Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization

Abstract

Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced. Ex vivo expanded hEPCs may thus have utility as a "supply-side" strategy for therapeutic neovascularization.

Figures

Figure 1

In vitro differentiation of peripheral mononuclear cell subpopulation into EPCs. Endocytosis of acLDL (red fluorescence) and binding to UEA (green fluorescence) identified EPCs. (A) ×10 magnification; (B) ×40 magnification.

Figure 2

Ex vivo expansion of endothelial progenitor cells. After 7 days in culture, among 85% antigenically identified cells, >70% expressed endothelial-specific antigens KDR, VE-cadherin, and CD31, whereas contamination by hematopoietic lineage cells was not significant.

Figure 3

(A) Representative results of laser Doppler perfusion imaging (LDPI) performed immediately after development of hindlimb ischemia and again 28 days later. Examination of hindlimb perfusion by LDPI disclosed profound improvement in the limb perfusion within 28 days after hEPC transplantation. (B) Quantitative analysis of perfusion recovery measured by LDPI. At 3 days postoperatively, limb perfusion was severely reduced in all three groups. Over the subsequent 28 days, however, substantial blood flow recovery in mice receiving hEPCs returned perfusion of the ischemic hindlimb to levels that were similar to those recorded in the contralateral nonischemic hindlimb. In contrast, limb perfusion remained markedly depressed in mice receiving either HMVECs or culture media.

Figure 4

Heterologous hEPCs incorporate into sites of neovascularization. Phase contrast photomicrographs show anatomic localization of DiI-labeled hEPCs incorporated (red, arrowheads) into foci of neovascularization in ischemic mouse muscle stained with BS1-lectin specific for mouse EC (green) (A) and stained against mouse CD31 (PECAM) (B). Human-specific EC marker UEA-I lectin (C) and human CD31 identified transplanted hEPCs as endothelial lineage cells (green, arrows) (E). (D and F) Equivalent phase contrast pictures show anatomic localization of DiI-labeled hEPCs (red) in mouse muscle.

Figure 5

Histologic evaluation of neovascularization in ischemic hindlimb. Capillary density was significantly increased in mice receiving hEPC vs. culture media at 1 wk (P < 0.001 hEPC vs. culture media; P < 0.01 hEPC vs. HMVEC); at 2 wk (P < 0.0005 hEPC vs. culture media; P < 0.0001 hEPC vs. HMVEC); and at 4 wk postoperatively (P < 0.002 hEPC vs. culture media; P < 0.0003 hEPC vs. HMVEC). There were no statistically significant differences in capillary density between groups receiving culture media and HMVEC at the observed time points.

Figure 6

Administration of hEPCs leads to reduced limb loss and increased limb salvage. (A) Representative macroscopic photographs of mice showing three different outcomes observed in the study. (Left) Autoamputation, characterized by loss of the ischemic hindlimb. (Middle) Severe foot necrosis. (Right) Most favorable outcome, complete salvage of ischemic hindlimb with intact function. (B) Percent distribution of above outcomes among mice receiving control media, HMVECs, and hEPCs. The differences in outcome were statistically significant (hEPC vs. HMVEC, P = 0.006; hEPC vs. culture media, P = 0.003, HMVEC vs. culture media, P = NS).