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Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy - PubMed

Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

Quiling Wu et al. J Cell Mol Med. 2009 Sep.

Abstract

Cell surface receptors play major roles in the mobilization and homing of progenitor cells from the bone marrow to peripheral tissues. CXCR4 is an important receptor that regulates homing of leucocytes and endothelial progenitors in response to the chemokine stromal cell-derived factor-1 (SDF-1). Ionic calcium is also known to regulate chemotaxis of selective bone marrow cells (BMCs) through the calcium-sensing receptor, CaR. Here we show that calcium regulates CXCR4 expression and BMC responses to SDF-1. CaCl(2) treatment of BMC induced a time- and dose-dependent increase in both the transcription and cell surface expression of CXCR4. BMC subpopulations expressing VEGFR2(+), CD34(+) and cKit(+)/Sca-1(+) were especially sensitive to calcium. The effects were blocked by calcium influx inhibitors, anti-CaR antibody and the protein synthesis inhibitor cycloheximide, but not by the CXCR4 antagonist AMD3100. Calcium treatment also enhanced SDF-1-mediated CXCR4 internalization. These changes were reflected in significantly improved chemotaxis by SDF-1, which was abolished by AMD3100 and by antibody against CXCR4. Calcium pre-treatment improved homing of CD34(+) BMCs to ischemic muscle in vivo, and enhanced revascularization in ischemic mouse hindlimbs. Our results identify calcium as a positive regulator of CXCR4 expression that promotes stem cell mobilization, homing and therapy.

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Figures

Figure 1
Figure 1

Calcium induced CXCR4 surface expression on BMC. (A). BM cells were incubated at 37°C for 4 hrs in PBS or PBS plus CaCl2 (1 mM) and labelled with FITC-conjugated anti-CXCR4 mAb. CXCR4+ cells were detected by flow cytometry. (B). Time course of CXCR4 surface expression after calcium treatment. BM cells were incubated in PBS (white bar) or PBS with 0.5 mM CaCl2 (black bar) at 37°C for the specified time. Surface CXCR4 on BMC was analysed by FACS and expressed as MFI. (C). BMC were incubated in PBS with the specified concentration of CaCl2 at 37°C for 4 hrs. Surface CXCR4 on BMC was analysed by FACS. (D). Similar to (A), BMCs were labelled with different fluorescent conjugated Abs against VEGFR2, CD34, cKit, Sca-1, and CXCR4. CXCR4+ cells were detected by FACS in the different subpopulations either positive or negative for the surface markers.

Figure 2
Figure 2

Correlation of calcium influx with CXCR4 expression. Calcium influx into BMC was measured by flow cytometry with Fluo-4/AM staining. Arrows indicate the time when CaCl2 (A), or CaCl2+ BAPTA (B) were added. CXCR4 surface expression under the different conditions was measured by FACS (C). *P < 0.05 versus PBS group, #P < 0.05 versus calcium treated group.

Figure 3
Figure 3

CXCR4 expression at different conditions. (A) FACS analysis of CXCR4 surface expression. (B) FACS analysis of intracellular CXCR4. (C), Quantitation of CXCR4 expression at different conditions.

Figure 4
Figure 4

Internalization of CXCR4 after binding with SDF-1. BMC were incubated with PBS with or without calcium for 4 hrs and then mixed with SDF-1 for 1 hr at 37° C. The surface (A) and intracellular (B) CXCR4 were measured by FACS. Surface CXCR4 was decreased and intracellular CXCR4 was increase after binding with SDF-1. *P < 0.05.

Figure 5
Figure 5

BMC migration towards SDF-1. (A) BMCs were pre-incubated with PBS with or without CaCl2 for 4 hrs at 37°C and transferred to the upper chamber of inserts in a 24-well plate containing DMEM and 100 ng/ml SDF-1. Migrated cells were counted after 6 hrs incubation at 37°C. Inhibitors AMD3100, LY294002, L-NMMA and anti-CXCR4 antibody were added to the upper chamber along with BMCs. *P < 0.05 versus Calcium treated group; #P < 0.05 versus other groups. (B) Effect of the inhibitors on CXCR4 surface expression. BMC were incubated at 37°C for 4 hrs in PBS without or with 0.5 mM CaCl2 plus specified inhibitors, and subjected same FACS analysis as in Fig. 1. Amount of surface CXCR4 on BMC was expressed as MFI.

Figure 6
Figure 6

Homing of injected BMC in ischemic site. BMCs from GFP mice were incubated in PBS with or without CaCl2 at 37°C for 4 hrs, and then intravenously injected into WT BL6 mice with ischemic limb. SDF-1 protein (100 ng) was injected into the ischemic hindlimb muscle twice at consecutive days after the surgery. The hindlimb muscles were recovered 7 days after the cell injection, stained with DAPI for nucleus (blue), and examined for GFP cells incorporation under fluorescent microscopy. (A) Untreated BMC, (B) Ca-treated BMC, (C) BMC + SDF-1, (D) BMC/Ca + SDF-1. (E) The incorperated GFP cells were quantified as cells per high power field (HPF). *P < 0.05, and **P < 0.01 versus all other groups (n= 6).

Figure 7
Figure 7

Effect of BMC injection on angiogenesis. BL6 mice with ischemia limb were injected intravenously with BM cells that were treated with or without CaCl2. SDF-1 protein was injected into the ischemic hindlimb muscle after the surgery. Blood flow of the lower limbs was measured using an laser Doppler perfusion images analyser. (A). Representative laser Doppler perfusion images at day 0 and day 21 after ischemia and cell injection. (B) Capillaries in the ischemic muscle from the mice at day 21 were identified by alkaline phosphatase cryosectional staining. (C). CD34 immunostaining of paraffin sections obtained from the ischemic muscle of mice at day 7. Brown spots are CD34+ cells (arrow). Blue represents cells counter-stained with haematoxylin. (D). Quantitative measurement of perfusion ratio of ischemic limbs to that of normal limbs at day 21 (n= 6). (E) Quantification of capillary density on the tissue section, which is presented as the ratio of the number of capillaries to the number of muscle fibres. (F), Quantification of CD34+ cells around each muscle fibre. *P < 0.05 versus BMC/Ca group. #P < 0.05 versus other groups.

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