TGF-β in Skin Chronic Wounds | Encyclopedia MDPI

TGF-β in Skin Chronic Wounds: Comparison

Please note this is a comparison between Version 1 by Francisco J. Nicolás and Version 2 by Vivi Li.

Chronic wounds are characterized for their incapacity to heal within an expected time frame. Potential mechanisms driving this impairment are poorly understood and current hypotheses point to the development of an unbalanced milieu of growth factor and cytokines. Among them, TGF-β is considered to promote the broadest spectrum of effects. Although it is known to contribute to healthy skin homeostasis, the highly context-dependent nature of TGF-β signaling restricts the understanding of its roles in healing and wound chronification. Historically, low TGF-β levels have been suggested as a pattern in chronic wounds. However, a revision of the available evidence in humans indicates that this could constitute a questionable argument. Thus, in chronic wounds, divergences regarding skin tissue compartments seem to be characterized by elevated TGF-β levels only in the epidermis.

TGF-β
keratinocytes
chronic wounds
wound healing

1. Introduction

Chronic wounds are characterized by being unable to heal in an expected time frame. This is often related to defects in keratinocyte ability to epithelialize over recovered dermal tissue. In healthy unaltered epidermis, keratinocytes in the basal layer proliferate slowly to contribute to tissue homeostasis. They later differentiate while progressing into suprabasal layers until becoming mere keratin scales in the stratum corneum. Yet, since they are the main constituent of the exposed organism physical barriers, keratinocytes in the epidermis seem to be prepared to quickly respond to environmental injury. Across strata, living keratinocytes express cell mediators and showcase receptors ready to signal in case of harm. Amid other effects, signaling through these factors triggers keratinocyte activation in order to contribute to tissue repair. Upon the eventual re-establishment of epithelial continuity, keratinocyte phenotype would revert to the initial state, thus completing the keratinocyte activation cycle [1]. Alterations of this process could result in unsuccessful wound closure. This situation commonly associates with persistent inflammation, which perpetuates a non-healing state, thus promoting ulcer chronicity and frequent relapse along with the development of comorbid complications (i.e., infections) [2]. Although the etiologies leading to the development of chronic wounds are fairly well understood, whether in venous stasis ulcers, chronic pressure ulcers, diabetic ulcers, or massive traumatic wounds, the molecular events leading to failure of keratinocytes to help closure are, generally, poorly understood.

Throughout the diversity of growth factors and cytokines known to influence wound healing, TGF-β is considered to promote the broadest spectrum of effects ^[3][4][3,4]. Initially conceived as a rather simple linear non-amplified signaling pathway, accumulated research on TGF-β has demonstrated otherwise, with new implications still emerging. In developing embryos, members of the TGF-β superfamily, including bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), activins, and TGF-βs, mediate key intercellular communications necessary for adequate tissue development and for the arrangement of the overall body plan. Not by accident, the origins of the superfamily date back to non-bilaterian animal forms of life. Being one of the youngest within this superfamily, the rise of the TGF-β family itself is linked to the emergence of the anteroposterior/ventral–dorsal bilateral symmetry which defines most metazoans today, which originated between 635 and 541 million years ago during the Ediacaran Period [5]. Consequently, TGF-β signaling had quite enough time to evolve and finely specialize as well as to relate to other pathways. In adult mammals, responses to its signaling range from damaged tissue repair, extracellular matrix (ECM) maintenance, epithelial and endothelial cell growth, and differentiation to the regulation of immune responses [6]. This constitutes an evolutionary wonder which introduces great difficulty in understanding some cellular behaviors at the molecular level. In fact, that would be the case for TGF-β isoforms role in skin chronic wounds which, although regarded as relevant, remains elusive and is yet to be completely addressed [7].

2. TGF-β Signaling: A Context Dependent Mechanism

The TGF-β family comprises TGFB1, TGFB2, and TGFB3. All three genes are highly conserved across species and humans, in which their products share strong sequence similarity and also display nearly identical three-dimensional structures ^[8][9][8,9]. They signal through the same ubiquitously expressed transmembrane receptors, generally referred to as TβRI and TβRII, which develop a similar affinity for isoforms TGF-β1 and TGF-β3, whereas only TβRII binds with less intensity to TGF-β2 ^[10][11][10,11]. Thus far, the main element discriminating physiological roles for the three TGF-β isoforms might be differences in their spatial and temporal expression patterns. However, molecular recognition of TGF-β is not achieved via simple ligand–receptor interaction, but through a network of interactions deeply affecting the final outcome. For starters, TGF-β is usually regarded as a homodimer, yet heterodimeric configurations showing variable potency and binding affinity with TβRs have been also reported both in vivo and in vitro ^[12][13][12,13]. Once secreted, TGF-β can remain in a latent state for some time, allowing for stock build-up in the extracellular matrix (ECM). This arises from the presence of the latency associated peptide prodomain (LAP). Indeed, TGF-β is translated into single polypeptide chains containing both a monomer and its corresponding LAP whose maturation through the trans-Golgi network involves the formation of disulfide bonds resulting in dimer stabilization. The following proteolytic cleavage splits polypeptide chains; however, the association between LAP and TGF-β remains stable through non-covalent interaction. LAP removal constitutes a critical regulatory event for TGF-β activity achieved through diverse mechanisms, which range from conformational changes promoted by interaction with integrins to enzymatic digestion including matrix metalloproteinases ^[14][15][14,15]. In that sense, and in contrast to TGF-β isoforms, LAPs show four times greater sequence divergence, allowing for diversification of the activation dynamics [5]. Additionally, modulators already present on secretion, such as latent TGF-β binding proteins (LTBPs), or in the ECM, such as decorin, biglycan, or fibromodulin, bind and delay TGF-β activation ^[16][17][16,17]. Moreover, co-receptor molecules with the ability to modulate and which bind to TβRs have been described [18]. Furthermore, downstream to TβRs activation, intracellular transduction of the signal shows, again, possibilities for modulation by means of post-translational modifications ^[19][20][19,20] or the regulation of protein levels of the factors participating on either Smad-reliant canonical signaling [21] or MAP-kinase-dependent non-canonical signaling [22]. In that sense, the aspects of TGF-β signaling intracellular transduction and the crosstalk with other pathways have been the subject of extensive reviews ^{[23][24][25][26]}[23,24,25,26]. In the end, the ability of TGF-β stimulation to promote long-term modulation of gene expression is affected by concomitant circumstances, for instance, the epigenetic status or the microRNA profile of the targeted cell ^[27][28][27,28], among others. Altogether, the sum of mechanisms involved in TGF-β signaling implies countless possibilities for modulation at any level, making the final outcome highly dependent on cell type and context. This fact encourages the shift of the paradigm for TGF-β pathway from linear non-amplified signaling cascades to complex signaling networks, better explaining the variety of functional responses to TGF-β isoforms [5].

3. TGF-β Expression in Healthy and Healing Epidermis

In the skin, TGF-β isoforms are differentially expressed by nearly all classes of its constituent cells. For the case of healthy human epidermis, some TGF-β1 but mostly TGF-β3 expression has been described at the basal cellular layer ^[29][30][29,30]. This has been suggested to be constitutive and necessary for epithelial homeostasis ^[31][32][33][31,32,33]. In contrast, for the process of wound healing, most cells involved develop distinct spatial and temporal expression patterns of TGF-β [29]. Upon skin injury, TGF-β regional induction in the wound has been found to result in a double-peak availability pattern ^[34][35][36][34,35,36]. That pattern would emerge from the initial quick abundant platelet release, later building up on the aggregated contributions over time from endothelial cells, monocytes, fibroblast, and keratinocytes [37]. Specifically regarding the epidermis, it is acknowledged that TGF-β expression levels surge across strata during the course of acute wound healing; however, there is no clear description of the patterns for each TGF-β isoform [3]. Crosstalk with concurrent signaling might also affect the definite cellular responses characteristic of dermal regeneration, angiogenesis, and re-epithelization (reviewed in ^[38][39][38,39]). In this regard, it should be noted that growth factors like EGF, PDEGF, HIF, VEGF, and other cytokines expressed by the cells which constitute each tissue layer of the skin are known to affect responses at neighboring tissue compartments [40]. As a pertinent example of the previously mentioned crosstalk between tissue compartments, TGF-β produced by keratinocytes during acute wound healing is believed to induce the suppression of TGF-β release by fibroblasts [41]. Interestingly, that crosstalk has been described as necessary to revert keratinocytes to a basal state and complete the activation cycle [1].

4. TGF-β in Human Chronic Wounds

In spite of accumulated knowledge on the acute healing process, regarding human chronic skin lesions, few studies have provided meaningful evidence on TGF-β patterns according to tissue compartments. Interestingly, regardless of the type of ulcer, original research articles on this issue coincide in describing a noticeable lack of expression for all the TGF-β isoforms in the human chronic wound bed. These observations rely on results obtained by means of in situ hybridization, immunofluorescence, and immunohistochemistry techniques, complemented with PCR in some cases ^[30][42][43][30,81,82]. As mentioned before, this notion of reduced TGF-β levels in chronic wounds has prevailed in literature. Yet, although the dermal component constitutes the bulk in terms of the wound surface, these reduced levels would just circumscribe to it. In the same studies, observations regarding the epidermal compartment are contrasting. For decubitus sores, in situ hybridization studies have shown increased levels of both TGF-β1 and 3 in the hyperkeratotic epidermis surrounding the chronic ulcer, yet TGF-β2 expression was not detected [30]. Regarding venous stasis ulcers, immunofluorescent studies provide mixed evidence: some authors reported intense TGF-β1 expression in the epidermis next to the ulcer edge, along with reduced TGF-β3 and negligible TGF-β2 staining ^[42][81], in contrast with other authors, who reported moderate TGF-β2 and 3 levels with negligible TGF-β1 expression in biopsies that included epidermis next to the wound bed ^[43][82]. On the issue of diabetic foot ulcers, immunohistochemistry studies showed increased TGF-β3 levels across all epidermis layers. Interestingly, although the same studies showed modest TGF-β1 expression at the basal stratum in the ulcer edge, cells next to the wound edge clearly displayed intense staining ^[43][82]. Overall, this collective evidence suggests that augmented TGF-β levels are to be expected in the epidermis of human chronic wounds. Thus, while it is true that decreased TGF-β levels can be found in chronic wounds, a thorough review of aforementioned studies suggests that this statement would be rather inaccurate as it fails to convey the fact that high TGF-β levels are detected in the epidermis compartment.

5. Characteristic TGF-β Responses of Skin Cells

For fibroblasts and endothelial cells, TGF-β is known to exert chemotactic and pro-mitotic activities essential for the constitution of functional granulation tissue during the inflammatory and proliferative phases of wound healing ^[44][83]. Later, TGF-β ability to promote fibronectin synthesis and collagen deposition by fibroblasts becomes crucial for proper ECM replacement during the remodeling phase ^[45][46][84,85]. Interestingly, in human fibroblasts, these responses have been reported to evolve as TGF-β concentration varies. Higher levels are considered better at promoting migration, in contrast with lower levels, which are better at inducing proliferation ^[47][86]. Moreover, while TGF-β levels remain far from those achieved through platelet degranulation, the second TGF-β peak, resulting from the contribution of cells constituent of the skin, has been reported to specifically contribute to wound contraction by prompting fibroblasts to differentiate into myofibroblasts ^[48][49][87,88]. Also, the combined effects of TGF-β3 and TGF-β1 have been found capable of fine-tuning collagen deposition ^[50][51][89,90], with relevant implications in the development of scarring and fibrosis ^[52][91]. It is worth mentioning that fibroblasts isolated from chronic venous ulcers have shown non-functional TGF-β signaling due to decreased TβRII expression. This results in a number of critical fibroblast abnormalities, including reduced proliferation and impaired migration ^[53][92].

Distinct and particular effects have seemingly been described for keratinocytes and TGF-β signaling. Varying TGF-β levels have been reported to be involved in the exchange of keratins typical of keratinocyte activation and their reversal to a basal phenotype [1]. TGF-β has also been suggested to promote keratinocyte migration through a mechanism involving integrin induction and Smad-dependent signaling ^[54][93]. Moreover, TGF-β has been suggested to mediate proliferative responses in activated keratinocytes. TGF-β inoculated in vitro is well known to induce robust G1 cell cycle arrest in epithelial cell lines ^[55][94], including keratinocytes ^[56][95]. That kind of response seems to be channeled through the Smads, since loss of or aberrant canonical signaling is a common feature of squamous cell carcinomas derived from epidermal keratinocytes ^[57][96]. Interestingly, studies performed on HaCaT cells describe how these keratinocytes experience TGF-β induction, mainly TFG-β1, during differentiation ^[58][97]. Moreover, they show TβR upregulation and sensitization to autocrine TGF-β in response to an initial TGF-β1 treatment ^[59][98]. Altogether, this is in line with the TGF-β double-peak dynamics mentioned earlier [35] as well as with the evidence indicating that after injury, initial keratinocyte proliferation is restricted to cells distal to the leading edge, with recruitment of those cells next to the wound edges not earlier than 2–3 days post-wounding ^[60][61][99,100].

6. Reaction to Exacerbated TGF-β Levels in Wound Healing

Based on what has been discussed above, it is plausible that excessive TGF-β levels occurring only in the epidermal layer at the edge of the ulcer might contribute to the chronification of the wound. This notion is supported by concurrent evidence obtained from animal and human study models. Constitutive and high expression of integrin αvβ6 has been linked to a chronic wound state ^[62][101]. Moreover, overexpression of this integrin has been associated with elevated TGF-β1 levels and spontaneous ulceration in mice ^[62][101]. Studies using transgenic mice expressing different TGF-β1 constructs reveal skin alterations similar to those found in chronic wounds. The first model ever described implemented a construct expressing constitutively active TGF-β1 under the control of the human keratin-1 promoter. These animals showed an altered phenotype characterized by restricted mobility and impaired breathing and died within a day after birth. Analysis of their skin revealed hyperkeratosis and decreased proliferation in the epidermis ^[63][102]. Later models expanded on this evidence as those mice reached adulthood. This was achieved by implementing complete TGF-β1 sequences along with either human keratin-5 (hK5) or keratin-14 (hK14) promoters, which restrict transgene expression to the basal keratinocyte layer [31]. These advanced models differed greatly in their phenotype, ranging from hK14 shabby aspect ^[64][49] to hK5 scaly and erythematous skin similar to psoriatic erythroderma ^[65][48]. Notably, while TGF-β1 levels in unwounded skin of hK14 mice were similar to TGF-β1 levels in wild type ^[64][49], persistent levels similar to those occurring in acute wounds were reported for the hK5 models ^[65][66][48,50]. At the microscope, hK5 mice showed altered epidermis with signs of acanthosis (hyperplastic epidermis), diminished granular layer, and thickened stratum corneum, in some cases also showing abnormal basal and follicular keratinocyte proliferation in the form of hyperplasia and hyperkeratosis ^[65][66][48,50]. Moreover, some of these models developed spontaneous ulcerations in friction areas [36]. Interestingly, regardless of the promoter used, all these mice models developed significant delay in full-thickness wound recovery in comparison with non-transgenic mice ^{[36][64][67][68]}[36,49,65,103]. By contrast, results obtained from TGF-β1 knockout mice showed regular wound healing and, in some cases, accelerated recovery ^[69][70][69,104]. In sum, the evidence obtained in transgenic mice constitutively overexpressing TGF-β1 in keratinocytes supports the hypothesis that persistent TGF-β signaling might not benefit wound healing and might constitute a relevant element in the pathogenesis of chronic wounds.

7. Epithelial to Mesenchymal Transition During Wound Healing and the Involvement of TGF-β

Though devoid of proliferation, keratinocytes at the edge of acute wounds experience changes similar to epithelial–mesenchymal transition (EMT). The concept of EMT implies a dramatic phenotypical switch providing epithelial cells with unusual capacities, like high mobility, the ability to surpass basement membranes, and resistance to apoptosis ^[71][105]. These are considered unequivocal traits of advanced states in tumor transformation and are necessary for the progression of epithelium-derived cancer in terms of invasiveness and metastatic potential ^[71][105]. Diverse molecular processes engage in this phenomenon and contribute to its development. These processes include signals from growth factor and cytokines, including EGF, FGF, HGF, KGF, and TGF-β, promoting the activation of transcription factors, reorganization of cell architecture, expression of specific surface proteins, production of ECM-degrading enzymes, and modulation of specific microRNAs ^[71][105]. Biased response to TGF-β is considered a major mechanism for EMT in cancer, this being the subject matter of numerous reviews ^[72][73][106,107]. Indeed, biased responses affect the expression of key cell adherence and migration markers, among other known effects ^{[72][73][74][75][76]}[106,107,108,109,110]. These include Smad canonical signaling driving the expression of key transcription factors such as Slug or Snail ^[77][111], which mediate reduced E-cadherin detection and induction of mesenchymal markers such as vimentin, the two latter effects being recognized EMT hallmarks ^[78][112]. Concurrent signaling involving MAP-kinases has also been suggested to contribute to this phenomenon ^[79][113]. The transition phenomenon described above develops less intensely in skin wounds. In that context, keratinocytes are reported to experience temporal transdifferentiation involving cytoskeleton reorganization, loss of cell polarity, and partial dissociation of adhesion structures ^[80][114]. This is supposed to allow cells to minimize attachment to the basal membrane while elongating and acquiring motility, seeking to re-establish epithelial coherence ^[81][115]. While these transdifferentiation dynamics are fairly well understood, no consensus exists on how keratinocyte activities coordinate for re-epithelization. Several models have been proposed for the discussion on whether proliferation at the wound edge and migration occurs in the basal, suprabasal, or both layers ^[81][115]. Moreover, specifically for migration, the considerations in the available literature regarding keratinocyte transdifferentiation during wound healing are mostly based on what is known about TGF-β and EMT during development and cancer ^[38][80][82][38,114,116]. Interestingly, in light of the accumulated evidence, different review authors point to the existence of intermediate EMT states ^[83][84][117,118]. Though it is still being discussed, molecular characterization of these intermediate states might be helpful for wound healing research. As mentioned before, there is a lack of specific knowledge on TGF-β and EMT in skin wound healing. A recently published study (2019) demonstrated the implication of both canonical and non-canonical TGF-β1 signaling for proper keratinocyte transdifferentiation and successful wound closure in the axolotl (Ambystoma mexicanum), since exposure to selective inhibitors for each pathway resulted in delayed re-epithelization ^[85][119]. Compared to humans, the axolotl shows accelerated wound closure also characterized by no scarring, both achieved through a molecular mechanism which involves TGF-β signaling ^[86][120]. In fact, several studies show how TGF-β signaling is necessary for tissue regeneration in other lower vertebrate models, including Xenopus and zebrafish ^[87][88][121,122]. This evidence provides unique hints on the role of TGF-β in keratinocyte transdifferentiation; however, the axolotl model itself is distant from human skin. At the molecular level, this is evidenced by the fact that only TGF-β1, but not TGF-β2 or TGF-β3, is detected in its regenerating tissues ^[89][123]. Moreover, the axolotl develops shorter TGF-β1 induction and scarce leukocyte infiltration during wound healing ^[90][124]. Interestingly, it has been suggested that the regenerative capacities shown by some lower vertebrates might be related to their neotenic potential (i.e., retaining typical traits of early stages of life) ^[91][125]. To that extent, it is well established that early-gestation human skin wounds repair quickly and without scar formation ^[92][126]. The mechanisms leading to this resolution of fetal wounds remain unknown. However, reviewed evidence on this issue points to TGF-β as the main factor involved, as in fetal skin, only TGF-β3 expression is found to be increased while TGF-β1 levels remain steady, in clear contrast with what is found in adults ^[93][127]. In that sense, studies performed on mice and rats provide evidence of a contrasting TGF-β1/TGF-β3 ratio between the skin and the oral mucosa during wound healing ^[94][95][128,129]. This observation is highly interesting, as oral mucosal wounds are indeed known to heal faster and with minimal scarring in comparison with skin wounds ^[96][130]. Interestingly, although evidence available in humans is restricted, a recent study analyzing oral scars appearing after oral tumor removal suggests a pattern of increased TGF-β1 detection ^[97][131]. Indeed, the treatment of adult skin wounds with exogenous TGF-β3 or, alternatively, with neutralizing antibodies for TGF-β1 and TGF-β2 has been suggested to reduce scar formation and improve aesthetics after healing ^[50][98][89,132]. Moreover, treatment of fetal wounds with TGF-β1 results in scarification ^[99][133]. Altogether, this evidence suggests that relative fractions of TGF-β isoforms, rather than absolute amounts, may direct the evolution of the wound through their impact on the regulation of gene expression and the release of cell mediators driving leukocyte recruitment, keratinocyte activation, or ECM deposition.