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Mesenchymal stromal cells (MSCs) can be isolated from various tissues of healthy or patient donors to be retransplanted in cell therapies. Because the number of MSCs obtained from biopsies is typically too low for direct clinical application, MSC expansion in cell culture is required. However, ex vivo amplification often reduces the desired MSC regenerative potential and enhances undesired traits, such as activation into fibrogenic myofibroblasts. Transiently activated myofibroblasts restore tissue integrity after organ injury by producing and contracting extracellular matrix into scar tissue. In contrast, persistent myofibroblasts cause excessive scarring—called fibrosis—that destroys organ function. In this review, we focus on the relevance and molecular mechanisms of myofibroblast activation upon contact with stiff cell culture plastic or recipient scar tissue, such as hypertrophic scars of large skin burns. We discuss cell mechanoperception mechanisms such as integrins and stretch-activated channels, mechanotransduction through the contractile actin cytoskeleton, and conversion of mechanical signals into transcriptional programs via mechanosensitive co-transcription factors, such as YAP, TAZ, and MRTF. We further elaborate how prolonged mechanical stress can create persistent myofibroblast memory by direct mechanotransduction to the nucleus that can evoke lasting epigenetic modifications at the DNA level, such as histone methylation and acetylation. We conclude by projecting how cell culture mechanics can be modulated to generate MSCs, which epigenetically protected against myofibroblast activation and transport desired regeneration potential to the recipient tissue environment in clinical therapies. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis is a key aspect of heart failure, leading to reduced ventricular compliance and impaired electrical conduction in the myocardium. Various pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite growing evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Given the distinct natures of the ventricles, we hypothesized that LV- and RV-derived CFs would display baseline transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA sequencing of CFs isolated from healthy murine left and right ventricles indicated that LV-derived CFs may be further along the myofibroblast transdifferentiation trajectory than cells isolated from the RV. Single-cell RNA-sequencing analysis of the two populations confirmed that Postn+ CFs were more enriched in the LV, whereas Igfbp3+ CFs were enriched in the RV at baseline. Notably, following pressure overload injury, the LV developed a larger subpopulation of pro-fibrotic Thbs4+/Cthrc1+ injury-induced CFs, while the RV showed a unique expansion of two less-well-characterized CF subpopulations (Igfbp3+ and Inmt+). These findings demonstrate that LV- and RV-derived CFs display baseline subpopulation differences that may dictate their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform on whether LV and RV fibrosis require distinct treatments. [ABSTRACT FROM AUTHOR]

A β-catenin/T cell factor-dependent transcriptional program is critical during cutaneous wound repair for the regulation of scar size; however, the relative contribution of β-catenin activity and function in specific cell types in the granulation tissue during the healing process is unknown. Here, cell lineage tracing revealed that cells in which β-catenin is transcriptionally active express a gene profile that is characteristic of the myeloid lineage. Mice harboring a macrophage-specific deletion of the gene encoding β-catenin exhibited insufficient skin wound healing due to macrophage-specific defects in migration, adhesion to fibroblasts, and ability to produce TGF-β1. In irradiated mice, only macrophages expressing β-catenin were able to rescue wound-healing deficiency. Evaluation of scar tissue collected from patients with hypertrophic and normal scars revealed a correlation between the number of macrophages within the wound, β-catenin levels, and cellularity. Our data indicate that β-catenin regulates myeloid cell motility and adhesion and that β-catenin-mediated macrophage motility contributes to the number of mesenchymal cells and ultimate scar size following cutaneous injury., Introduction When the protective barrier of the skin is damaged, an intricate process of tissue repair is set in motion that involves multiple cell types and signaling pathways. Three percent [...]

Expression of [alpha]-smooth muscle actin ([alpha]-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify [alpha]-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-[micro]m-long 'supermature' focal adhesions (suFAs), which exert a stress approximately fourfold higher (~12 nN/[micro][m.sup.2]) on micropatterned deformable substrates than 2-6-[micro]m-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of [greater than or equal to] 11 kPa and on rigid micropatterns of 6-[micro]m-long classical FA islets confines [alpha]-SMA to the cytosol. Reincorporation of [alpha]-SMA into stress fibers is established by stretching 6-[micro]m-long classical FAs to 8.1-[micro]m-long suFA islets on extendable membranes; the same stretch producing 5.4-[micro]m-long classical FAs from initially 4-[micro]m-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of [alpha]--smooth muscle actin ([alpha]-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their N[H.sub.2]-terminal sequences; these conserved differences suggest different functions. When the N[H.sub.2]-terminal sequence of [alpha]-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The N[H.sub.2]-terminal peptide of [alpha]--skeletal actin has no effect on myofibroblasts, whereas the N[H.sub.2]-terminal peptide of [beta]--cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing [alpha]-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of [alpha]-SMA in wound contraction. The [alpha]-SMA--FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed [alpha] V [beta] 3-integrin dynamics in migrating cells using a green fluorescent protein-tagged [beta] 3-integrin chain. At the cell front, adhesion sites containing [alpha] V [beta] 3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of [beta] 3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast [beta] 3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.