The role of PDGF includes all stages of healing, from the mediation of inflammation, angiogenesis, proliferation of fibroblasts and formation of granulation tissue. ²

PDGF family consists of five distinct homodimeric isoforms of glycoproteins (PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD and PDGF-AB), which: PDGF-AA is synthesized by epithelial, muscle and neural cells; PDGF-BB by endothelial cells and megakaryocytes; PDGF-CC by epithelial, endothelial cells and neurons and PDGF-DD by fibroblasts and vascular smooth muscle. ⁸

The five isoforms are also synthesized by platelets, macrophages, vascular endothelial cells, fibroblasts and keratinocytes, and has a mitogenic effects over mesenchymal cells through proteolytic reactions mediated by receptors platelet-derived growth factor alpha and beta (PDGFR-α and PDGFR-β). ^{8 – 10}

In normal skin or chronic wounds, PDGF levels are almost absent. ¹¹ However, during healing, PDGF act on fibroblast proliferation and chemotaxis ^{9 , 12} mediate inflammation, angiogenesis and form the granulation tissue. ^{8 , 9 , 13}

IL-1 is a pro-inflammatory cytokine found in two forms: interleukin 1 alpha (IL-1α) and interleukin 1 beta (IL-1β). Both act on keratinocytes proliferation and migration, angiogenesis control and MECs remodeling. ^{14 – 16} During the inflammatory phase, IL-1 is highly produced by keratinocytes, in the epidermis ^{15 , 16} ; high levels of IL-1α and IL-1β are related to negative changes in the prognosis of skin inflammatory and proliferative events ¹⁷ and to the synthesis and regulation of other inflammatory mediators. ¹⁸

Antagonists receptors recombinant human (IL-1Ra) and mouse (IL-36Ra) inhibit the expression of high levels of IL-1 and other cytokines during intense skin inflammatory and fibrotic responses. ¹⁹ Inhibition of IL-1 by IL-1Ra results in a decrease in pro-inflammatory cytokines (IL-1α, IL-1β, IL-12 and IFN-γ) and increase of IL-10, M2 macrophages, endothelial cells and granulation tissue, confirming their role over cell differentiation and proliferation. ²⁰

Regarding, IL-1β Inhibition signaling pathway leads to a decrease in the pro-inflammatory M1 macrophages, an increase in the anti-inflammatory macrophages M2 and growth factors resulting in optimization of skin wounds healing. ²¹

TNF-α is a pro-inflammatory cytokine synthesized by several cells (fibroblasts, keratinocytes, monocytes, macrophages, eosinophils and T cells), especially in the initial phase of healing, ²² which acts on the regulation of immune, inflammatory and proliferative responses (fibroblasts and keratinocytes) through type 1 (TNFR1 - p55) and type 2 (TNFR2 - p75) receptors, which trigger internal signals in target cells, leading to NF-kB activation and these responses. ²³

Rapid skin healing occurred in the absence of the TNFR1-p55, with reduction of cytokines and inflammatory infiltrates, and increase of angiogenesis and collagen I ²⁴ ; suggesting that TNFR1-p55 can negatively affect repair by inducing leukocyte infiltration at the wound site and decreasing reepithelization. ^{23 – 25}

Low levels of TNF-α stimulate the inflammatory process and synthesis of growth factors by macrophages, optimizing healing. However, at high levels, TNF-α impairs repair by increasing synthesis and activity of MMPs and decreasing synthesis of their inhibitors (TIMPs), which results in degradation of the ECM, inhibition of cell migration and collagen deposition and, consequent, delay in the process of healing, that is, chronification of the wound. ¹⁴

The TNF-α deficiency leads to less granulation tissue formation and slower reepithelization. ²⁶

Keratinocyte growth factor is poorly synthesized in intact skin tissue but becomes highly active in fibroblasts and keratinocytes after epithelial injury. Both, KGF-1 and KGF-2, can modulate the proliferation and migration of keratinocytes, resulting in reepithelization and epidermis regeneration. ²⁷

KGF-1, highly synthesized by fibroblasts at the beginning of healing, binds to the fibroblast growth factor receptor 2 (FGFR2-IIIb) in keratinocytes, regulating migration, proliferation and differentiation. ^{28 – 32}

KGF-2 has a mitogenic effect similar to KGF-1, but binding to the specific KGFR receptor. ^{33 , 34} During wound healing, KGF also acts in the formation of granulation tissue in addition to protecting cells from toxicity induced by active oxygen species. ³⁵

TGF-β family consists of the TGF-β1, TGF-β2 and TGF-β3 isoforms ^{36 , 37} and is synthesized by platelets, macrophages, fibroblasts and keratinocytes, and constitute an important antiproliferative mediator to control the different stages of healing, ^{14 , 37} by recruiting inflammatory cells, inhibiting proliferation and migration of endothelial cells and, consequently, angiogenesis, inhibiting fibroblasts and keratinocytes migration and ECM production (collagen and fibronectin), promoting remodeling and adequate repair of epidermis. ^{36 – 42}

TGF-β1 is synthesized at high levels prior to reepithelialization and is related to cellular hypertrophy ^{36 , 43} ; it can later inhibit the MMPs synthesis leading to an increase in collagen fibers. ³⁶ TGF-β3 is synthesized during the initial stage of healing and can improve repair, preventing fibroplasia. ^{39 , 43}

The main mechanism by which TGF-β inhibits and/or controls cell growing occurs through the interaction between both type 1 (TGF-βRI) and type 2 (TGF-βRII) receptors, promoting specific pathway signaling (TGF-β/SMAD), phosphorylation of R-Smads 2 or 3 and binding to Smad4, and, consequently, growth inhibition by regulation of target genes. ^{42 , 44 , 45}

Neovascularization, mediated by the VEGF family, is an important event in the skin repair and can occur mediated by endothelial progenitors (EPCs). ^{46 , 47}

The VEGF family consists of six isoforms of pro-angiogenic glycoproteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PIGF), VEGF-A121, VEGF-A165, VEGF-A189 and VEGF-A206. ^{41 , 48 , 49}

VEGF-A is the main pro-angiogenic growth factor related to wound healing, ^{41 , 46 , 49} being expressed mainly by endothelial cells and platelets, but also by fibroblasts, keratinocytes and macrophages, ^{50 , 51} once their specific tyrosine kinase 1 receptor Flt-1 (VEGFR-1) and Flk/KDR-1 (VEGFR-2) are also present in these cells. ^{46 , 52 , 53}

According to Ref. 54 an increase in angiogenesis can be mediated by VEGF-A synthesized by monocytes and macrophages in chronic venous wounds. Such a process is possible because of the interactions between endothelial and inflammatory cells.

VEGF-A increases vascular permeability in the early stages of skin repair ⁵¹ and stimulates quiescent endothelial cells to interact with adjacent cells in full state of proliferation and migration, promoting vascular growth. ^{46 , 55} The balance in VEGF-A levels is also important to promote normal or delayed dermis’ healing, re-epithelialization and wound contraction. ^{46 , 56 , 57}

VEGFR-2 receptor is also quite important to control angiogenesis, since binding to VEGF activates the signaling pathways of protein kinase B, resulting in inhibition of apoptosis and induction of cell proliferation. ⁵⁸

Different types of matrix metalloproteinases (MMPs), such as collagenases, gelatinases, stromelysins, are present in several tissues performing specific functions regulated by different metalloproteinase inhibitors. ^{59 , 60}

MMP-9 or gelatinase B, one of the 25 existing isoforms, is synthesized by basal cells and keratinocytes ^{60 , 61} and has an important role in the skin healing, ⁶² regulating the acute and chronic inflammation ^{63 , 64} through the activation and control of cytokines and chemokines, ^{65 , 66} controlling the release of growth factors, ⁵⁹ angiogenesis ⁶⁷ and migration/proliferation of keratinocytes ⁶⁸ ; regulating communication cells-ECM; promoting ECM reorganization and, consequently, tissue reepithelization. ^{63 , 64 , 66}

The deficiency of MMP-9 in rat skin results in an inflammatory process, with a high deposit of IL-1α and disorganization of basement membrane and ECM ⁶⁴ ; as well as the absence of MMP-9 results in decrease of angiogenesis and delay in reepithelization, with scarcity of keratinocyte migration at the edges of the wound. ^{68 , 69}

MMP-9 is inhibited by tissue metalloproteinase inhibitor-1 (TIMP-1) and the balance between them promotes adequate skin repair ⁶³ ; high levels of MMP-9 and decrease in TIMP-1 result in slow healing in wounds of diabetic rats, for example. ⁶⁹

TIMPs (tissue inhibitor of metalloproteinase), TIMP-1, TIMP-2, TIMP-3 and TIMP-4, regulate several cellular mechanisms, depending or not on MMPs. ^{70 – 72}

In wound healing, TIMP-1 plays an important role in inflammation by modulating the expression of cytokines and growth factors (PDGF and TGF-β1); regulating cell differentiation, angiogenesis and apoptosis. ^{71 – 76} It also acts in the remodeling of the ECM, stimulating synthesis and degradation of protein. ^{72 , 73}

TIMP-1 increases proliferation of fibroblasts, ⁷⁴ protects quiescent endothelial cells ⁷⁵ and epithelial cells ⁷⁶ against TNF-α induced apoptosis through activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways and Akt mitogen-activated kinase (ERK/MAPK), respectively.

Together with Glycosylphosphatidylinositol (TIMP-1-GPI), TIMP-1 stimulates migration and proliferation of epidermal keratinocytes, reducing dermal myofibroblasts and the secretion of TGF-β, improving skin repair. ⁷⁶

IFN-γ is a pro-inflammatory cytokines (Type I: IFN-α and IFN-β; Type II: IFN-γ) produced by several cells, mainly by T-lymphocytes (Th1) and natural killer cells in response to viral infections and events involving inflammatory and mitogenic activities. ^{77 , 78} IFN-γ responds to type 1 (IFNGR1) and type 2 (IFNGR2) receptors ⁷⁹ and acts by signaling the JAK1/2 STAT pathway and tyrosine kinase 2 (TYK2). ⁸⁰

This glycoprotein has anti-proliferative and anti-fibrotic effects, inhibiting fibroblast proliferation, collagen, cytokines IL-4 and TGF-β synthesis ^{77 , 81} ; acts in the activation of macrophages and control the collagenase synthesis by these cells ⁸¹ ; regulates the immune activity of stem cells of different tissues. ⁸²

The presence of IFN-γ, at high levels, can delay or impair skin repair, ^{80 , 83 – 85} by decreasing angiogenesis, synthesis, deposition, organization and stability of collagen in ECM, and controlling cell proliferation. ^{86 , 87}

However, reduction or absence of IFN-γ leds to early angiogenesis, by increasing VEGF and decreasing cytokine CXCL10 levels, attenuating inflammation, proliferating keratinocytes and fibroblasts, increasing expression of FGF-7, IGF and EGF, improving reepithelization, collagen deposition and synthesis of TGF-β1, accelerating, consequently, healing and wound contraction. ^{82 , 88 , 89}

NF-kB family (p50/p105, p65/RelA, c-Rel/RelB and p52/p100) regulates genes involved in a variety of cellular processes, including skin healing, by activating and controlling the inflammatory response, the proliferative and migratory cellular activities, the expression of MMPs, the release and activity of growth factors, TGF-β and collagen, resulting in reepithelization ^{90 – 92} and ECM remodeling. ⁹³

The NF-kB can be activated through several cellular stimuli, such as the phosphorylation of the IkB kinase complex (IKK), decreasing the secretion of cytokines (TNF-α, IL-I and IL-6) in human dermal fibroblasts. ⁹³

However, the inhibition of the NF-kB signaling pathway by the IkB kinase inhibitor (IKK) complex resulted in marked tissue inflammation due to high expression of cytokines. In keratinocytes, the inhibition of NF-kB leads to skin inflammation, through TNF-α high expression, and epidermal hyperplasia. ⁹⁴

Growth factors and cytokines are essential mediators to initiate the skin healing process, which starts by an inflammatory phase, followed by epidermis and dermis proliferation and remodeling. ^{1 – 6}

During the inflammatory phase keratinocytes, in the epidermis, ^{15 , 16} produce IL-1. Like a positive-feedback, IL-1α and IL-1β act in the proliferation, differentiation and migration of keratinocytes, ^{14 – 16} resulting in reepithelization. ^{90 – 92}

Antagonists receptors IL-1Ra and IL-36Ra, generally act by inhibiting IL-1 in the presence of intense inflammation and/or cutaneous fibrosis, ¹⁹ accelerating the inflammatory phase and consequently closing the wound. ⁹⁵ IL-1Ra accelerated reepithelialization, ⁹⁶ leading to earlier skin healing in rats. ^{95 , 97} IL-1 also plays an important role in this phase of healing activating neutrophils and macrophages infiltration. ⁹⁵

As well described by the literature, PDGFs activate some inflammatory mediators, ^{9 , 12} induce ^{8 , 9 , 13} and control ⁹⁸ the granulation tissue formation.

Associated to KGF-1, PDGF-BB mediate the proliferation of keratinocytes from mesenchymal cells derived from adipose tissue during healing, inducing faster reepithelialization, resulting in an epidermis similar to that found in normal skin. ⁹⁹

PDGF-BB is also important for fibroblasts growing and collagen deposition, ¹⁰⁰ suggesting its effectiveness in repairing acute and chronic wounds, ⁹⁸ promoting ECM remodeling. ⁹⁴ In addition, PDGF-BB acts in the oxidative balance, decreasing (3rd day) and increasing (7th day) the levels of nitric oxide, ¹⁰¹ controlling the rate of wound contraction. ¹⁰²

The synthesis of KGF by fibroblasts is essential for modulating the proliferation and differentiation of keratinocytes. These cells interfere with the fibroblast response by modulating genes linked to ECM, changing phenotypes, synthesis of IL-1 and MMPs during healing. The balance between keratinocyte synthesis and apoptosis mediated by KGF is fundamental for the prevention of cutaneous fibrosis. ¹⁰³

Kerotinocytes and fibroblasts also activate TGF-β family, ^{36 – 42} VEGF and EPCs ^{47 , 48 , 98} improving formation of granulation tissue. ¹⁰³

TGF-β1 contributes to epidermis and dermis thickness, ¹⁰⁴ acting as an antiproliferative mediator in the healing phases, inhibiting migratory events and controlling cellular hypertrophy, usually caused by myofibroblast clusters. The suppression of myofibroblast synthesis and consequent inhibition of smooth muscle α-actin and collagen deposition is important in the control of hypertrophic healing. ¹⁰⁵

VEGF promotes angiogenesis, increases rate of wound contraction and, consequently, early skin healing. ¹⁰⁶ Low levels of VEGF and PDGF were found both in healing and in the intact skin of mice. ¹⁰⁷ Both VEGFR-1 and VEGFR-2 receptors ⁵⁸ are targets for VEGF binding, which induces cell proliferation. ¹⁰⁸

During healing, the persistent presence of pro-inflammatory M1 macrophages in the granulation tissue impairs the repair, however this event can be regulated by the signaling of the VEGFR-1 receptor, which acts on the balance of the levels of pro-inflammatory M1 and anti-inflammatory macrophages M2 to promote angiogenesis. ¹⁰⁸

The activation of VEGFR-2 increases expression of IL-10, which reduces macrophages and, consequently, inflammation and differentiation of myofibroblasts in cutaneous wounds, as well as increases density of angiogenesis, improving the quality of repair. ¹⁰⁹

Other pro-inflammatory cytokines synthesis, such as TNF-α ²² and NF-kB ²³ were activated by IL-1 and fibroblasts. TNF-α acts on the inflammatory process and on the proliferative and remodeling events of ECM during healing. ^{14 , 26} Its imbalance interferes in tissue repair, since at low levels TNF-α positively modulates inflammation, inducing proliferation of keratinocytes ¹¹⁰ and reepithelization ¹⁴ ; at high levels TNF-α inhibits cell migration, increases MMPs synthesis and ECM degradation. ¹⁴ Persistent inflammation, with M1 macrophage and neutrophil infiltration, TNF-α and TIMP-1 were observed in chronic skin wounds. ^{111 – 113}

NF-kB also acts on inflammation, proliferation and cell migration during skin repair, through the mediation of growth factors, collagen synthesis and MMPs, promoting re-epithelialization ⁹¹ and tissue remodeling. ⁹³

Keratinocytes also contribute to the regulation of TIMPs/MMPs activities, modulating ECM by inactivating MMPs, increasing TIMP-1. ¹¹⁴ Myofibroblasts also contribute to the regulation of TIMPs/MMPs during skin repair. ¹¹⁵

MMP-9 is involved in the inflammatory, ^{65 , 66} proliferative ^{67 , 68} and remodeling phases during skin healing. ^{63 , 64 , 66} Therefore, the balance between MMP-9 synthesis and degradation by TIMP-1 is essential for adequate repair. ⁶³ Both high levels of MMP-9 and very low levels impair the migratory activity of keratinocytes and consequently the reepithelialization and wound remodeling of epidermis. ¹¹⁶

Exogenous MMP-9 decreases collagen IV, delaying wound healing in rats, suggesting that MMP-9 interferes with the composition of basement membrane proteins, which prevent keratinocyte migration, accession and restructuring of the epidermis. ¹¹⁷

Just like the MMP-9, TIMP-1 also acts during the healing phases by modulating events related to ECM inflammation, proliferation, migration and remodeling ^{73 – 76} ; and the balance in their expression levels contributes to the prevention of fibrosis and to a better skin repair. ¹¹⁴

An increase in TIMP-1 and a decrease in MMP-9, ^{115 , 118} improved angiogenesis, wound contraction ¹¹⁵ and reduced inflammation. ¹¹⁸ TIMP-1-GPI blocked the secretion of MMPs by altering the association of MMPs with the cell surface, improving the proliferation of dermal fibroblasts and reducing the expression of fibrotic genes, suggesting that this complex may help control fibrosis during cutaneous healing. ¹¹⁹

As the proliferative phase is established, IFN-γ regulates the neutrophilic inflammatory responses ¹²⁰ and the immune activity of skin stem cells, ⁸² initiating the remodeling phase, ^{81 – 83 , 85 – 88} and the anti-proliferative and anti-fibrotic events, ^{78 , 83} as inhibits the proliferation of fibroblasts and, consequently, the synthesis of collagen, ¹²⁰ cytokines and TGF-β. ^{77 , 81} In complete absence of IFN-γ, collagen deposition and wound resistance to traction were reduced. ¹²¹