Biology subjects

Watkins, C. M.

Publications and source records attributed to Watkins, C. M..

2 recordsLinked to original sources

bioRxiv2025

Tuning Scaffold Degradation with Non-Natural Peptidomimetics to Control Human Umbilical Vein Endothelial Cell Morphology and Vessel Formation

The in vitro vascularization of 3D tissue constructs, such as hydrogels, remains a paramount challenge in tissue engineering. Extracellular matrix degradation and remodeling are key parts of the vascularization process; however, it is difficult to isolate the effects of degradability in both natural and synthetic matrix models. Naturally-derived matrices typically couple degradability to other material properties, whereas synthetic matrices rely on short peptide sequences to impart degradability, which typically exhibit substrate overlap to many proteases. Here, we present a method to independently and broadly tune 3D hydrogel degradation using crosslinkers with non-natural peptoid (N-substituted glycine) substitutions. Increased peptoid substitutions reduced hydrogel degradability to collagenases without altering hydrogel modulus, swelling ratio, or crosslinker length. Using this approach, human umbilical vein endothelial cells (HUVECs) encapsulated in more degradable hydrogels proliferated more, formed more vessels, exhibited higher metabolic activity, and secreted more extracellular matrix than HUVECs encapsulated in less degradable or non-degradable hydrogels. Interestingly, HUVECs encapsulated in the least degradable hydrogels secreted significantly higher matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) than HUVECs encapsulated in the most degradable hydrogels, suggesting higher MMP secretion to compensate for the reduced matrix degradability. Overall, this work highlights the importance of protease-mediated remodeling on vascularization and suggests that peptoid substitutions are effective for tuning hydrogel degradability for a variety of 3D cell applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/684851v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@c22b3corg.highwire.dtl.DTLVardef@1a4ae14org.highwire.dtl.DTLVardef@a0ad1forg.highwire.dtl.DTLVardef@c5def5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Fluorescent peptomer substrates for differential degradation by metalloproteases

Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide-peptoid hybrids) to probe the impact of non-natural residues on MMP specificity for a MMP peptide consensus sequence. Peptoids are non-natural, N-substituted glycines with a large side chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offer N-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography-mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well-tolerated in the P3 and P3 substrate sites and that the identity of the peptoid in these sites displays moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with sidechain identity than sidechain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as "fingerprints" for the proteases and yielded separation by multivariate data analysis. To further demonstrate practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.

biochemistry↗