Fig. 3

Heterologous (A-L), homologous (M), or autologous (N, O) EC progenitors incorporate into sites of angiogenesis in vivo. (A, B) CD34⁺ mononuclear peripheral blood cells (MB^CD34+) (red, arrows), labeled with the floursecent dye DiI, between skeletal myocytes (M), including necrotic (N) myocytes 1 week after injection; most are colabeled with CD31 (green, arrows). Note pre-existing artery (small A), identified as CD31-positive, but DiI-negative. (C and D) Evidence of proliferative activity among several DiI-labeled MB^CD34+-derived cells (red, arrows), indicated by coimmunostaining for Ki67 antibody (green). Proliferative activity is also seen among DiI-negative, Ki67-positive capillary ECs (arrowheads); both cell types comprise neovasculature. (E) DiI (red) and CD31 (green) in capillary ECs (arrow, arrowhead) between skeletal myocytes, photographed through double filter 1 week after DiI-labeled MB^CD34+ injection. (F) Single green filter shows CD31 (green) expression in DiI-labeled capillary ECs, integrated into capillary with native (DiI-negative, CD31-positive) ECs (arrow, arrowhead). (G) Immunostaining 1 week after MB^CD34+ injection showing capillaries comprised of DiI-labeled MB^CD34+-derived cells expressing Tie-2 receptor (green). Several MB^CD34+-derived cells (arrows) are Tie-2 positive and are integrated with some Tie-2-positive host capillary cells (arrowheads) identified by the absence of red fluorescence. (H) Phase-contrast photomicrograph of same tissue section shown in G indicates corresponding DiI-labeled (arrows) and -unlabeled (arrowheads) capillary ECs. (I, J) Six weeks after administration, MB^CD34+-derived cells (red) colabel for CD31 in capillaries between preserved skeletal myocytes. (K, L) One week after injection of MB^CD34−, isolated MB^CD34−-derived cells (red, arrows) are observed between myocytes, but do not express CD31. (M) Immunostaining of β-galactosidase in tissue section harvested from ischemic muscle of B6, 129 mice 4 weeks after administration of MB^{Flk-l +} isolated from transgenic mice constitutively expressing β-gal. (Flk-1 cell isolation was used for selection of EC progenitors because of a lack of a suitable anti-mouse CD34 antibody.) Cells overexpressing β-gal (arrows) have been incorporated into capillaries and small arteries; these cells were identified as ECs by anti-CD31 and anti-BS-1 lectin. (N, O) Sections of muscles harvested from rabbit ischemic hindlimb 4 weeks after administration of autologous MB^CD34+. Red fluorescence indicates localization of MB^CD34+-derived cells in capillaries, seen (arrows) in phase-contrast photomicrograph (O). Each scale bar indicates 50 µm.