Human Adventitial Fibroblast Phenotype Depends on the Progression of Changes in Substrate Stiffness

Abstract

Adventitial fibroblasts (AFs) are major contributors to vascular remodeling and maladaptive cascades associated with arterial disease, where AFs both contribute to and respond to alterations in their surrounding matrix. The relationships between matrix modulus and human aortic AF (AoAF) function are investigated using poly(ethylene glycol)‐based hydrogels designed with matrix metalloproteinase (MMP)‐sensitive and integrin‐binding peptides. Initial equilibrium shear storage moduli for the substrates examined are 0.33, 1.42, and 2.90 kPa; after 42 days of culture, all hydrogels exhibit similar storage moduli (0.3–0.7 kPa) regardless of initial modulus, with encapsulated AoAFs spreading and proliferating. In 10 and 7.5 wt% hydrogels, modulus decreases monotonically throughout culture; however, in 5 wt% hydrogels, modulus increases after an initial 7 days of culture, accompanied by an increase in myofibroblast transdifferentiation and expression of collagen I and III through day 28. Thereafter, significant reductions in both collagens occur, with increased MMP‐9 and decreased tissue inhibitor of metalloproteinase‐1/‐2 production. Releasing cytoskeletal tension or inhibiting cellular protein secretion in 5 wt% hydrogels block the stiffening of the polymer matrix. Results indicate that encapsulated AoAFs initiate cell‐mediated matrix remodeling and demonstrate the utility of dynamic 3D systems to elucidate the complex interactions between cell behavior and substrate properties.

Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-β/ALK5 signaling

“…Although we observed a marked decrease in MVEC viability over time in the RGD-containing hydrogels (Fig 1, S4 Fig), this phenomenon was similarly observed for other matrices evaluated in this study and such decreases are consistent with previously reported behaviors of MVECs in 3D monoculture [51]. As we and others have utilized PEG-based materials to support growth of various vascular cell types in monoculture with a high degree of viability in each case [33,34,52], it is likely that MVECs simply require the support of mural cells, like AoAFs. Indeed, our studies unambiguously demonstrate the beneficial impact of AoAFs in sustaining MVEC viability and initiating cell spreading (Fig 2).…”

“…Interestingly, not all AoAFs aligned adjacently to MVEC tubules and a portion of these unaligned cells also expressed αSMA, suggesting that the matrix itself stimulates transdifferentiation of a small percentage of AoAFs towards a myofibroblastic phenotype. Indeed, we have previously observed transdifferentiation of AoAFs into myofibroblasts in 3D PEG-based hydrogels [34,36]. As pericyte populations share developmental origins with fibroblasts [63,64] and are able to transition into myofibroblasts under pathological conditions [47], the expression of αSMA by both perivascular cells and myofibroblasts is not surprising.…”

“…The bis-maleimide (MI), end-functionalized, enzymatically-degradable crosslinker peptide (MI-GGPQGIRGQGK(MI)G; PQ-MI 2 ) and monomaleimide-functionalized RGD peptide (MI-KGGRGDSPG; RGD-MI) were synthesized using a Focus Xi automated peptide synthesizer (AAPPTec, Louisville, KY) with standard Fmoc chemistry, at 0.5 mmol scale using Rink-Amide resin (ChemPep Inc, Wellington, FL), as in prior studies [34,36]. PQ-MI 2 and RGD-MI were synthesized with Fmoc-Lys(Dde)-OH (ChemPep Inc).…”

“…In particular, poly(ethylene glycol) (PEG)-based hydrogels are biologically inert and serve as a highly tailorable blank canvas while also offering initial autonomous control over mechanical and biochemical properties [28]. Importantly, PEG-based substrates have been fabricated to comprise cues important for supporting vasculogenesis and angiogenesis [29,30], to study cell-cell and cell-ECM interactions [31,32], and to investigate important signaling pathways involved in vascular cell function [33,34].…”

Colonization versus encapsulation in cell-laden materials design: porosity and process biocompatibility determine cellularization pathways

“…In this sense, the development of relevant cellularization techniques as discussed above is essential to maximize their physiological relevance. Nowadays, these materials are obtained using colonization of hydrogels [252] or three-dimensional printed constructs [253], or by cell encapsulation in hydrogels [254,255] with tuneable mechanical properties [252,256,257], three-dimensional printed gels [258] or microbeads [259]. Moreover, recent bioinspired scaffolds based on cell encapsulation in hydrogels have enabled to model angiogenesis [260], the osteochondral environment [261], bone [262], brain [263], gastric mucosa [264], and the liver [265].…”