P504Effects of cardiac nerve growth factor overexpression on bone marrow and peripheral blood progenitor cells after myocardial infarction

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Abstract

Purpose

Mobilization of progenitor cells (PCs) from the bone marrow (BM) requires many complex signals, including activation of osteoclasts and release of proteolytic enzymes that allows PCs to be detached from the BM endosteal niche and to egress into the circulation. We recently demonstrated that nerve growth factor (NGF) gene transfer to the mouse peri-infarct myocardium promotes the expansion of cardiac lin-/c-kit + PCs. Here, we investigate whether NGF can additionally stimulate the mobilization of BM-derived PCs.

Methods

Myocardial infarction (MI) was induced in male CD1 mice and the peri-infarct zone was injected with adenoviruses carrying the human NGF gene (Ad.hNGF, 10^8 p.f.u.) or an empty vector (Ad.Null) as control. Sham-operated mice receiving Ad.Null were used as reference. Bones (femurs and tibias) were collected at 1, 2 and 3 days post-MI for histological evaluations. A transwell in vitro migration assay was performed to evaluate the migratory capacity of either mouse BM cells or human peripheral blood (PB) mononuclear cells (MNCs) isolated from acute MI (aMI) patients towards NGF.

Results

Immunohistochemical analyses showed that intra-myocardial Ad.hNGF increases the number of TRAP + activated osteoclasts lining the bone endosteal surface (27.3 ± 11 and 24.3 ± 10 vs 11.4 ± 6 and 13.7 ± 8 cells/cm2 in Ad.Null at 1 and 2d post-MI, respectively; P < 0.05 for both comparisons). MMP9 + cells were also increased in the BM of Ad.hNGF-treated mice at 2 and 3d post-MI (P < 0.05 vs MI/Ad.Null for both comparisons). In addition, at 3d post-MI, Ad.hNGF increased the number of CD45 + /c-kit + PCs in the BM endosteal niche (2.0 ± 0.6 vs 1.5 ± 0.2 cell/10^5 total BM cells in MI/Ad.Null) and BM parenchyma (39.4 ± 9 vs 26 ± 5 cell/10^5, P < 0.05 for both comparisons). Interestingly, transwell in vitro migration assay showed that NGF (50 and 100 ng/mL) attracted both mouse BM cells and human PB-MNCs. Moreover, migration of human PB-MNCs towards NGF enriched for both TrkA + cells and ckit + cells (P < 0.05 for both comparison vs 0.1% BSA). Finally, FACS analyses of PB-MNCs isolated from aMI patients showed an increased number of CD34 + /c-kit + cells co-expressing the NGF high-affinity TrkA receptor (P < 0.05 vs healthy controls).

Conclusions

These results provide evidence that post-MI intra-myocardial NGF gene therapy induces activation of osteoclasts and proteolytic enzymes in the mouse BM, thus increasing the number of ckit + PCs in the BM compartments. The increased number of circulating CD34 + /c-kit + PCs expressing the TrkA receptor in aMI patients might suggest that NGF is a relevant factor for PCs mobilization.

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