Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo.
OBJECTIVE
Our laboratory and others recently provided evidence indicating that endothelial progenitor cells (EPCs) participate in postnatal neovascularization. However, the extent to which EPCs contribute to adult neovascularization remains unclear. To address this issue, we investigated the quantitative contribution of EPCs to newly formed vascular structures in an in vivo Matrigel plug assay and corneal micropocket assay.


METHODS
Lethally irradiated FVB mice were transplanted with bone marrow (BM) mononuclear cells from transgenic mice constitutively expressing beta-galactosidase (beta-gal) encoded by the lacZ gene regulated by an endothelial-specific tie-2 promoter. Reconstitution of the transplanted BM leads to the expression of lacZ in mice, which is restricted to BM cells expressing tie-2.


RESULTS
Four weeks after BM transplantation (BMT), tie-2/lacZ/BMT mice were implanted with either Matrigel containing fibroblast growth factor-2 subcutaneously or with a vascular endothelial growth factor pellet into the cornea. After 7 days, the Matrigel plug or the cornea was removed and analyzed by X-gal staining or immunostaining for beta-gal. X-gal staining of the Matrigel plug identified 5.7% +/- 1.2% of endothelial cells (ECs) as cells originated from BM-derived EPCs, whereas the more sensitive technique of immunofluorescence identified 26.5% +/- 0.9% of ECs. Similarly, EPC-derived cells comprised 5.0% +/- 2.4% and 17.7% +/- 3.6% of the ECs in corneal neovascularization identified by X-gal staining and immunohistochemistry, respectively. Ki67 staining of the corneal tissue documented that the majority of EPC-derived cells were actively proliferating in situ.


CONCLUSIONS
These findings suggest that BM-derived EPCs make a significant contribution to angiogenic growth factor-induced neovascularization that may account for up to 26% of all ECs.
Murayama
Toshinori
T
Division of Cardiovascular Research, Department of Medicine, St. Elizabeth's Medical Center of Boston, Tufts University School of Medicine, Brighton, Mass 02135-2997, USA.
Tepper
Oren M
OM
Silver
Marcy
M
Ma
Hong
H
Losordo
Douglas W
DW
Isner
Jeffery M
JM
Asahara
Takayuki
T
Kalka
Christoph
C
D000818:Animals; D015415:Biomarkers; D016026:Bone Marrow Transplantation; D002455:Cell Division; D003315:Cornea / Q000098:blood supply*; D004343:Drug Implants; D016228:Endothelial Growth Factors / Q000008:administration & dosage / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000008:administration & dosage / Q000494:pharmacology*; D017930:Genes, Reporter; D006801:Humans; D019394:Ki-67 Antigen / Q000032:analysis; D007763:Lac Operon; D008222:Lymphokines / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D008822:Mice, Transgenic; D009363:Neoplasm Proteins / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000139:chemically induced*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D011828:Radiation Chimera; D042787:Receptor, TIE-2; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D015415:Biomarkers; D004343:Drug Implants; D016228:Endothelial Growth Factors; D019394:Ki-67 Antigen; D008222:Lymphokines; C105373:MEN1 protein, human; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2; D042787:Receptor, TIE-2