points after induction of vascular injury or sham operation (day 0, day 1, day 3, day 7, day 14). SDF-1 determination in peripheral blood and bone marrow samples was performed by SDF-1 enzyme-linked immunosorbent assay (ELISA) kit. EPCs were quantified in peripheral blood samples by flow cytometry. EPCs were also examined in peripheral blood samples of mice in subgroups, in which blocking SDF-1 monoclonal antibody or IgG1 isotype control was injected after vascular injury. Moreover, reendothelialization was determined by Evans blue staining in a whole vessel preparation 14 days after induction of endothelial cell damage in subgroups.In part three, mice received either 1x106 Dil-Ac-LDL–labeled spleen-derived EPCs or 1x106 CXCR4-blocked EPCs by direct percutaneous intracardial injection after induction of arterial injury. Control animals received a corresponding amount of normal saline. Thirty minutes before euthanasia, mice received FITC-labeled lectin intravenously. A subset of these animals received an injection of Evans blue dye 10 minutes before tissue harvesting. Carotid arteries were harvested 14 days after wire injury. Some of them were sectioned for fluorescent microscopic analysis to investigate Dil-Ac-LDL positive cells and lectin expression. A subset of carotid arteries was opened longitudinally for en face microscopy to examine Dil-Ac-LDL positive cells or to measure the area of reendothelialization. H&E staining was performed for morphometric analyses, and Lucia Measurement software was used to measure external elastic lamina, internal elastic lamina, and lumen circumference as well as medial and neointimal area of carotid arteries in all sections.3. Results:Part one:After 7 days in endothelial cell selection medium, bone marrow mononuclear cells turned into spindle-shaped, endothelial cell-like cells. Most of them showed uptake of ac-LDL and lectin binding, demonstrating endothelial cell characteristics. These cells were characterized further by demonstrating the expression of the mouse stem-cell marker Sca-1 as well as the endothelial cell lineage antigen VEGFR-2 by flow cytometry. Endothelial cell lineage antigen CD31, eNOS and vWF were also expressed by most of these cells in immunohistochemical analysis. Counting double fluorescent staining positive cells revealed that incubation of bone marrow mononuclear cells with SDF-1αincreased the number of differentiated, adherent EPCs in a concentration-dependent manner. Data in MTT assay showed that the proliferative activity of bone marrow-derived EPCs was improved concentration-dependently by SDF-1α. Pre-exposed to SDF-1α, the adhesive activity of EPCs was increased in a concentration-dependent manner. A dose-dependent decrease of apoptosis, which was induced by paclitaxel or serum absent media, was noticed as EPCs was incubated with SDF-1α. Data revealed that SDF-1αcould mediate obvious EPCs migration in a dose-dependent manner.Part two:Complete removal of the endothelium was achieved with a non-microscopical surgery method. Detected with Scanning electron microscopy, a platelet monolayer covered the denuded surface of injured carotid arteries. 2 weeks later, obvious neointimal hyperplasia was noticed in the injured carotid arteries. The results in RT-PCR and western blotting showed that up-regulation of SDF-1αmRNA and protein were already evident at 1day, and peak expression was achieved at 3 days after arterial injury. In enzyme-linked immunosorbent assay, an obvious rise in plasmatic concentration of SDF-1αwas also noticed 1 day, 3 days and 7 days after carotid injury, compared with the sham operation group (P<0.01). In contrast, a significant reduction of SDF-1αbone marrow concentration was examined 1 day, 3 days and 7 days after carotid injury, compared with the sham operation group (P<0.01). The results in fluorescence-activated cell sorting analysis showed that the amount of circulating EPCs was increased markedly 1 day, 3 days and 7 days after induction of vascular injury, compared with the sham operation group (P<0.01). In a subgroup in which blocking SDF-1αmonoclonal antibody was injected, the amount of circulating EPCs was increased lightly only at 1 day after induction of vascular injury, compared with the sham operation group (P<0.05). Interestingly, Evans blue staining showed that the reendothelialization area in the SDF-1 mAb-treated group was reduced significantly 14 days after induction of endothelial cell damage, compared with the IgG1 isotype control group (P<0.01). Surprisingly, a reduced reendothelialization in the SDF-1 mAb-treated group was not associated with an increased neointima formation, compared with the IgG1 isotype control group (P>0.05).Part three:In modified Boyden chamber assay, pretreatment of bone marrow-derived EPCs with 10 ng/ml AMD3100 almost completely blocked cell migration in response to 100 ng/mL SDF-1α, compared with the control (P>0.05). En face microscopy 14 days after induction of injury revealed that infusion of CXCR4-free EPCs resulted a more number of cells attached at the injury site, forming islets of transplanted cells within the deendothelialized area, compared with the group treated with CXCR4-blocked EPCs (P<0.01). Immunohistochemical analysis revealed that transfused CXCR4-free EPCs were predominantly found at the injury site, lining the intraluminal margin of the neointima. Lectin staining revealed that the attaching cells at the site of endothelial injury were lectin-positive. Overlay experiments clearly demonstrated the endothelial phenotype of transfused cells. In the CXCR4-blocked EPCs transfusion group, only few Dil-Ac-LDL- and lectin-positive cells were present, and the endothelial cell line of the neointima appeared disrupted at day 14 after induction of injury compared with the CXCR4-free EPCs treatment group. In the placebo group, a few endogenous (lectin-positive, Dil-Ac-LDL–negative) endothelial cells were present at the site of endothelial denudation, and no endothelial cell line was formed. Data in Evans blue staining revealed that transfusion of CXCR4-free EPCs was associated with a significant increase in reendothelialization area compared with the placebo group (P<0.01). But no significant increase in reendothelialization area was noted in the group of CXCR4-blocked EPCs transfusion, compared with the placebo group (P>0.05). Computer-based morphometric analysis showed that the accelerated reendothelialization after transfusion CXCR4-free EPCs was associated with a significant reduction of neointima formation compared with the placebo group (P<0.01); no change in neointima formation was detected in CXCR4-blocked EPCs treated group compared with the placebo group (P>0.05).4. Conclusions:1. SDF-1αcould induce EPCs migration in a dose-dependent manner, and improved the proliferative and adhesive activity of EPCs, and inhibited EPCs apoptosis in the same manner. Moreover, SDF-1αcould induce differentiation of hematopoietic stem cells toward EPCs.2. Up-regulated SDF-1 expression after vascular injury participateded in the mobilization of EPCs through changing the gradient of SDF-1 concentration between the peripheral blood and the bone marrow. Blocking EPCs mobilization mediated by SDF-1 after vascular injury could inhibit reendothelialization of injured arteries.3. Local SDF-1 expression in injured arteries could mediate EPCs home to the site of endothelial denudation and participate in reendothelialization of neointimal lesions.4. Bone marrow-derived EPCs of mice can be obtained through culturing bone marrow mononuclear cells in endothelial cell selection medium.5. Carotid artery injury model of mouse can be established by a non-microscopical surgery method.