The interplay of fibroblasts, the extracellular matrix, and inflammation in scar formation

Abstract

Various forms of fibrosis, comprising tissue thickening and scarring, are involved in 40% of deaths across the world. Since the discovery of scarless functional healing in fetuses prior to a certain stage of development, scientists have attempted to replicate scarless wound healing in adults with little success. While the extracellular matrix (ECM), fibroblasts, and inflammatory mediators have been historically investigated as separate branches of biology, it has become increasingly necessary to consider them as parts of a complex and tightly regulated system that becomes dysregulated in fibrosis. With this new paradigm, revisiting fetal scarless wound healing provides a unique opportunity to better understand how this highly regulated system operates mechanistically. In the following review, we navigate the four stages of wound healing (hemostasis, inflammation, repair, and remodeling) against the backdrop of adult versus fetal wound healing, while also exploring the relationships between the ECM, effector cells, and signaling molecules. We conclude by singling out recent findings that offer promising leads to alter the dynamics between the ECM, fibroblasts, and inflammation to promote scarless healing. One factor that promises to be significant is fibroblast heterogeneity and how certain fibroblast subpopulations might be predisposed to scarless healing. Altogether, reconsidering fetal wound healing by examining the interplay of the various factors contributing to fibrosis provides new research directions that will hopefully help us better understand and address fibroproliferative diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis.

Keywords: extracellular matrix; fetal and adult wound healing; fibrin; fibroblast; fibronectin; integrin; interleukin; macrophage; mast cell; transforming growth factor beta.

Figure 1

Phases of wound healing and temporal expression of healing factors in fetal scarless and adult wounds.A, hemostasis takes place right after injury. Platelets stop blood loss by activating, releasing their granules, and forming an early provisional matrix made up of fibrin and fibronectin. B, approximately 2 h after injury, inflammatory signals released by platelets and tissue-resident immune cells, such as macrophages, and mast cells recruit neutrophils from circulation to the site of injury. Macrophages at this stage are activated in their proinflammatory state, involving secretion of interleukins IL-6 and IL-8. C, at the beginning of repair, the early provisional matrix being used to produce a more mature late provisional matrix made up of collagen and fibronectin. Keratinocytes undergo a proliferative burst to re-epithelialize the wound. Dermal fibroblasts migrate into the wound. First, they secrete fibronectin, then enter an activated state named myofibroblasts, characterized by the expression of alpha-smooth muscle actin and secrete collagen as well. Afterward, keratinocytes migrate over the newly produced ECM and release vascular endothelial growth factor. The ensuing angiogenesis and neovascularization, needed to support myofibroblastic presence, leads to the formation of granulation tissue, characterized by high density of myofibroblasts, macrophages, capillaries, and loosely organized collagen bundles. D, remodeling canonically begins once myofibroblasts begin contraction. Myofibroblasts increase expression of actin stress fibers and integrins, in order to produce the contraction needed to realign the excessively deposited ECM, mostly collagen at this stage. Activated fibroblasts continue to support the mechanical load until the ECM is crosslinked, creating striated scar tissue, which in skin becomes paler and paler as time goes by and vascularization is lost. A–D, adapted from “Wound Healing,” by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates. E, the duration of expression or upregulation of select genes and transcription factors in fetal wound healing are represented in the top half, whereas the bottom represents the duration of the same molecules in adult scarring healing. Hyaluronan (HA) is highly expressed in the wound site for 3 weeks and 1 week in fetal and adult conditions, respectively. c-jun and c-fos, transcription factors for activator protein-1, are quickly upregulated for up to 2 h in fetal wound, whereas in adults, they last past 12 h. Proinflammatory interleukins IL-6 and IL-8 are increased in response to wounds in adults for up to 72 h, but they disappear after just 12 h in fetal scarless wounds. Transforming growth factor beta 1 (TGF-β1, profibrotic) is released and upregulated for 18 h since the wound in the fetus, while its expression is sustained for weeks in the adult. In the case of fibrotic development, TGF-β1 does not subside. Its antifibrotic isoform TGF-β3 is expressed as soon as 10 min after injury in fetal conditions, whereas in adult wounds, it appears after 1 week. Tenascin C (TNC) is also expressed in the fetal wound bed as soon as 1 h after injury, whereas this does not take place until after 24 h in adults.

Figure 2

Fibronectin structure and fibroblast integrins that bind it.Top, schematic of a single 220 kDa subunit of fibronectin. Type III repeats making up the integrin-binding domain are in blue, extra domain type III repeats that are part of the cellular fibronectin (cFn) isoform are in gray. Green type III (7th and 15th) domains where glutathionylation may occur under conditions that unfold the protein domains. Bottom, the fibroblast integrins binding Fn are the following: α9β1 and α4β1 bind the extra domain A present in cell secreted Fn, whereas αvβ3 and α5β1 bind the canonical integrin-binding domain. While αvβ3 binds the RGD motif on the 10th type III repeat (and other proteins), integrin α5β1 requires the “synergy” PHSRN peptide sequence on the ninth type III to be in close proximity. Cell-generated forces on Fn fibers can unfold the type III repeats, increasing the distance between RGD and the synergy site, inhibiting α5β1 engagement. Under those conditions, only αvβ3 can properly bind the integrin-binding domain of Fn. This phenomenon, first predicted via steered molecular dynamics, has been named “integrin switch.”

Figure 3

Main differences between fetal, scarless (green, left side), and adult scar-producing wound healing. The fetal ECM is softer than in the adult or postnatal conditions. The predominant macrophage activation state is anti-inflammatory. Macrophages are known to transition between inflammatory, anti-inflammatory, and tissue remodeling. Furthermore, inflammatory macrophages can interact and push fibroblasts to become activate as myofibroblasts (right) and secrete the profibrotic isoform TGF-β1. In fetal conditions, the predominant isoform is TGF-β3, which has an antifibrotic effect. Integrin α1β1 binds collagen III, the more abundant collagen isoform in fetal ECM, leading to a downregulation of collagen production. Because of the softer environment, α5β1 can properly bind the integrin-binding domain of fibronectin and maintain fibroblast homeostasis. During adult wound healing, collagen I, a ligand for α2β1, is the most common collagen isoform. Increased cell contractility and ECM mechanics inhibit α5β1 binding of Fn causing an increase in αvβ3 fibroproliferative signaling. Although dendritic cells are present in fetal and adult wound healing, in the fetus, they suppress TNFα secretion, thus subduing inflammation. On the other hand, in the adult and postnatal conditions, neutrophils are recruited to the injury site and are much more likely to release their neutrophil extracellular traps, exacerbating inflammation and leading to downstream scar formation.

Figure 4

Summary of TGF-β signaling and interplay with Wnt. Mechanical cues from the extracellular matrix potentiate TGF-β activation from the latency-associated peptide. In most cases, cells can access TGF-β by releasing it from the complex via αv class integrin-mediated forces that are transmitted only at certain levels of ECM stiffness. Alternatively, BMP-1 cleaves TSP-1 and enhances its capability to liberate TGF-β. Gremlin can not only antagonize this BMP-1 activity but also activate SMAD2/3 in the absence of TGF-β. TGF-β ligates its receptors, leading to phosphorylation and formation of Smad2/3/4 complexes that then translocate to the nucleus on chromatin Smad response elements. LEM domain–containing protein 3 (LEMD3) (purple) antagonizes this TGF-β/Smad2/3 signaling by complexing with Smad2/3 both in the nuclear envelope and inhibits the mechanical response of cells to TGF-β. Cytosolic LEMD3 fragments are post-translationally generated, separating the nuclear-localizing LEM domain and the Smad2/3-interacting RRM domain (RRM in cytosol, purple). Both nuclear and cytosolic LEMD3 activities are inhibited by F-actin polymerization, which is driven by mechanical cues from the matrix, thereby connecting ECM mechanics to inhibition of an inhibitor of Smad2/3 signaling. TGF-β signaling downregulates Dkk-1 expression, which is an antagonist for Wnt. This soluble factor engages its receptor Frizzled, enabling accumulation of β-catenin in the cytosol and thus the latter translocation into the nucleus where it acts as a pluripotent transcription factor. In studies attempting to recapitulate scarless healing, CXXC5 (a zinc finger catenin inhibitor) reduced αSMA and collagen I expression in fibroblasts. Inhibitory relationships are marked with red block-end arrows. Wnt, wingless type.