Targeting Fibrotic Signaling: A Review of Current Literature and Identification of Future Therapeutic Targets to Improve Wound Healing

Abstract

Fibrosis is a consequence of aberrant wound healing processes that can be debilitating for patients and often are associated with highly morbid disease processes. Myofibroblasts play an important role in determining an appropriate physiologic response to tissue injury or an excessive response leading to fibrosis. Specifically, “supermature” focal adhesions, α-smooth muscle actin, and the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway likely play a significant role in the differentiation and survival of myofibroblasts in fibrotic lesions. Thus, targeting each of these and disrupting their functioning could lead to the development of therapeutic options for patients suffering from fibrosis and other sequelae of dysregulated wound healing. In this paper, we review the current literature concerning the roles of these three constituents of fibrotic signaling pathways, work already done in attempting to regulate these processes, and discuss the potential of these biomolecular constituents as therapeutic targets in future translational research.

Introduction

Dysregulated wound healing resulting in sequelae including fibrosis continues to be problematic in patient care.¹ Fibrosis affects every major organ system through a variety of diseases, and it is estimated to contribute to about 33% of deaths globally and 45% of deaths in industrialized nations,² while skin-associated fibrotic complications, including hypertrophic scars and keloids, remain significant causes of suboptimal healing outcomes that negatively impact patient function and satisfaction following surgical procedures. Therefore, improved treatments for fibrotic complications carry the potential to have a major impact that could benefit almost any patient undergoing surgery.

Fibrosis is characterized by the overproduction of collagenous material in the extracellular matrix (ECM) leading to scar and contracture.³ A contractile form of fibroblasts, known as myofibroblasts, is thought to be the main mediator of this process.⁴ Myofibroblast differentiation is thought to be largely facilitated by TGF-β1 and/or mechanical stress.⁵ Though it seems an optimal balance of myofibroblast activation leads to proper wound healing, the exact mechanisms leading to improper wound healing remain elusive. It is clear, however, that the process of wound healing involves a specific and intricate balance among growth factors, cell-ECM contacts, and cell-cell contacts.⁶ Specifically, α-smooth muscle actin (SMA), “supermature” focal adhesions, and the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway appear to be a few of the important players mediating wound healing and thus the development of fibrosis.^7–9 In this article, we review these components’ roles in the development of fibrosis individually then discuss their interconnection, and how they may offer potential avenues for future research into wound healing therapeutics.

α-Smooth Muscle Actin (SMA)

An important component of myofibroblasts is α-smooth muscle actin (SMA). Indeed, it is routinely the molecule used to identify myofibroblasts—though not all cells expressing SMA are myofibroblasts.^10,11 While the exact function of SMA in cells of connective tissues is not well defined, it has been suggested that it plays an important role in generating mechanical strain. High levels of SMA have been found in stress fibers and focal adhesions, both of which are highly associated with tensile force generation.^12,13 Furthermore, it is the expression and incorporation of SMA into stress fibers that enhances the contractility of activated fibroblasts.⁸ SMA can also help mediate external force on the ECM through various actin binding proteins (vinculin, talin, filamin A, paxillin, α-actinin), integrins, and focal adhesions.¹⁴ Thus, SMA is important in generating both intracellular and extracellular mechanical stress.

It has also been suggested that SMA expression level can be regulated by external mechanical stress. SMA expression increases in the presence of exogenously generated stress and decreases with a reduction in exogenous tension.¹⁵ The influence of both internal and external force on SMA expression can lead to a positive feedback loop, which is one theory for the exaggerated myofibroblast phenotype in fibrotic diseases (Figure 1). Specifically, increased expression of SMA results in increased intracellular tension, which through cell-ECM contacts produces more extracellular tension. The increased extracellular tension in turn exerts its effects on the cell leading to further increased SMA expression, starting the feedback loop anew. For a more comprehensive review on SMA and its role in mechanotransduction, see Wang et al. 2006.¹⁴

Figure 1.

α-smooth muscle actin (SMA) Positive Feedback Loop.

Due to its importance in myofibroblast differentiation and contraction, SMA has been targeted in studies focused on optimizing wound healing and hypertrophic scar prevention. Fibroblasts treated with Palmyitoyl-pentatpeptide (Pal-KTTKS) showed reduced SMA expression and myofibroblast differentiation in a dose-dependent manner through a mechanism not yet fully established.¹⁶ The dose dependence was important because wound healing was accelerated at higher doses of Pal-KTTKS, accompanied by increased SMA expression. This is in accordance with another study showing that faster wound healing led to more contraction and hypertrophic scar.¹⁷ Furthermore, treatment of wounded primary human dermal fibroblasts with C-phyocyanin (C-pc) decreased SMA expression by 70% and reduced collagen contraction by 29% in a dose-dependent manner, likely through an inflammatory pathway involving the inhibition of TGF-β1 and/or the upregulation of activity of the prostaglandin PGE₂.¹⁸ Another group showed that decreased cell-cell contact on wound edges led to an increase in SMA expression, suggesting that decreased cell-cell contacts can lead to increased tensile force on cells generated from cell-matrix contacts.¹⁹ Clearly, a balance between cell-cell contact, cell migration speed, and tensile forces are important for proper wound healing.

“Supermature” Focal Adhesions

As mentioned above, “supermature” focal adhesions are thought to be an important mediator of SMA exerting mechanical force on the ECM. Focal adhesions are specialized cell-ECM contact sites consisting of integrins and cytoplasmic actins that are commonly found in fibroblasts.²⁰ As fibroblasts transition to myofibroblasts following increases in intra/extracellular mechanical tension, these focal adhesions develop into larger “supermature” focal adhesions.¹³ These “supermature” focal adhesions create stronger adhesive forces and are associated with elevated SMA expression. Indeed, it has been shown that increases in intracellular force by increased SMA expression induces transformation of focal adhesions into “supermature” focal adhesions.¹²

A focal adhesion-associated protein of particular importance is the focal adhesion kinase (FAK). In response to TGF-β1, the activation of FAK by phosphorylation of integrins in focal adhesions induces SMA expression and myofibroblast differentiation.²¹ FAK could be important in transferring mechanical signals, independent of TGF-β1, to increase the expression of SMA.²² Taken together, it is clear that FAK and “supermature” focal adhesions play important roles in controlling SMA expression downstream of both TGF-β1 and mechanical stress signaling in myofibroblasts. Indeed, keloid fibroblasts were found to have increased focal adhesion complex formation.²³

Two components of the “supermature” focal adhesions that have been therapeutically targeted are integrins and FAK signaling. One study examined the importance of αv integrin (ITGAV) in skin fibrosis. Using ITGAV knockout mice, the researchers showed that the proliferation of dermal fibroblasts and FAK phosphorylation were decreased. In addition, a FAK inhibitor reduced the expression of collagens and collagen tissue growth factor (CTGF) in mice dermal fibroblasts. Furthermore, the expression of integrin β3, commonly associated with ITGAV, was decreased in the knockout mice. Based on these data, it was proposed that the balance between the α and β chains of integrins is important in the control of collagen expression and dermal thickness.²⁴

Targeting FAK has displayed promising therapeutic results. FAK is activated following cutaneous injury and skin scarring.²⁵ Though complete FAK gene knockout was lethal in mice, fibroblast-specific FAK knockout mice had less fibrogenesis than the wild-type mice in a hypertrophic scar model. ^24,26,27 It has also been shown that FAK inhibition results in the downregulation of TGF-β-induced pro-fibrotic genes including SMA.²⁸ A recent study showed that an inhibitor of FAK delivered by hydrogel not only decreased activation of myofibroblasts in excisional and burn wounds in mice leading to scar reduction, but also accelerated wound closure, indicating the complex relationships that exist among fibrosis and wound healing signaling pathways. ²⁹

Myocardin-Related Transcription Factor/ Serum Response Factor (MRTF/SRF) Pathway

MRTF, and its associated molecule SRF, function as transcriptional co-activators of many genes, including SMA and collagen, to promote the transformation of fibroblasts into the myofibroblast phenotype.³⁰ Mechanical stiffness or TGF-β1 stimuli associated with fibrosis have been found to promote the nuclear transport of MRTF.³⁰ Furthermore, MRTF/SRF are downstream mediators of the Rho family GTPases and the Rho associated kinase (ROCK), which play important roles in myofibroblast differentiation through actin polymerization and stress fiber formation.^30,31 Indeed, MRTF signaling is regulated by actin polymerization. When actin is depolymerized, MRTF is bound to the globular form of actin and remains in the cytoplasm. When actin is polymerized and stress fiber formation occurs, MRTF is unbound and can translocate to the nucleus.³² Additionally, MRTF, also known as megakaryoblastic leukemia 1 (MKL1), transcriptionally regulates B-cell lymphoma-2 (BCL-2), which inhibits the intrinsic mitochondrial apoptotic pathway and promotes myofibroblast survival.³³ In summary, the MRTF/SRF pathway starts with a stimulus, namely mechanical stress/stiffness and/or TGF-β1, which leads to changing actin dynamics and contraction through Rho/ROCK signaling. The changes in actin polymerization in turn lead to the release of MRTF and translocation into the nucleus. MRTF then upregulates the expression of SMA and BCL-2, which promote myofibroblast differentiation and survival, respectively (Figure 2).

Figure 2. The MRTF/SRF Pathway.

TGF-β1: transforming growth factor-β1, Rho: Rho family GTPases, ROCK: rho associated kinase, SMA: α-smooth muscle actin, BCL-2: B-cell lymphoma-2.

The MRTF/SRF pathway has been shown to be an important mediator in fibrosis for several organ systems, including ocular, hepatic, pulmonary, renal, skin, colon, and cardiac.¹⁰ Furthermore, targeting this pathway has led to experimental findings associated with decreased fibrosis: Silencing the MRTF/SRF pathway significantly decreased matrix contraction, fibroblast protrusive behavior, matrix degradation, and matrix metalloproteinase gene expression in fibrotic human conjunctival fibroblasts.³⁴ Inhibiting the actin cytoskeleton by disruption of MRTF in hepatic stellate cells inhibited Type 1 collagen synthesis and fibrogenesis in hepatic fibrosis.³⁵ Inhibitors of Rho/ROCK have been shown to decrease fibrosis and increase myofibroblast apoptosis in vitro and in vivo for pulmonary fibrosis.³³ Furthermore, MRTF/SRF signaling inhibition decreased pulmonary fibrosis and increased myofibroblast apoptosis in two distinct animal models.³² MRTF/SRF inhibitors have also been shown to decrease scar formation in a preclinical model.³⁶ Clearly, disruption of this pathway has produced encouraging results in decreasing fibrosis in various organ systems, which suggests that it is a promising target for developing therapeutic options for improved wound healing and decreased scar formation.

Therapeutic Targets for Future Research

It is clear that “supermature” focal adhesions, SMA, and the MRTF/SRF pathway are interconnected and intimately related. Broadly speaking, “supermature” focal adhesions can be thought of as upstream, SMA in the middle, and MRTF downstream. The relationship is not simply a linear one, however, as SMA can directly respond to and influence tension within and without the myofibroblast. “Supermature” focal adhesions transmit mechanical tension information from SMA to the ECM. They also receive information from the ECM and relay it to SMA through FAK signaling, which has been shown to regulate Rho family GTPases.³⁷ These Rho GTPases and ROCK influence the polymerization of actin and formation of stress fiber bundles containing SMA, which directly influences the ability of MRTF to translocate to the nucleus and upregulate expression of genes found in myofibroblasts, including SMA and BCL-2 (Figure 3).

Figure 3. Communication among ECM (extracellular matrix),

“Supermature” focal adhesions, SMA (α-smooth muscle actin), MRTF/SRF (myocardin-related transcription factor/serums response factor) pathway, and the nucleus. Double arrows refer to communication in both directions. Single arrows refer to communication in one direction. “Upstream” and “downstream” refer to the relative locations of each component in the cascade.

Thus, there are three obvious targets to disrupt this signaling cascade: focal adhesions, SMA, and MRTF transcriptional activation. SMA seems least promising of the three as it is non-specific for myofibroblasts and most likely plays important roles in other cells in the body. Perhaps development of a topical SMA “inhibitor” that could be placed on areas of cutaneous hypertrophic scarring or fibrosis may make use of the non-specificity of SMA. Targeting focal adhesions may be more promising. As keloid fibroblasts have been found to have increased focal adhesion complex formation, an agent that blocks their formation may help prevent keloid formation.²³ Possible targets unique to focal adhesions could be those integrins specific to “supermature” focal adhesions of myofibroblasts or FAK signaling. As FAK signaling is important for both mechanical stress and TGF-β1 induced upregulation of SMA, blocking the signal should decrease the expression of SMA and the transformation of myofibroblasts. Likely the most promising target, however, is MRTF/SRF signaling, as this seems to be the most downstream mediator of dysregulated wound healing. As described in the previous section, there have been multiple studies suggesting that inhibiting this pathway leads to decreased collagen formation, impaired myofibroblast differentiation and their compromised survival. Agents that downregulate the expression of MRTF/SRF, prevent its translocation to the nucleus, or directly inhibit its transcriptional activities on the chromatin seem promising. Inhibitors directed at Rho/ROCK signaling seem to be another encouraging avenue as an inhibitor has already been shown to decrease fibrotic processes associated with pulmonary fibrosis.³³

However, rather than relying on the development of a single agent, the most effective therapeutic approach may be a combinatorial one where manipulation of “supermature” focal adhesions, SMA expression, and the MRTF/SRF pathway to varying degrees will provide the most therapeutic benefit. The research literature to date provides tantalizing evidence that altering the relative dynamics of these three components will alter fibrosis in a clinically meaningful way, suggesting that it is possible to develop novel therapeutic approaches that will have a major impact on surgical outcomes for patients.

Conclusion

Dysregulated wound healing is characteristic of fibrosis and continues to present challenges for clinicians. Myofibroblasts play a key role in determining an appropriate physiologic response to tissue injury or an excessive response leading to fibrosis, and an optimal balance of myofibroblast activation is necessary for proper wound healing. Optimizing the process of wound healing also involves achieving a balance among growth factors, cell-ECM contacts, and cell-cell contacts. Specifically, SMA, “supermature” focal adhesions, and the MRTF/SRF pathway appear to be a few of the important biomolecular constituents mediating fibrotic wound healing. As is the case with many disease processes, a multi-pronged intervention aimed at disrupting each of these main factors may provide the best therapeutic results for addressing and preventing fibrotic processes in surgical patients.