Unraveling cross-talk between macrophages and (myo)fibroblasts for in situ tissue engineering

General introduction

Arteriovenous (AV)-grafts ensure vascular access for hemodialysis treatment in end-stage renal disease patients. However, the currently used AV-grafts easily occlude due to thrombosis and intragraft neo-intimal hyperplasia. In situ tissue engineering is a promising new approach to reduce this vascular access dysfunction. We aim to develop an off-the-shelf, synthetic, biodegradable, AV-graft that will develop in vivo into a living, self-healing vascular access graft.

Upon implantation of the graft, host immune cells infiltrate the fibrous graft. Especially macrophages are important in attracting (myo)fibroblasts to initiate tissue formation. The interplay between macrophages and (myo)fibroblasts leads to either healthy, functional tissue formation or adverse remodeling. Although the feasibility of vascular in situ tissue engineering has been proven, the endogenous remodeling processes are not well understood. The aim of this project is to gain insight in the scaffold-induced regenerative processes.

BEP Project 1: Migration of (myo)fibroblasts towards chemokines secreted by macrophages

Histology reveals that the intimal hyperplasia in PTFE AV-shunts is mainly composed of myofibroblasts that migrate towards the intima of the injured vessel 1–3. We hypothesize that the migration of myofibroblasts is dependent on macrophage polarization state. Therefore, the goal of this project is to elucidate the effect of macrophage conditioned medium on myofibroblasts migration. Monocytes will be isolated and differentiated into macrophages. Macrophage polarization will be induced and the medium supernatant from these macrophage cultures will be collected. Myofibroblasts (human vena saphena cells-HVSC) will be exposed to the conditioned media and migration will be studied using a Transwell migration assay/Boyden chamber assay and/or scratch assay.

Employed techniques:

• Human PBMC isolation from whole blood
• Monocyte isolation
• Cell culturing
• Histology
• Cell migration assays (scratch assay, boyden chamber)

BEP Project 2: Differentiation of (myo)fibroblasts by macrophage-secreted cytokines

The outcome of wound healing and regeneration is dependent on myofibroblasts activation state. We hypothesize that this state is dependent on the inflammatory milieu that is mediated by the macrophages. Therefore the goal of this project is to study the differentiation of (myo)fibroblasts by macrophage-secreted cytokines. Fibroblasts are incubated for different timeframes with supernatant medium derived from polarized macrophage-culture. Gene expression is determined of main myofibroblast markers (alpha smooth muscle actin, fibroblast specific protein) as well as the expression of ECM core components and ECM remodeling enzymes (collagen I and III, matrix metalloproteinases, tissue inhibitors or metalloproteinases).

Employed techniques:

• PBMC isolation from whole blood
• Monocyte isolation
• Cell culturing
• qPCR
• Stainings

References

1. Brahmbhatt A, Remuzzi A, Franzoni M, Misra S. The molecular mechanisms of hemodialysis vascular access failure. Kidney Int. 2016;89(2):303-316.
2. Riella MC, Roy-Chaudhury P. Vascular access in haemodialysis: strengthening the Achilles’ heel. Nat Rev Nephrol. 2013;9(6):348-357.
3. Li L, Terry CM, Blumenthal DK, et al. Cellular and morphological changes during neointimal hyperplasia development in a porcine arteriovenous graft model. Nephrol Dial Transplant. 2007;22(11):3139-3146.
4. Kramer N, Walzl A, Unger C, et al. In vitro cell migration and invasion assays. Mutat Res Mutat Res. 2013;752(1):10-24.
5. Stenmark KR, Yeager ME, El Kasmi KC, et al. The adventitia: essential regulator of vascular wall structure and function. Annu Rev Physiol. 2013;75:23-47.