Deciphering the relative roles of matrix metalloproteinase‐ and plasmin‐mediated matrix degradation during capillary morphogenesis using engineered hydrogels

Abstract

Extracellular matrix (ECM) remodeling is essential for the process of capillary morphogenesis. Here we employed synthetic poly(ethylene glycol) (PEG) hydrogels engineered with proteolytic specificity to either matrix metalloproteinases (MMPs), plasmin, or both to investigate the relative contributions of MMP‐ and plasmin‐mediated ECM remodeling to vessel formation in a 3D‐model of capillary self‐assembly analogous to vasculogenesis. We first demonstrated a role for both MMP‐ and plasmin‐mediated mechanisms of ECM remodeling in an endothelial‐fibroblast co‐culture model of vasculogenesis in fibrin hydrogels using inhibitors of MMPs and plasmin. When this co‐culture model was employed in engineered PEG hydrogels with selective protease sensitivity, we observed robust capillary morphogenesis only in MMP‐sensitive matrices. Fibroblast spreading in plasmin‐selective hydrogels confirmed this difference was due to protease preference by endothelial cells, not due to limitations of the matrix itself. In hydrogels engineered with crosslinks that were dually susceptible to MMPs and plasmin, capillary morphogenesis was unchanged. These findings highlight the critical importance of MMP‐mediated degradation during vasculogenesis and provide strong evidence to justify the preferential selection of MMP‐degradable peptide crosslinkers in synthetic hydrogels used to study vascular morphogenesis and promote vascularization. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2507–2516, 2019.

Hydrogel Network Dynamics Regulate Vascular Morphogenesis

“…While multiple studies have demonstrated the safety and cellbased formation of vasculature by UV crosslinked hydrogels (Beamish et al, 2019;Chen et al, 2012;Moon et al, 2010), we carefully examined and compared the cytotoxicity of D-hydrogels and N-hydrogels to eliminate the possible impact on ECFCs. No significant change in cell viability was observed in samples treated with various gelation precursors of D-and (F and G) Significant increase in stress relaxation time over 3 days in culture in EGM-2 (F) but half-stress relaxation time, t 1/2 , is maintained lower than in N-hydrogel along the 3-day culture period (G).…”

“…For example, Moon et al (2010) developed protease-sensitive poly(ethylene glycol) (PEG) hydrogels with selected moduli for blood vessel formation from human umbilical vein endothelial cells (ECs). Beamish et al (2019) engineered another PEG-based hydrogel with MMP-sensitive crosslinkers for capillary morphogenesis from encapsulated ECs. These studies have revealed that with a suitable stiffness, the robust in vitro vascular bed could be engineered in the synthetic elastic hydrogels.…”

Cell-mediated matrix stiffening accompanies capillary morphogenesis in ultra-soft amorphous hydrogels

“…All reagents were obtained from Thermo Fisher Scientific (Waltham, MA) unless specified. Endothelial and stromal cell sources in this study were chosen to provide continuity with our prior work [5,19]. Human umbilical vein endothelial cells (ECs) were harvested from fresh umbilical cords (obtained from the University of Michigan's Mott's Children's Hospital via an IRB-exempt process, without any identifying information provided to the researchers) and cultured in fully supplemented Vasculife VEGF Endothelial Medium (Lifeline Cell Technology LLC, Frederick, MD) as previously described [24].…”

“…Hydrogels were functionalized with CGRGDS peptide ("RGD", Genscript, Piscataway, NJ) and crosslinked with Ac-GCRDVPMS↓MRGGDRCG-NH2 ("VPMS", Genscript, cleavage site indicated by ↓), which contains an N-terminal acetylation and a C-terminal amidation. These peptides were chosen to provide continuity with our previous work [5]. All reagents were prepared in batches of single-use aliquots.…”

“…Synthetic, proteolytically degradable poly(ethylene glycol) (PEG) hydrogel ECMs also have attracted interest because of their biocompatibility and versatile macromer chemistry [4]. However, the extent of capillary morphogenesis is significantly restricted in PEG hydrogels compared with biologically-derived ECMs [5][6][7]. To overcome this limitation, a range of strategies have emerged to modulate initial hydrogel properties [8,9] and to incorporate proangiogenic biological cues into PEG hydrogels to enhance their angiogenic potential [7,[10][11][12].…”