| Background:Bone marrow derived cells (BMCs) contribute to angiogenesis by differentiating into endothelial cells (ECs), smooth muscle cells, and pericytes, and by delivering angiogenic proteins that support vessel growth and maturation. The chemotactic cytokine stromal cell derived factor-1 (SDF-1) plays a key role in mobilizing cells from the bone marrow and promoting homing of the cells to target tissues. SDF-1 acts by binding and activating the cell surface receptor CXCR4 on target cells. CXCR4 is essential for homing and maintenance of hematopoietic stem cells (HSCs) in distinct stromal cell niches within the marrow. CXCR4 surface expression varies between cell types. Most mesenchymal stem cells (MSCs) do not express CXCR4 but endothelial progenitor cells (EPCs) express relative high surface CXCR4. Only 5% of freshly isolated CD34+ cells from peripheral blood express CXCR4 but the expression can be increased> 10-fold by exposing cells to appropriate culture in vitro. Our previous study shows that calcium treatment can increase the surface expression of CXCR4 on BMCs.Accumulating evidence indicates that the level of CXCR4 surface expression on BMCs determines the efficiency of homing and the subsequent angiogenic response within target tissues. Over-expression of CXCR4 in MSCs or CD34+ cells enhances the migration of these cells towards SDF-1 in vitro and increases the efficiency of bone marrow transplant in vivo, probably due to increased cell survival and homing. Infusions of autologous progenitor cells have been shown to promote revascularization of muscle in animal models of peripheral ischemia but this has not been translated well into clinical applications. One reason may be that bone marrow of aged or diseased subjects contain stem cell progenitors that are defective for therapy and repair. EPC level and function are reported to be adversely impacted by aging, diabetes, ischemic heart disease and atherosclerosis in animals and humans. Angiogenesis is also impaired in aging. The molecular aspects of these defects are not known.We hypothesize that defective CXCR4 expression develops during aging, decreasing the number of CXCR4-positive progenitor cells of both the bone marrow and the circulation. This results in reduced progenitor cell presentation to the ischemic tissue and reduces angiogenesis.Objectives:The objectives of this study is to determine that aging defects bone marrow cells both on the decreased population of CXCR4+ progenitor cells and on the impaired homing capacity and angiogenetic ability and to explore possible mechanisms.Methods:To evaluate the aging impact on CXCR4 expression from gene transcription to protein synthesis and internalization, surface and intracellular CXCR4 expression on BMCs from old or young mice were analyzed with FCM. And multi-color FCM was used to examine the CXCR4 expression on the surface of different subpopulations of BMCs. The mRNA of CXCR4 was detected by real-time PCR and normalized to hypoxanthine phosphoribosyl transferase 1(HPRT1) mRNA levels. To compare the inducibility of CXCR4 expression in response to environmental change between BMCold and BMCyoung,1 mM CaCl2 was used as an extracellular stimuli to treat BMCs for 4 hours. To examine how BMCold are defect on calcium-induced CXCR4 surface expression, calcium influx was analyzed by measuring the increase of intracellular calcium after BMCs were mixed with CaCl2. Western blot analysis was performed to quantify the SDF-1-mediated phosphorylation of Akt kinase and Extracellular signal-regulated kinases 1 and 2 (ERK1/2). To imitate the in vivo process of BMCs homing from the CXCR4+ BMCs capture by SDF-1, close adhesion to vascular endothelial cell, to the trans-endothelial cell migration to injured tissue in vitro, cell-cell adhesion assays were used to detect the SDF-1 mediated adhesion of BMCs to the HUVEC monolayer, boyden chamber assays and vertical Collagen Gel invasion assay were used respectively to quantify the migration and trans-endothelial migration of BMCs from young and old mice in response to a gradient of SDF-1. We also established a mouse hindlimb ischemia model, injected BMCs via the tail vein, then detect proliferation, homing, and angiogenesis of the BMCs in ischemic tissue using immunohistochemistry and laser Doppler perfusion imaging (LDPI), to study whether aging impaired CXCR4 expression on BMCs would impact the cell functions. To study the effect of the micro-environment on BMC CXCR4 expression, BMCs of young mice were replaced with BMCold and vice versa after receiving lethal irradiation. After a 2-month reconstitution, mice were sacrificed to recover BMCs from femurs and tibias. The BMC surface CXCR4 expression before infusion and after recovery was determined by flow cytometry.Results:BMCold have less number of cells expressing CXCR4 and less CXCR4 on the surface of each cell as compared to BMCyoung. CXCR4 expression on BMCyoung could be enhanced by calcium, but CXCR4 surface expression in cells from old mice increased significantly less then BMCyoung. And it partly because of the defective calcium influx in BMCold which reduced the CXCR4 gene transcription, consequently lead to impaired responses to calcium-induced CXCR4 surface expression. There were no significant differences in the different BMC subpopulations, except the lineage negative (Lin-) subset, was observed between BMCyoung and BMCold. BMCs from young mice have significantly more Lin-cells than old mice. Surface CXCR4 expression in Lin-Sca-1+ subpopulation from young mice is significantly higher than that of old mice, while CXCR4 expression on CD34+Flkl+ subset and Gr-1+CD11b+ subset was not significantly different between BMCs from old and young mice. When the subpopulations of BMCs were grouped according to cell size, the "small cell" subpopulations from both young and old mice have higher CXCR4 surface expression than the other cells with bigger size. In cells from young mice, CXCR4 expression in all subpopulations was significantly increased by calcium treatment. However, such increase was significant less in the subpopulations of BMCs from old mice.SDF-1 binding causes internalization of the CXCR4 receptor. In BMCs from old mice, this CXCR4 receptor internalization responds normally to SDF-1, and the main difference is lower basal CXCR4 expression and absence of a response to calcium. Furthermore, BMCyoung are more sensitive to calcium stimulation to enhance intracellular signaling through SDF-1/CXCR4 interaction, suggesting that the SDF-1/Akt pathway is repressed in BMCs from old mice. On the other hand, the ERK1/2 signaling pathway is not altered in BMCold. BMCold showed weaker adhesion, lower mobility and lower trans-endothelial migration in vitro, and this was not enhanced by calcium pretreatment. After reciprocal bone marrow transplants, we found the changes due to transplantation were not significantly different in either case. Similarly transplantation did not change basal or calcium-stimulated mobility toward SDF-1. And also, aged BMCs have the impaired SDF-1-mediated homing capacity and angiogenic response to ischemia in vivo, with the defective calcium response. Conclusions:Compared to BMCs from young mice, BMCs from aged mice had:(1) reduced both surface CXCR4 expression and intracellular CXCR4 concentration. (2) diminished stimulation of CXCR4 surface expression by extracellular adjuvant like calcium; (3) significant lower Ca-influx and loss of calcium-stimulated CXCR4 mRNA accumulation; (4) dramatic reduction of SDF-1-mediated CXCR4 internalization both in the presence and absence of calcium stimulation; (5) Nearly complete abrogation of Akt phosphorylation in response to SDF-1 treatment.These effects of aging on CXCR4 expression in BMCs translated into the following functional impairments:(1) BMCs failed to migrate in an SDF-1 gradient; (2) Calcium failed to enhance this migration; (3) BMCs failed to home in vivo to the ischemic tissue in response to SDF-1 chemoattraction in the ischemic hindlimb; (4) Angiogenesis was markedly impaired despite injected SDF-1 in the ischemic muscle. |
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