A single injection of AMD3100 after MI increases BM EPC incorporation into ischemic tissue. WT mice transplanted with Tie2-LacZ BM received single s.c. injections of AMD3100 (AMD) or saline 1 h after MI. (A) BM EPCs were identified in the LV by X-gal staining (blue-green) for LacZ expression, and (B) the number of BM EPCs in the ischemic border zone was quantified (n = 4). (C) BM EPC incorporation in the ischemic border zone on day 28 was evaluated by staining for β-gal (green) and CD31 (red) expression and quantifying the number of double-positive cells (yellow) (n = 3). HPF, high-power field. *, P < 0.05; ***, P < 0.01. (Scale bar: 100 μm.)

The beneficial effects associated with a single injection of AMD3100 are dependent on MMP-9 expression. (A) WT mice, (B) MMP-9 knockout (KO) mice, or (C and D) WT and MMP-9 KO mice received single s.c. injections of AMD3100 (AMD) or saline 1 h after MI. (A) MMP-9 expression in PB or BM MNCs was evaluated via quantitative RT-PCR; measurements were normalized to 18S rRNA and expressed as the fold-change from levels before MI (n = 4). (B) PB EPC counts were determined in MMP-9 KO mice via flow-cytometry analyses for coexpression of Sca1 and Flk1 (n = 4). (C) Capillary density on day 28 after MI was evaluated by staining the vasculature via in vivo BS-1 lectin perfusion before killing, and then quantifying the number of BS-1 lectin–positive (brown) vessels (n = 5). (D) Fibrosis was quantified as the ratio of the length of fibrosis to the LV circumference; fibrotic tissue appears blue-gray (n = 5). *, P < 0.05; NS, not significant.

AMD3100 administration immediately after MI increases VEGF expression in PB and BM MNCs 3 to 7 d later. VEGF activity is required for AMD3100-induced MMP-9 expression. (A and B) Mice received single s.c. injections of AMD3100 (AMD) or saline 1 h after MI and then VEGF mRNA expression in (A) PB and (B) BM MNCs was measured via quantitative RT-PCR, normalized to endogenous 18S rRNA expression, and expressed as the fold-change from levels before MI. *, P < 0.05; n = 5. (C and D) AMD3100- and saline-treated mice received i.p. injections of a VEGF-neutralizing antibody (antiVEGF) or an IgG antibody after surgically induced MI. Five days later, MMP-9 mRNA expression was measured in (C) PB and (D) BM MNCs. mRNA measurements were normalized to endogenous 18S rRNA expression and expressed as the fold-change from measurements obtained in uninjured, untreated mice. *, P < 0.05; ***, P < 0.01; n = 5.

A single injection of AMD3100 after MI mobilizes EPCs, improves survival, reduces adverse cardiac remodeling, and increases capillary density. Mice received single s.c. injections of AMD3100 (AMD) or saline 1 h after MI. (A) Peripheral blood (PB) EPC counts were evaluated via the EPC culture assay and costaining for acLDL uptake and BS-1 lectin (n = 5). (B–D) To ensure suitable mortality for statistical analyses, MI was induced at the most proximal site of the coronary artery. (B) Survival rate of mice treated with AMD (solid line, n = 48) or saline (broken line, n = 23); subsequent assessments were performed in mice that survived through day 28. (C) Fibrosis was quantified as the ratio of the length of fibrosis to the LV circumference (AMD: n = 12; saline: n = 5); fibrotic tissue appears blue-green. (D) Capillary density was evaluated by staining the vasculature via in vivo BS-1 lectin perfusion before killing, and then quantifying the number of BS-1 lectin–positive (brown) vessels (AMD: n = 12; saline: n = 5). HPF, high-power field. *, P < 0.05; **, P < 0.03.

Continuous AMD3100 infusion increases adverse cardiac remodeling, decreases capillary density, and impairs BM EPC incorporation. (A and B) WT mice or (C) WT mice transplanted with Tie2-LacZ BM received single injections of AMD3100 (AMD) or were implanted with osmotic pumps that continuously infused AMD3100 or saline for 2 weeks after MI. (A) Fibrosis was quantified as the ratio of the length of fibrosis to the LV circumference (n = 5); fibrotic tissue appears blue. (B) Capillary density was evaluated by staining the vasculature via in vivo BS-1 lectin perfusion before killing, and then quantifying the number of BS-1-lectin–positive vessels (green) (n = 5). (C) BM EPC incorporation in the ischemic border zone on day 14 after MI was quantified as the number of β-gal-positive cells (red) (n = 3). HPF, high-power field; i, ischemic tissue; n, nonischemic tissue. *, P < 0.05; ***, P < 0.01.

The beneficial effects associated with a single injection of AMD3100 are dependent on BM MMP-9 expression, but not on MMP-9 expression in the ischemic tissue. MMP-9 KO mice transplanted with WT BM and WT mice transplanted with MMP-9 KO BM received single s.c. injections of AMD3100 (AMD) or saline 1 h after MI. (A and B) PB EPC counts were evaluated via flow-cytometry analysis for coexpression of Sca1 and Flk1. (A) n = 3 at all time points. (B) Before MI, n = 9; day 1, n = 3 for AMD and 4 for saline; day 3, n = 3; day 7, n = 4 for AMD and 3 for saline; day 28, n = 4. (C and D) LV fractional shortening was evaluated echocardiographically. (C) Before MI, n = 3; day 7, n = 5; day 14, n = 10; day 28, n = 11 for AMD and 9 for saline. (D) Before MI, n = 5; day 7, n = 4; day 14, n = 10; day 28, n = 10 for AMD and 11 for saline. (E) Fibrosis on day 28 after MI was quantified as the ratio of the length of fibrosis to the LV circumference. MMP-9 KO/WT_BM, n = 7 for saline and 10 for AMD; WT/MMP-9 KO_BM, n = 11 for saline and n = 6 for AMD. (F) Capillary density on day 28 was evaluated by staining the vasculature via in-vivo BS-1 lectin perfusion before killing, and then quantifying the number of BS-1 lectin–positive vessels. MMP-9 KO/WT_BM, n = 6 for saline and 7 for AMD; WT/MMP-9 KO_BM, n = 14 for saline and 7 for AMD. HPF, high-power field. *, P < 0.05; **, P < 0.03; ***, P < 0.01; NS, not significant.