P297Function-based enrichment of pro-angiogenic cells for cardiovascular repair

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Abstract

Purpose

Cell therapy holds great promise for the treatment of cardiovascular disease. However, all current strategies including those based on immunoselection do not allow the separation of viable from damaged cells as well as functionally competent from incompetent cells. Recently, we have developed a method to enrich for a subfraction of peripheral blood mononuclear cells (PB-MNC) on the basis of their ability to migrate towards the B2R agonist bradykinin (BK). Here, we propose to use migration as a rapid method for selection of functional and possibly therapeutically advanced cells for treatment of cardiovascular disease.

Methods

MNC extracted from PB and used for migration in modified Boyden chambers.

Results

We found that the migrating cell population (BKmig) is depleted for lymphocytes (0.6 ± 0.05-fold of PB-MNC, P < 0.001) and enriched for monocytes (3.7 ± 0.8-fold of PB-MNC, P < 0.001). In addition, migration towards BK allowed the marked enrichment for CD133 + and CD34 + circulating progenitor cells (CPC). Furthermore, significantly more migrating CPC (29.9 ± 8.1%) than non-migrating CPC (6.5 ± 1.9%, P=0.01 vs. BKmig) express the receptor for SDF-1, CXCR4, which is involved in the recruitment of pro-angiogenic cells during ischaemia. BKmig cells furthermore exhibit a variety of functional characteristics, indicating their regenerative and pro-angiogenic potential, such as their increased generation of nitric oxide (NO) and reduced production of superoxide (O2-*) in comparison with BKnon cells. BKmig cells released larger amounts of angiogenic factors, whereas BKnon cells generated more RANTES, a chemokine that plays an active role in recruiting leukocytes to inflammatory sites. Consistently, BKmig cells supported the formation of network structures by mature endothelial cells (EC) in a matrigel assay (an in vitro model of angiogenesis) while BKnon did not. The non-migrating population harbours a three- to four-fold higher number of dead cells. This finding underlines another aspect of function-based cell enrichment: the depletion of apoptotic or necrotic cells, which might perpetuate the inflammatory response if they remain among the transplanted cells.

Discussion

MNC selected on the basis of their migratory capacity, exhibit cellular functions that are considered to support angiogenesis and tissue regeneration. This indicates that within the heterogeneous CPC population, whose overall function is reportedly impaired, a subset of progenitor cells retaining functional integrity can be selected by migration. Our results can have a significant impact in the optimization of cell therapy.

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