Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention

Abstract

Significance: Myofibroblasts are responsible for wound closure that occurs in healed acute wounds. However, their actions can result in disfiguring scar contractures, compromised organ function, and a tumor promoting stroma. Understanding the mechanisms regulating their contractile machinery, gene expression, and lifespan is essential to develop new therapies to control their function.

Recent advances: Mechanical stress and transforming growth factor beta-1 (TGF-β1) regulate myofibroblast differentiation from mesenchymal progenitors. As these precursor cells differentiate, they assemble a contractile apparatus to generate the force used to contract wounds. The mechanisms by which mechanical stress promote expression of contractile genes through the TGF-β1 and serum response factor pathways and offer therapeutic targets to limit myofibroblast function are being elucidated.

Critical issues: Emerging evidence suggests that the integration of mechanical cues with intracellular signaling pathways is critical to myofibroblast function via its effects on gene expression, cellular contraction, and paracrine signaling with neighboring cells. In addition, while apoptosis is clearly one pathway that can limit myofibroblast lifespan, recent data suggest that pathogenic myofibroblasts can become senescent and adopt a more beneficial phenotype, or may revert to a quiescent state, thereby limiting their function.

Future directions: Given the important role that myofibroblasts play in pathologies as disparate as cutaneous scarring, organ fibrosis, and tumor progression, knowledge gained in the areas of intracellular signaling networks, mechanical signal transduction, extracellular matrix biology, and cell fate will support efforts to develop new therapies with a wide impact.

Figures

Livingston Van De Water, PhD

Figure 1.

Morphology of the myofibroblast. (A, B) Cartoons depicting the morphology and cytoskeletal components of a fibroblast (A) versus a myofibroblast (B). For more detail, see text. (C, D) Immunofluorescence images showing the stress fiber organization (red-phalloidin staining) and focal adhesion proteins (A, green-Hic-5 immunostaining; B, green-vinculin immunostaining) of a myofibroblast on a glass coverslip (C) or in a collagen gel (D). Scale bars: (C) 50 μm; (D) 40 μm.

Figure 2.

Type I collagen mRNA expression in normal dermis versus wound tissue. Paraffin sections of normal rat skin (A) or an 8-day wound (B) were reacted with an 35S-labeled transcribed RNA probe reactive with Type I Collagen. Note that normal dermal fibroblasts (arrows) in the reticular dermis (A) are modestly labeled with the probe while fibroblasts in the wound (B) are heavily labeled. Photographic emulsion was exposed for the same length of time. Scale bar: 50 μm.

Figure 3.

Trichrome stain of normal rodent wound and human HTS. Paraffin sections of a normal rat 14 day wound (A) or human HTS (B) were stained with Massons Trichrome. Note that red color that predominates in the wound bed (A) represents a cell-rich and less pronounced ECM. The deep blue color in the HTS is a result of prominent collagen deposition. Also, the cells within the wound (A, arrow) are linearly arrayed, while in the HTS they are organized into a nodule (dotted line). Scale bars: 100 μm. ECM, extracellular matrix; HTS, hypertrophic scar.

Figure 4.

Burn injury resulting in scar contracture across the wrist. Existence of myofibroblasts following a burn injury can deposit excessive ECM and contract the resulting scar leading to a pathologic contracture. As observed in this individual, the wrist is held in permanent flexion by the contracture that resulted from a burn injury. (Courtesy of Dr. Jaysheela Mudera, University College, London. Reprinted with permission from Tomasek et al.)

Figure 5.

Model of myofibroblast differentiation and wound contraction. (A) In normal tissues, fibroblasts are shielded from “routine” external mechanical perturbations in the skin by the collagen-rich ECM that they have assembled, such that the organization of a contractile cytoskeleton is not stimulated (light pink area of dermis). Following a full-thickness dermal injury, the wound is filled with a provisional matrix comprised of fibrin and fibronectin and a complement of newly released growth factors and cytokines. Fibroblasts, along with blood vessels, are stimulated to migrate into this pro-migration microenvironment and over time replace the provisional matrix with an ECM comprised of collagen and cellular FNs to form granulation tissue. (B) Tractional forces accompanying fibroblast migration are responsible for local areas of increased stiffness in newly made collagen. Focal adhesion assembly is increased with increasing stiffness and this is accompanied by clustering of integrins within focal adhesions and increased stress fiber assembly resulting in fibroblast acquisition of the proto-myofibroblast phenotype. Tensional forces and growth factors stimulate proto-myofibroblasts to secrete transforming growth factor-beta1 (TGF-β1) and increased levels of ED-A FN. (C) In response to TGF-β1, a threshold of mechanical stiffness and the presence of specific isoforms of FN proto-myofibroblasts differentiate into myofibroblasts. Feed-forward pathways, including TGF-β1, the mechanical environment, actin dynamics, SRF/MRTF transcriptional activation, and Hic-5, are responsible for myofibroblast function and persistence. At the same time, differentiated myofibroblasts deposit collagen and other ECM components, and produce proteases. This complex process of remodeling results in shortening of the collagen matrix with corresponding wound closure. (D) During normal acute wound healing, these feed-forward mechanisms are temporally limited and myofibroblast numbers diminish as a result of apoptosis and/or senescence. (E) In pathological situations, such as HTS formation, these feed-forward pathways presumably persist allowing for continued myofibroblast presence resulting in continued ECM deposition and remodeling. In conclusion, myofibroblasts, far from being a “bad” cell type, are functionally essential cells. It is their dysregulation that is the cause of tissue dysfunction. (Modified from Fig. 9, Tomasek et al.). FN, fibronectins; SRF, serum response factor; MRTFA/B, myocardin-related transcription factor A/B.

Figure 6.

Dermal fibroblasts generate contractile force sufficient to deform silicone substrates when stimulated with serum. Normal primary dermal fibroblasts were cultured on a deformable silicone substrate (PDMS). Serum stimulation is sufficient to generate wrinkling (A); wrinkling is lost upon removal of serum (B), demonstrating reversibility of contractile forces. Cells are viewed under phase microscopy. Scale bars: 100 μm.

Figure 7.

Decorative scarring. Ornate welts of raised scar tissue are a mark of beauty on the back of an individual from Mozambique. (Photo by Volkmar K. Wentzel/National Geographic Stock; all rights reserved.) To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 8.

Feed-forward pathways that support myofibroblast persistence. Active TGF-β1 induces many pathways but we highlight here Rho-dependent processes that include increases (light gray arrows left) in actin polymerization, dissociation of MRTF-A from G-actin, and translocation of MRTF-A to the nucleus where with SRF induces cytoskeletal genes, some focal adhesion proteins, including Hic-5 (black arrow, left), and Type I collagen synthesis (black arrow, right). The result is increased (light gray arrows, right) collagen synthesis and deposition, increased contractile force (intracellular tension) via the actin-myosin II containing stress fibers. SM α-actin promotes increased contractile force that in turn supports focal adhesion growth and stress fiber assembly (dark gray arrow, right). Hic-5 has been shown to be necessary for latent TGF-β1production and mechanical contraction promotes latent TGF-β1 activation (dark gray arrows, left). SM α-actin, smooth muscle alpha-actin.

Figure 9.

Mechanical environment regulates transcriptional activity of MRTF-A via actin dynamics. Stress fibers and filamentous actin (F-actin) are stabilized under high intracellular tension resulting in the liberation of MRTF-A from globular actin (G-actin). Free to shuttle to the nucleus from the cytoplasm, MRTF-A drives expression of contractile genes promoting further increases in intracellular tension. (Adapted from Crider et al.) To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound