Type I Collagen Matrix Activates the Hedgehog Pathway in CD34+ Cells and Enhances Their Function

Background. Acute myocardial infarction (MI) is a leading cause of death in the western world. Cell therapy using the CD34⁺ fraction of circulating endothelial progenitor cells (EPCs) is one approach under investigation for restoring blood flow and function to the ischemic heart. However, the number of CD34⁺ circulating cells is inversely proportional to the severity of cardiovascular disease and age. Therefore, there is a need to increase their numbers and/or function for therapy. One possibility is to deliver the therapeutic cells in combination with a biomaterial. We have previously demonstrated that the therapeutic potency of EPCs is improved by their culture on a type I collagen-based matrix. The aim of this study was to investigate the ability and mechanism through which the collagen matrix may enhance CD34⁺ cell function.

Methods/Results. Human blood from healthy donors was used to generate EPCs by 4-day culture on fibronectin. CD34⁺ cells were sorted from the heterogeneous EPC population and re-seeded onto fibronectin or collagen matrix for up to another 4 days and then used for in vitro experiments. CD34⁺ cells from matrix culture demonstrated increased migration towards VEGF (by 3.2-fold) in a Boyden chamber assay versus fibronectin-cultured cells (p=0.03). Also, the number of CD34⁺ cells derived from matrix that incorporated into capillary-like structures in an in vitro angiogenic assay was 3.1-fold greater than  cells from fibronectin cultures. Furthermore, survival upon exposure to serum deprivation and hypoxia for 48h was enhanced by approximately 15% in matrix-derived CD34⁺ cells compared to fibronectin (p=0.02). In contrast, the adhesion potential of CD34⁺ cells was not affected by matrix culture, as the ability to adhere to fibronectin or collagen matrix substrates was not different between matrix- and fibronectin-derived cells. Other studies have identified the importance of the Hedgehog (Hh) signaling pathway in the therapeutic potency of CD34⁺ cells. Therefore, we assessed the expression of known Hh pathway members by performing qRT-PCR. CD34⁺ cells within unsorted EPCs cultured on matrix for up to 96h had increased mRNA expression of Gli1 (28.9-fold), CyclinD1 (10.4-fold) and Hes1 (6.2-fold), compared to fibronectin-cultured cells (p< 0.05 for each).

Conclusion. In the present study, we demonstrated that the interaction of CD34⁺ progenitor cells with our collagen matrix enhances their migratory potential, angiogenic properties and survival. Our data also indicates the possible involvement of the Hh pathway in these functional changes as key downstream targets are up-regulated in matrix-derived CD34⁺ cells. This work provides insight into novel mechanisms for the observed matrix-enhanced function of therapeutic CD34⁺ cells.