A reductionist PEG-based system to elucidate the role of macrophages in vascular development

摘要

Introduction: Biomaterial systems which provide a high degree of control over cell-matrix interactions, such as regulating cell-receptor binding events or limiting proteolytic degradation sensitivity, offer an attractive foundation to study complex cell-cell interactions during tissue development. Moreover, recent work has hypothesized that macrophages can play a beneficial role during vasculature development as these cells are found at sites of active angiogenesis. Thus, there is a need to better characterize the effects of macrophages during vessel development. Through the use of our reductionist biomimetic hydrogel, we have created a biomaterial system which allows the study of macrophages during vessel development within a tightly controlled matrix. Within this system, we have demonstrated macrophage interactions with endothelial cells vary over time and speculate this variation is dependent on macrophage function during vessel development. Materials and Methods: Mouse macrophages and endothelial cells (ECs) are used in this work. To create a biomimetic hydrogel, two peptides are incorporated: RGDS, for cell adhesion and GGGPQGIWGQGK, (abbreviated as "PQ") for cell-mediated degradation. Incorporation of these peptides occurs via primary amine substitution when acryl-PEG-SVA is reacted with RGDS/PQ in HEPES buffer. The resulting product is PEG-RGDS or PEG-PQ-PEG. A precursor solution containing these polymers and Eosin Y is combined with macrophages and ECs. Gelation occurs upon exposure to white light. Immunocytochemistry using primary antibodies CD31, iNOS, and MMR were used. The Zeiss 510 Inverted Confocal was used for imaging. Results and Discussion: Within our PEG-based hydrogels, macrophages interacted specifically with ECs. Moreover, the macrophage interactions varied as a function of time. These interactions occurred at a greater frequency during earlier stages of vessel development than at later stages suggesting macrophages altered their function as the vessel structures developed. Additionally, two major types of interplay were noticed during earlier time points: interactions with tip cells and interactions that mimicked pericyte support cells (in which alignment with the tubule occurred) (fig. 1 A). Towards later stages, the macrophage interactions were primarily limited to alignment (fig. 1C). We speculate that this shift in interactions indicated an altered function of the macrophage during vessel development. Conclusion: Through the use of our PEG-based biomimetic platform, a reductionist in vitro system capable of studying macrophage interactions with ECs was created. Over time, a shift is seen in macrophage interactions indicating an interactive role in vessel development. This reductionist biomaterial model helps clarify in vivo characteristics of macrophage-endothelial cell interactions by providing a platform with specific control over cell-matrix interactions. Additionally, this work presents a foundation to explore regenerative aspects of macrophages within vasculature.