Fractal heterogeneity in minimal matrix models of scars modulates stiff-niche stem-cell responses via nuclear exit of a mechanorepressor

Abstract

Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that co-polymerization of collagen-I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fiber bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally – as in scars – while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the ‘scar marker’, smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, while cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a ‘mechanical memory’ of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.

Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo

“…Fibrotic tissue has a particularly heterogeneous collagenous matrix that often aligns with blood vessels and results from acute injury such as heart attack [36] as well as chronic diseases such as muscular dystrophy and liver cirrhosis [47]. Fibrosis is often referred to as a scar, which forms locally in most or all tissues of higher animals and is compositionally characterized by an abundance of crosslinked collagen-I fibers heterogeneously distributed within a fibrotic tissue.…”

“…Fibrosis is often referred to as a scar, which forms locally in most or all tissues of higher animals and is compositionally characterized by an abundance of crosslinked collagen-I fibers heterogeneously distributed within a fibrotic tissue. A scar tends to be locally stiff and long-lasting [36, 47]. Scar matrix seems to be made largely de novo , and a major role in the development of organ fibrosis has recently been ascribed to ubiquitous MSCs, which reside in perivascular niches of many organs including heart, liver, kidney, lung, and of course bone marrow [48].…”

“…To begin to clarify the effects of fibrotic, heterogeneous matrix on MSCs, minimal matrix models of scars (MMMS) were developed by mixing soluble collagen-I subunits with acrylamide monomers plus bisacrylamide crosslinker and then polymerizing the mix into a gel [47]. Upon initiation of polymerization, collagen-I fibers phase separate from pre-gelation clusters of PA, leading to highly branched fractal fiber bundles that segregate as islands heterogeneously entrapped at the subsurface of the hydrogel – as revealed by staining with a fibrosis dye commonly used in histology (Fig.…”

“…With the proper mixing ratios, a surface coverage of collagen fiber bundles of ∼30% approximates the extent of fibrosis seen, for example, in muscle cross sections [49, 50]. Importantly, collagen in the subsurface fiber bundles is not accessible for cell adhesion [47]. A uniform over-coating of matrix ligand is therefore provided for cell attachment, producing a highly heterogeneous structure with homogeneous ligand.…”

“…Although mechanisms of nuclear/cytoplasmic translocation of these factors remain unclear and do not seem to apply to normal tissue [7], these transcriptional regulators tend to favor entry into the nucleus in response to increased mechanical stress or stiffness in vitro . In contrast, rigid matrices promote nuclear exit of NKX-2.5, which is a crucial transcription factor in early cardiogenesis that also represses genes such as smooth muscle actin that contribute to high-tension, non-cardiogenic states [47]. Despite this handful of mechanosensitive signal transducers identified in recent years, much more can be learned about the mechanisms by which matrix stiffness and intracellular stress activate or repress specific transcriptional programs in cardiomyocyte maturation.…”