Proteome analysis of secretions from human monocyte-derived macrophages post-exposure to biomaterials and the effect of secretions on cardiac fibroblast fibrotic character.

The use of exogenous biomolecules (BM) for the purpose of repairing and regenerating damaged cardiac tissue can yield serious side effects if used for prolonged periods. As well, such strategies can be cost prohibitive depending on the regiment and period of time applied. Alternatively, autologous monocytes/monocyte-derived macrophages (MDM) can provide a viable path towards generating an endogenous source of stimulatory BM. Biomaterials are often considered as delivery vehicles to generate unique profiles of such BM in tissues or to deliver autologous cells, that can influence the nature of BM produced by the cells. MDM cultured on a degradable polar hydrophobic ionic (D-PHI) polyurethane has previously demonstrated a propensity to increase select anti-inflammatory cytokines, and therefore there is good rationale to further investigate a broader spectrum of the cells' BM in order to provide a more complete proteomic analysis of human MDM secretions induced by D-PHI. Further, it is of interest to assess the potential of such BM to influence cells involved in the reparative state of vital tissues such as those that affect cardiac cell function. Hence, this current study examines the proteomic profile of MDM secretions using mass spectrometry for the first time, along with ELISA, following their culture on D-PHI, and compares them to two important reference materials, poly(lactic-co-glycolic acid) (PLGA) and tissue culture polystyrene (TCPS). Secretions collected from D-PHI cultured MDM led to higher levels of regenerative BM, AGRN, TGFBI and ANXA5, but lower levels of pro-fibrotic BM, MMP7, IL-1β, IL-6 and TNFα,  when compared to MDM secretions collected from PLGA and TCPS. In the application to cardiac cell function, the secretion collected from D-PHI cultured MDM led to more human cardiac fibroblast (HCFs) migration. A lower collagen gel contraction induced by MDM secretions collected from D-PHI was supported by gene array analysis for human fibrosis-related genes. The implication of these findings is that more tailored biomaterials such as D-PHI, may lead to a lower pro-inflammatory phenotype of macrophages when used in cardiac tissue constructs, thereby enabling the development of vehicles for the delivery of interventional therapies, or be applied as coatings for sensor implants in cardiac tissue that minimize fibrosis. The general approach of using synthetic biomaterials in order to induce MDM secretions in a manner that will guide favorable regeneration will be critical in making the choice of biomaterials for tissue regeneration work in the future. STATEMENT OF SIGNIFICANCE: Immune modulation strategies currently applied in cardiac tissue repair are mainly based on the delivery of defined exogenous biomolecules. However, the use of such biomolecules may pose wide ranging systemic effects, thereby rendering them clinically less practical. The chemistry of biomaterials (used as a potential targeted delivery modality to circumvent the broad systemic effects of biomolecules) can not only affect acute and chronic toxicity but also alters the timeframe of the wound healing cascade. In this context, monocytes/monocyte-derive macrophages (MDM) can be harnessed as an immune modulating strategy to promote wound healing by an appropriate choice of the biomaterial. However, there are limited reports on the complete proteome analysis of MDM and their reaction of biomaterial related interventions on cardiac tissues and cells. No studies to date have demonstrated the complete proteome of MDM secretions when these cells were cultured on a non-traditional immune modulatory ionomeric polyurethane D-PHI film. This study demonstrated that MDM cultured on D-PHI expressed significantly higher levels of AGRN, TGFBI and ANXA5 but lower levels of MMP7, IL-1β, IL-6 and TNFα when compared to MDM cultured on a well-established degradable biomaterials in the medical field, e.g. PLGA and TCPS, which are often used as the relative standards for cell culture work in the biomaterials field. The implications of these findings have relevance to the repair of cardiac tissues. In another aspect of the work, human cardiac fibroblasts showed significantly lower contractility (low collagen gel contraction and low levels of ACTA2) when cultured in the presence of MDM secretions collected after culturing them on D-PHI compared to PLGA and TCPS. The findings place emphasis on the importance of making the choice of biomaterials for tissue engineering and regenerative medicine applied to their use in cardiac tissue repair.