Matrix metalloproteinase-mediated degradation governs angioarchitecture within poly(ethylene Glycol) hydrogels

Current tissue engineering strategies struggle to recapitulate the full diversity of tissue-specific vascular niches. Here, matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels are used to define design principles governing vascular morphogenesis. Vasculogenesis was modeled by encapsulating human umbilical vein endothelial cells (HUVECs) in PEG hydrogels co-cultured with human mesenchymal stem cells (hMSCs), enabling the formation of 3D vessel networks over two weeks. Angiogenesis was modeled by entrapping HUVEC-hMSC spheroids within PEG hydrogels and assessing endothelial sprouting behavior. In parallel, comparative kinetic parameters for peptide degradation were calculated for vasculogenic (k_vasc) and angiogenic (k_ang) conditions; k_vasc values for IPES↓LRAG (8.8 × 10^-7 hr^-1 cell^-1) and VPLS↓LYSG (1.4 × 10^-6 hr^-1 cell^-1) were three-to-five-fold higher than GPQG↓IWGQ (2.9 × 10^-7 hr^-1 cell^-1), indicating greater relative susceptibility to vasculogenic proteolysis. Consistent with these results, HUVECs encapsulated in hydrogels crosslinked with IPES↓LRAG or VPLS↓LYSG formed vessel networks with over two-fold greater diameter, length, and density compared to networks formed in GPQG↓IWGQ-crosslinked hydrogels under vasculogenic conditions, driven by greater overall degradability. In contrast, degradability influenced early sprouting dynamics under angiogenic conditions but did not affect overall network development. Additionally, vascular networks formed in non-degradable and GPQG↓IWGQ-crosslinked hydrogels exhibited compensatory upregulation of MMP and tissue inhibitor of metalloproteinase (TIMP) expression. Together, these findings elucidate how engineered extracellular matrix (eECM) degradability regulates vascular morphogenesis via cellular proteolytic feedback mechanisms, establishing a framework for designing eECM biomaterials that support tissue-specific microvascular modeling and improved vascular engraftment for regenerative medicine. STATEMENT OF SIGNIFICANCE: The microvasculature supplies nutrients and oxygen to nearly all tissues, yet current tissue engineering approaches struggle to recapitulate the diversity of tissue-specific vasculature. Poly(ethylene glycol) (PEG) hydrogels crosslinked with enzymatically degradable peptides were used to investigate how extracellular matrix degradability directs vessel development under vasculogenic and angiogenic conditions. Peptide crosslinkers with higher degradability promoted the formation of vessel networks with increased diameter, density, and length in vasculogenic conditions. Under angiogenic conditions, degradability regulated early sprouting without altering network properties. Additionally, the expression of matrix metalloproteinases and their inhibitors was elevated in slowly- or non-degradable hydrogels, suggesting a cellular attempt to overcome matrix constraints. These findings establish a biomaterial design strategy to engineer tissue-specific vasculature for disease modeling and regenerative medicine.