Mechanisms underlying pathological scarring by fibroblasts during wound healing

Abstract

Pathological scarring is an abnormal outcome of wound healing, which often manifests as excessive proliferation and transdifferentiation of fibroblasts (FBs), and excessive deposition of the extracellular matrix. FBs are the most important effector cells involved in wound healing and scar formation. The factors that promote pathological scar formation often act on the proliferation and function of FB. In this study, we describe the factors that lead to abnormal FB formation in pathological scarring in terms of the microenvironment, signalling pathways, epigenetics, and autophagy. These findings suggest that understanding the causes of abnormal FB formation may aid in the development of precise and effective preventive and treatment strategies for pathological scarring that are associated with improved quality of life of patients.

1. INTRODUCTION

Wound healing is a highly intricate process that involves the activation of various cell types, alterations in the wound microenvironment, and activation of various molecular signalling pathways. Physiological wound repair occurs in three main phases: haemostasis and inflammation proliferation and granulation tissue formation, and maturation and remodelling. Pathological scarring is an abnormal outcome of wound healing, which often manifests as excessive proliferation and transdifferentiation of fibroblasts (FBs), and excessive deposition of the extracellular matrix (ECM). It includes hypertrophic scarring (HS) and keloid (KD). HS is a wide, thickened, raised scar that is formed at the site of skin injury, can be pruritic, usually does not extend beyond the boundaries of the original wound, and will spontaneously resolve within 1–2 years. KD is often considered a benign tumour and is accompanied by itching and pain; it occurs predominantly in the earlobes, face, chest, and upper back as raised irregular growths that extend beyond the borders of the original skin injury site and invade adjacent tissues. Therefore, the lesion size may extend well beyond the edges of the initial trauma. ¹ The pathogenesis of HS and KD is not completely understood, and they cannot be completely cured. The quality of life of patients with HS or KD is reduced because of the loss of joint mobility due to scar contraction, emotional distress, and mental health problems due to cosmetic disfigurement. ²

As the most important effector cell involved in wound healing and scar formation, ³ FBs are mainly involved in wound contraction, granulation tissue formation, collagen (COL) production, and remodelling processes involved in wound healing. The mechanisms underlying pathological scarring and currently available treatment modalities involve FB proliferation and function to varying degrees. FBs are markers for scar formation and healing; however, there are few systematic analyses and summaries of factors that affect FB abnormalities. This review provides an in‐depth summary of the factors that affect the formation of pathological scars by FBs in terms of the microenvironment, signalling pathways, epigenetics, and autophagy. This review also discusses the molecular mechanism of scar formation to provide a basis for the future development of new therapeutic approaches (Figure 1).

FIGURE 1.

Schematic diagram of the factors affecting fibroblast abnormalities. The initiating factors such as inflammation, mechanical stress, hypoxia, and dysregulation of autophagy during wound healing affect fibroblast proliferation, migration, transdifferentiation, collagen production, and apoptosis by regulating the TGF‐β/smad pathway, wnt‐β/catenin pathway, Notch pathway, PI3K/Akt/mTOR pathway, TLR4/MyD88/NF‐κB pathway, and ncRNA. NICD: Notch intracellular domain；miRNA: microRNA； DNMT: DNA methyltransferase； HAT: Histone acetyltransferase； HDAC: Histone deacetylase.

2. PHYSIOLOGICAL WOUND HEALING PROCESS

The initial phase of physiological wound healing is called haemostasis, in which blood vessels constrict, platelets are activated and aggregated, and fibrin clots form to stop bleeding, prevent bacterial invasion, provide a scaffold for incoming immune cells, and store cytokines and growth factors in preparation for the inflammatory phase. ² The formation of a stable clot and the onset of leukocyte migration marks the initiation of the inflammatory phase of wound healing. During the inflammatory phase, resident immune cells, such as mast cells (MCs) and Langerhans cells are activated and release chemokines and pro‐inflammatory cytokines that recruit neutrophils, monocytes, and macrophages, thus activating the downstream inflammatory pathways. ⁴ Many experts believe that prolonged infection as well as chronic inflammation are key factors that contribute to pathological scar formation. ⁵

The proliferative phase follows the hemostatic/inflammatory phase and occurs between days 3 to day 21. FBs play a major role in this phase. This phase is characterised by the formation of granulation tissue. During the proliferative phase of healing, there is extensive activation of keratin‐forming cells, fibroblasts, endothelial cells, and macrophages to coordinate wound closure, stromal deposition, and angiogenesis.

The final phase of wound healing is the remodelling phase, which begins around three weeks after the injury and can last for up to one year or more. During this phase, matrix proteins (such as fibronectin and fibronectin) are degraded, COLIII is gradually replaced by COLI, macrophages, FBs, and endothelial cells undergo apoptosis, and finally wound healing is completed with scar formation. The remodelling phase is the final phase of wound healing, and pathological scars often form during this period. This is also the phase when patients often present for treatment. Therefore, the remodelling phase is a critical period for anti‐keloid treatment.

3. DEFINITION AND PHYSIOLOGICAL ROLE OF FBS

FBs are polymorphic cells and are generally called FBs in the resting state. They are the primary repair cells for wound healing and are one of the most important effector cells responsible for scar formation. ³ FBs typically appear three days after trauma and rapidly increase, wander toward the trauma site, proliferate, and synthesise extracellular matrix proteins, such as COL. Myofibroblasts (mFB) are a transient cellular state whose activation is a key event in physiological and pathological tissue repair. ⁶ MFBs which are primarily derived from TGF‐β1‐induced fibroblast transformation can also be derived from epithelial cells via epithelial‐mesenchymal transition, ⁷ epidermal stem cells, ⁸ bone marrow‐derived mesenchymal stem cells, ⁹ and endothelial‐mesenchymal transition process from endothelial cell transition. ¹ MFBs are the main ECM‐secreting cells during wound healing and fibrosis and are primarily responsible for the contractility of scar tissue during its maturation. The persistent presence of mFB that express α‐SMA is a typical feature of HS. ¹⁰ In addition, FBs may be involved in the regulation of inflammation, producing a variety of cytokines and growth factors such as VEGF, FGF2, EGF, TGF‐β, MMP, and tissue inhibitors of MMP. These molecules can affect their own functions as well as those of endothelial cells, keratin‐forming cells, and immune cells. ¹¹ , ¹²

4. ABNORMAL MICROENVIRONMENT IN PATHOLOGICAL SCARRING

4.1. Dysregulated inflammatory response

An unbalanced or persistent inflammatory response is a key factor that contributes to pathological scar formation. ⁵ During the remodelling phase of normal wound healing, matrix proteins (such as fibronectin and fibronectin) are degraded, COLIII is gradually replaced by COLI, and immune cells, FBs, and endothelial cells undergo apoptotic, ¹³ eventually completing wound healing. Long‐term inflammation can accelerate the transformation of FBs into mFBs. ¹⁴ If the injury stimulus or local factors are not removed in time and inflammation persists, macrophages, and mast cells (MCs) are constantly activated to reduce apoptosis, which affects the conversion of FBs and the deposition of COL through direct or indirect actions.

Macrophages are key players in wound repair and provide critical signalling molecules to coordinate wound healing. Activated macrophages are divided into two subtypes: those associated with inflammatory responses (M1) and those associated with tissue remodelling and anti‐inflammatory responses (M2). M1 macrophages phagocytose pathogens and cellular and tissue debris from the wound surface, secrete pro‐inflammatory factors and are involved in the inflammatory phase of wound healing. In comparison, M2 macrophages are predominantly involved in the proliferation and remodelling phases, participating in granulation tissue formation, remodelling, and termination of inflammation. ¹⁵ Sustained activation of M1 leads to chronic inflammation and delayed wound healing. In contrast, excessive activation of M2 leads to excessive fibrosis and scar formation. The number of M2 macrophages is significantly increased in keloid compared to normal skin. ¹⁶ M2 can manipulate the composition of the ECM, differentiate into mFBs, ¹⁷ and secrete a large number of pro‐fibrogenic factors, such as TGF‐β, CTGF, arginase, and IGF‐1. ⁹ , ¹⁰ CTGF acts as a downstream mediator of the TGF‐β1 signalling pathway, and CTGF acts through the STAT3/AKT/ ERK1/2 pathway to mediate the proliferation and migration of FBs. Additionally, it assists TGF‐β1 in promoting scar formation. ¹⁸ Chen et al. found that lncRNA‐ASLNCS5088 in M2 macrophage exosomes may regulate glutaminase by inhibiting the function of miR‐200c‐3p in FBs, which in turn affects FB transformation. ¹⁹ Normal wound healing depends largely on the balance of pro‐inflammatory M1 and anti‐inflammatory M2 macrophages.

MCs drive FB activation and COL remodelling. ²⁰ MCs are involved in all phases of wound healing. Early in wound healing, MCs are massively aggregated, activated, and degranulated at the injury site, which increases vascular permeability and recruits inflammatory cells. Then, MCs promote wound granulation, cell migration, angiogenesis, and COL maturation. ²¹ Finally, MCs promote COL deposition, and remodelling by regulating FB activity. ²² MCs have a greater role in influencing COL maturation and remodelling than in COL production. ²³ The number of MCs and quantities of secreted active transmitters, such as histamine and trypsin‐like enzymes, are significantly increased in pathological scarring, ²⁴ implying that MCs play a role in pathological scar formation. MCs can influence the actions of FBs by interacting directly with them directly in a paracrine manner or through heterocellular gap junctions. MC‐derived mediators, such as proteases, histamines, cytokines, and growth factors, enhance FB activation, transformation, and ECM and growth factor production. ²⁵ The MC stabilisers (tyrosine kinase inhibitors) inhibit the functional effects of MCs, which in turn inhibit inflammation, fibrosis, wound contraction, and COL deposition to form less dense and thinner collagenous protofibrils, ²⁶ which provides new avenues for neo‐scar treatment.

4.2. Abnormal mechanical stress during wound healing

Abnormal mechanical stress in the wound microenvironment induces hypertrophic scar formation by activating mechanotransduction pathways. YAP/TAZ is a key regulator of FB mechanical activation and fibrogenic function. In contrast to normal FBs, Keloid fibroblasts (KFBs) have higher levels of YAP/TAZ expression in primary culture. ²⁷ Mechanotransduction signalling can cause nuclear translocation of the transcription factors YAP and TAZ to activate Engrailed‐1 and the target gene SERPINE1 (encoding PAI‐1), ²⁸ leading to scarring or fibrosis that impairs organ function. Targeted inhibition of the YAP/TAZ pathway can promote wound regeneration through Engrailed‐1 spectrum‐negative fibroblasts (ENF) while restoring skin attachment, ultrastructure, and mechanical strength. ²⁹ It also has the potential to be used in the treatment of KD. The integrin‐focused adhesion kinase (FAK) pathway plays a central role in regulating the mechanical signalling in the skin. Alterations in mechanical signalling during wound healing and scar formation activate the FAK pathway, affecting downstream factors (such as PI3K and MAPK kinases) that mediate fibrotic responses. ³⁰ Piezo1 channels are novel mechanically activated cation channels. Mechanosensitive molecules, such as FAK, ERK, and YAP, that affect scar formation are Piezo1‐mediated Ca2+ pathway downstream effectors. Piezo1 channels may be key mediators involved in mechanical signalling to induce HS development. ³¹ Purposeful modulation of the pathways associated with mechanical tension or mechanotransduction signals at the wound surface during wound healing can reduce scar formation.

4.3. Hypoxic environment of the trauma surface

Hypoxia is considered to be an important cause of pathological scar formation. Abnormal and excessive microvascular formation is one of the prominent features of fibrosis. ¹⁴ Transient loss of vascular structures usually occurs after tissue injury, leading to a hypoxic state and upregulation of the hypoxia‐inducible factor 1 (HIF‐1) and PI3K‐AKT pathways. Activations of these pathways modulate FB proliferation, migratory invasion, and COL synthesis, as well as inhibit apoptosis, ³² which facilitates wound repair. The expression level of HIF‐1α is increased in KD. ¹⁴ HIF‐1α can promote KD development through the TGF‐β/Smad and TLR4/MyD88/NF‐κB pathways. ³³ The TGF‐β/Smad pathway and HIF‐1α can synergistically upregulate the VEGF expression in FBs, which drives endothelial cell proliferation and in turn leads to non‐functional blood vessel formation. Hypoxia also promotes FB glycolysis and increases COL production. ³⁴ In addition, the rapid proliferation of FB and excessive COL deposition in KD aggravate local tissue hypoxia, further exacerbating inflammation and hypoxia. This in turn, further increases the HIF‐1α expression, creating a malignant cycle. During the remodelling phase of wound healing, preventing hypoxia or inhibiting HIF‐1α expression on the wound surface is an effective method to prevent scar formation.

5. SIGNALLING PATHWAYS ASSOCIATED WITH PATHOLOGICAL SCAR FORMATION

5.1. TGF‐β/Smad

The TGF‐β‐Smad pathway is the most important regulator of the wound healing process and the one most strongly correlated with the formation of pathological scarring. TGF‐β binds to TβR‐II to phosphorylate TβR‐I, which promotes the formation of Smad2/Smad3/Smad4 complexes that translocate to the nucleus and bind to Smad‐binding elements (SBE) to regulate target genes such as ColI and ColIII. Phosphorylation of Smad2/3 by the TGF‐β receptor complex can be antagonised by Smad6 and Smad7. ³⁵ TGF‐β is synthesised and secreted mainly by inflammatory cells and FBs. It is upregulated in the early granulation tissue and is involved in several processes, such as inflammation in wound healing, stimulation of angiogenesis, proliferation of FB, and deposition and remodelling of ECM. ³⁶ Abnormalities of these processes can lead to pathological scar formation. Different TGF‐β types play different roles in wound healing. TGF‐β1 is considered to be the most potent cytokine that induces the conversion of FBs to mFBs and is upregulated in hypertrophic scar tissues, ³⁷ whereas elevated levels of TGF‐β3 promote scar‐free healing in fetal wounds. TGF‐β1 is a powerful chemoattractant for monocytes and macrophages as well as FBs. ³⁸ Smad3 and Smad4 bind directly to the Smad3‐binding element in the upstream region of the ASMA promoter, where it regulates the transcription of the α‐SMA gene. ³⁹ Smad3 interacts with the transcriptional co‐activator p300 and CREB‐binding proteins. It binds to the CAGACA sequence to stimulate CoLIII transcription ⁴⁰ and regulate ECM. TGF‐β1 interacts with calpain to promote platelet‐derived growth factor‐dependent COL deposition in FBs. ⁴¹ TGF‐β1 upregulates the expression of autophagy‐related proteins and inhibits fibroblast apoptosis through the endoplasmic reticulum (ER) stress pathway. ³⁷ Most of the pro‐fibrotic factors such as hypoxia and chronic inflammation can affect the function of FBs by differentially affecting the TGF‐β expression and secretion. The TGF‐β/Smad pathway plays a key role in pathological scar formation and is often used as a therapeutic target. However, TGF‐β inhibitors can attenuate fibrosis but cannot achieve scar‐free healing.

5.2. Wnt/β‐catenin

The Wnt/β‐catenin pathway is one of the major pathways that regulate angiogenesis and epithelial remodelling, which plays a critical role in epidermal stem cell maintenance, hair follicle development, regeneration, and FB‐mediated scar formation. ⁴² Wnt/β‐catenin signalling activation is characterised by stabilisation and nuclear translocation of β‐ catenin. Pathological activation of the Wnt signalling is associated with abnormal wound healing, leading to various fibrotic diseases. β‐catenin expression is significantly increased in KD tissues. ⁴³ Wnt overexpression enables mFB proliferation as well as ab initio transcription of ECM and matrix metalloproteinase genes. ⁴⁴ Human KD exhibits Wnt‐3a overexpression and induces the transformation of endothelial‐derived FBs into mesenchymal cells, which in turn leads to COL accumulation. ⁴⁵ Wnt3a administration increases postnatal mouse FB proliferation and CoLI production. ⁴⁶ The negative regulators of the Wnt/β‐catenin pathway (e.g., CXXC5, Dkk‐1, and IWR‐1) inhibit normal FB and KFB proliferation and migration, as well as CoLI production and secretion, and increase the expression of MMPs to further reduce CoL. ⁴⁷ Adenoviruses that express Wnt decoy receptors inhibit cytoplasmic β‐catenin stabilisation and reduce Wnt/β‐catenin signalling, and promote extracellular matrix degradation in pathologically scarred FBs. ⁴⁸ Crosstalk exists between the Wnt/β‐catenin and TGF‐β pathways, with β‐catenin stimulating the secretion of TGF‐β from FBs and TGF‐β causing upregulation of the Wnt/β‐catenin pathway. FB proliferation and its differentiation into mFBs through the activation of the TGF‐β signalling pathway occurs in a β‐linked protein‐dependent manner. ⁴⁷ Control of the Wnt/β‐catenin pathway may be an effective method to target and regulate the TGF‐β pathway in dermal fibrosis.

5.3. Notch

The Notch signalling pathway plays a key role in embryonic development, angiogenesis, wound healing, proliferation, and regulation of tissue homeostasis. Its dysregulation of this pathway may lead to cellular transformation and tumorigenesis. ⁴⁹ Activation of the classical Notch pathway usually requires binding between Notch receptors and Notch ligands exposed on adjacent cells. This binding triggers secondary enzymatic cleavage, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and binds to the CSL (RBP‐J) and MAML families to form the Notch ICD/CSL/MAML complex. This complex regulates the transcription of downstream genes and thus mediates a variety of biological effects. ⁵⁰ NICD is highly expressed in KFBs and HFBs, while it is low or absent in normal FBs. ⁵¹ JAG‐1 and Notch signalling pathway activation or inhibition significantly alter primary KFB in cell migration, invasion, and angiogenesis. ⁵² NICD‐SiRNA‐treated NFBs and HFBs have reduced FB viability and migration, inhibited fibrogenic factor production, including ColI, ColIII, α‐SMA, and TGF‐β1 production, and increased apoptosis. ⁵³ He et al. ⁵⁴ found that RBP‐J knockout mice had significantly inhibited COL deposition after wound healing and suppressed inflammatory cytokine expression in LPS‐stimulated activated macrophages. Patel et al. ⁵⁵ found that the endothelial cells in transgenic mice with loss of Notch signalling were rapidly converted to mesenchymal cells, which stimulated scar tissue formation. Notch signalling can also regulate FB proliferation and affect pro‐inflammatory macrophage polarisation, ⁵⁶ activation of keratin‐forming cells, and by affecting NF‐κB and TGF‐β/Smad signalling pathways. ⁵⁷

5.4. Other pathways

The PI3K/Akt/mTOR pathways are important targets in KD pathogenesis. Syed et al. found mTOR hyperactivation in KD compared to corresponding extra‐lesional regions. ⁵⁸ Activation of the PI3K/Akt/mTOR pathway enhances KFB migration and proliferation and promotes inflammation, angiogenesis, and COL deposition. ⁵⁹ CD26 can act as an effective biomarker of KFBs, CD26 + FBs exhibit enhanced proliferation and invasion. CD26 increases the level of phosphorylation of S6 kinase and 4 E‐binding protein through the IGF‐1‐induced PI3K/AKT/mTOR signalling pathway to increase KF proliferation and invasion. ⁶⁰ Inhibition of the PI3K pathway by dipeptidyl peptidase 4 inhibitors leads to anti‐fibrotic effects in KD progression. ³ mTOR is a regulator of COL expression in dermal FBs. Rapamycin reduces ECM deposition by inhibiting mTOR. ⁶¹ The PI3K/Akt/mTOR pathway leads to ECM overproduction in KD, and targeting the mTOR pathway is a potential therapeutic approach to eradicate KD.

The TLR4/MyD88/NF‐κB pathways play an important role in scar formation. Toll‐like receptor (TLR) is a transmembrane protein that recognises multiple types of pathogen‐associated molecular patterns. TLR acts as an anti‐inflammatory immunomodulator by activating the MyD88‐dependent pathway, which leads to the activation of NF‐κB and ultimately to the release of inflammatory mediators and cytokines. The transcription factor NF‐κB participates in the progression of immune, tumorigenic, and fibrotic processes. ⁶² In contrast to NSFs, TLR4 is overexpressed in HFs at both mRNA and protein levels. ⁶³ FBs regulate the immune and inflammatory responses through LPS‐activated TLR4, leading to NF‐κB activation as well as cytokine and costimulatory molecule expression. These changes result in inflammation during HS formation. ⁶⁴ The anti‐inflammatory factor IL‐10 can modulate the TLR4/NF‐κB pathway in dermal fibroblasts to attenuate LPS‐induced skin scar formation and pro‐fibrogenic factor expression. ⁶⁵

6. EPIGENETIC ALTERATIONS ASSOCIATED WITH PATHOLOGICAL SCAR FORMATION

6.1. Non‐coding RNA

The non‐coding RNAs (ncRNAs) include miRNAs, lncRNAs, and CircRNAs. A large number of studies have demonstrated the important roles of ncRNAs in the trauma healing process. MiRNAs are short ncRNAs with an average length of 22 nucleotides. Mature miRNAs are incorporated into RNA‐induced silencing complexes and bind to the 3′ untranslated region of the target mRNA, resulting in translational repression or degradation of the target mRNA. ⁶⁶ During pathological scar formation, the target mRNA can be COL, a‐SMA gene expression product, or the transcriptional products of the TGF‐β/Smad pathway, WNT pathway, NOTCH pathway, or other fibrotic signalling pathways. MiRNAs are involved in transcriptional and post‐transcriptional regulation of gene expression in skin fibrotic diseases, such as Squamous cell carcinoma and KD. Many miRNAs are involved in regulating FB activity, including aberrant proliferation, mFB activation, autophagy, apoptosis, migration, and COL production. Table 1 summarises the alterations and mechanisms of miRNAs in scar formation. LncRNAs are a long‐stranded group of ncRNA molecules, with an average length of 200 nucleotides and limited protein‐coding potential. LncRNAs are involved in the regulation of gene transcription, ⁶⁷ and lncRNAs have functional interactions with miRNAs. Dysregulated expression of lncRNAs is essential during keloidogenesis. CircRNAs consist of covalently closed contiguous loops of ncRNAs that have the ability to encode proteins and regulate gene transcription. CircRNAs have abundant miRNA binding sites. ⁶⁸ Aberrant expression of circRNAs plays a vital regulatory role in the development and progression of fibrotic diseases, such as liver fibrosis, cardiac fibrosis, and KD. LncRNAs and circRNAs can drive pathological scar formation by regulating the miRNAs in FBs. ⁶⁹ , ⁷⁰ Existing studies on the mechanisms of lncRNAs and circRNAs in pathological scar formation are mainly based on the indirect role of miRNAs. Their direct effects on transcription and translation products in scar FBs should be further investigated in the future.

TABLE 1.

Expression and mechanism of Non‐coding RNA (ncRNA) in pathological scarring.

miRNA	Expression in pathological scarring	Correlation with scar growth	Target genes	Effects of miRNA	Pathological scar types	LncRNA/CirRNA and its effects
miR‐29a	Downgrade	Negative	COL3A1、COL1A2	Decrease the expression of ColI and ColIII of KFB and inhibit the viability, proliferation, migration, and invasion of fibroblasts	KD	H19 is upregulated in KD and promotes fibroblast proliferation and metastasis by sponging downstream miR‐29a and COL1A1	84 ； 85
miR‐196a	Downgrade	Negative	COL1A1、 COL3A1	Resulting in decreased levels of type I/III collagen secretion	KD		86
miR‐124‐3p	Downgrade	Negative	TGFβR1、Smad5	Targeted regulation of TGFβR1 and Smad5 to inhibit fibroblast proliferation, migration and trigger apoptosis, and reduce collagen production	KD	HOXA11‐AS inhibits apoptosis and promotes angiogenesis by sponging miR‐124‐3p, and promotes Smad5 signalling to induce ColI synthesis, thereby promoting KD formation	87 , 88
miR‐637	Downgrade	Negative	Smad3	Inhibits cyclin D1, suppresses MMP2, and HKF cell metastasis, regulates KFBs proliferation and metastasis	KD		89
miR‐3141	Downgrade	Negative	TGF‐β1	Inhibits the proliferation and migration of KFBs and promotes apoptosis	KD	LINC01116 is upregulated in KD, sponging miR‐3141 regulates TGF‐β1/SMAD3 signalling, promotes KFBs proliferation and migration, and promotes apoptosis.	90
miR‐203	Downgrade	Negative	EGR1、FGF2、SMAD5	Inhibits proliferation, invasion, and ECM production of KFBs and triggers apoptosis	KD	LINC01116 promotes the progression of KD formation by targeting miR‐203 to regulate the expression of SMAD5	91 , 92
miR‐196b‐5p	Downgrade	Negative	FGF2	Inhibited the viability, migration and ECM protein production of KFBs	KD		93
miR‐205‐5p	Downgrade	Negative	VEGF	Reduced VEGF expression‐mediated PI3K/Akt signalling impairs cell viability, induces apoptosis, and inhibits the cell invasion and migration ability of KFBs to inhibit keloid formation	KD		94
miR‐133a‐3p	Downgrade	Negative	IRF5	Inhibition of TGF‐β/Smad2 pathway through downregulation of IRF5 inhibits fibrosis, promotes apoptosis and reduces the proliferation of KFs	KD		95
miR‐200c	Downgrade	Negative	ZNF217	Targeting ZNF217 regulates the autocrine secretion of TGF‐β	KD	lncRNA‐ATB controls TGF‐β2 autocrine secretion in KFs by reducing the expression level of ZNF217 through miR‐200c	96
miR‐138‐5p	Downgrade	Negative	CDK6	Decreased CDK6 expression and significantly increased apoptosis	KD	circ_101238 is upregulated in KD and regulates the miR‐138‐5p/CDK6 axis to promote inhibition of apoptosis in KFBs	97
miR‐4417	Downgrade	Negative	CyclinD1	Inhibition of proliferation, apoptosis, migration and invasion of KFBs	KD		98
miR‐194‐3p	Downgrade	Negative	RUNX2	Inhibition of CDK4 and MMP2 to suppress the proliferation and migration of KFBs	KD		99
miR‐1‐3p，miR‐214‐5p	Downgrade	Negative	TM4SF1	Inhibition of TM4SF1 expression, activation of AKT/ERK signalling pathway, cell proliferation, migration and induction of apoptosis in HKFs	KD		100
miR‐34a	Downgrade	Negative	SATB1	Inhibition of proliferation, migration and invasion of KFBs	KD		101
miR‐141‐3p	Downgrade	Negative	GAB1	Inhibition of GAB1 expression to reduce proliferation and migration of keloid fibroblasts	KD		102
miR‐148b‐3p	Downgrade	Negative	IGFBP5	Inhibits KFBs, migration and triggers apoptosis	KD	HOXA11‐AS promotes keloid progression, promotes cell proliferation, migration and inhibits apoptosis via miR‐148b‐3p/IGFBP5 axis	103
miR‐194‐5p	Downgrade	Negative	NR2F2	Inhibits proliferation, apoptosis, migration and invasion of KFBs	KD		104
miR‐205	Downgrade	Negative	THBS1	Antagonistic effect on proliferation and invasion of KFDs and promotion of apoptosis	KD	CACNA1G‐AS1 upregulates and targets antagonism of miR‐205 in KD as well as KDFs	105
miR‐1587、miR‐2392	Downgrade	Negative	ZEB2	Inhibition of proliferation and migration of KFBs and promotion of apoptosis and autophagy	KD		106
MiR‐21	Upgrade	Positive	FasL、PTEN、Smad7	Inhibits FasL‐induced caspase‐8 and mitochondria‐mediated apoptotic signalling pathways to regulate KF apoptosis; regulates PTEN/AKT signalling pathway to promote KFs transdifferentiation, MMP expression and secretion, and migration; negatively regulates Smad7 to promote Col1A1 and Col3A1 expression in KFB; upregulates α‐SMA and downregulates E‐calmodulin	KD		107 , 108 , 109
miR‐21‐5p	Upgrade	Positive	Smad7	Activation of Wnt pathways to accelerate the growth of KFBs	KD	circPTPN12 is downregulated in KD, sponging miR‐21‐5p	110
miRNA‐31	Upgrade	Positive	HIF1AN	Regulation of proliferation, apoptosis and cell cycle of KFBs by mediating the HIF1AN/VEGF signalling pathway	KD		111
miR‐152‐3p	Upgrade	Positive	FOXF1	Promotes cell proliferation, invasion and ECM expression, including ColI, ColIII and fibronectin	KD		112
MiR‐181a	Upgrade	Positive	PHLPP2	MiR‐181a targets PHLPP2 to enhance AKT signalling and regulate human KF proliferation and apoptosis	KD		113
miR‐98	Downgrade	Negative	Col1A1	Inhibition of HSFBs migration and collagen expression	HS		114
miR‐145‐5p	Downgrade	Negative	Smad2/3	Inhibits the activation and proliferation of HSFs, induces apoptosis of HSFs, prevents the development of fibrosis and reduces the formation of HS	HS		115
miR‐152‐5p	Downgrade	Negative	Smad3	Inhibits cell migration and promotes apoptosis, reduces ColIII production	HS		116
miR‐205‐5p	Downgrade	Negative	smad2	Induction of apoptosis in HSF cells, prevention of AKT phosphorylation‐mediated effects on ECM production‐related proteins	HS		117
miR‐155	Downgrade	Negative	HIF‐1a	Inhibition of collagen expression in vitro and inhibition of collagen fibre alignment and FB proliferation in vivo	HS		118
miR‐9‐5p	Downgrade	Negative	PPARβ	Inhibition of extracellular matrix‐associated genes (α‐SMA, waveform protein, COL1A) levels in HSF, induction of HSF apoptosis	HS		119
miR‐10a	Downgrade	Negative	PAI‐1	Reduction of ColI expression and upregulation of MMP1	HS		120
miR‐137	Downgrade	Negative	PTN	Inhibition of cyclin B1 by PTN to inhibit the proliferation of HSFBs and inhibition of MMP9 to inhibit the metastasis of HSFBs	HS		121
miR‐143‐3p	Downgrade	Negative	CTGF	Regulation of proliferation and apoptosis of HSFs, inhibition of hypertrophic scar formation by targeting CTGF to inhibit the expression of ECM production‐related proteins and inhibition of the Akt/mTOR pathway	HS		122
miR‐205	Downgrade	Negative	THBS1	Inhibits the growth and migration of HSFs, hinders HS growth and collagen formation	HS		123
miR‐495	Downgrade	Negative	FAK	Inhibition of HSF growth, inhibition of COL1A expression, stimulation of pro‐apoptotic gene expression, promotion of FB apoptosis	HS		124
miR‐519d	Downgrade	Negative	SIRT7	Targeting SIRT7 inhibits the expression of ECM‐related genes, reduces the proliferation of HSF and induces apoptosis	HS		125
miR‐3613‐3p	Downgrade	Negative	ARGLU1	inhibited the expression of ECM production‐related proteins and promoted the activation of Caspase‐3 and Caspase‐9 in HSF	HS		126
miR‐21	Upgrade	Positive	PTEN	Targeting the PTEN/PI3K/AKT signalling pathway regulates hTERT expression and thus controls HSFs growth	HS		127
miR‐181a‐5p	Upgrade	Positive	PTEN	Inhibition of PHLPP2 expression promotes cell proliferation and suppresses apoptosis, leading to activation of the AKT pathway and thus accelerating pathological scar formation.	HS	lncRNA TRHDE‐AS1 acts as a molecular sponge for miR‐181a‐5p to regulate PTEN expression	128
miR31‐5p	Upgrade	Positive	FIH	Activates the HIF‐1α fibrosis regulatory pathway in HSFs and stimulates HSFs proliferation and ECM synthesis, ultimately leading to fibrosis and scar formation	HS		129
miR‐145	Upgrade	Positive	KLF4	Promotion of α‐SMA expression in primary FB, contractility and migration of mFB, promotion of ColI and TGF‐β1 production, and	HS		130
miR‐181b	Upgrade	Positive	DCN	Promote TGF‐β1‐induced down‐regulation of DCN in HSFs and up‐regulation of upregulation of m‐differentiation	HS		131
miR‐181c	Upgrade	Positive	uPA	Increased type I collagen expression and down‐regulation of MMP1	HS		119

Abbreviations: IRF5: Interferon regulatory factor 5; ZNF217: Zinc finger gene 217; CDK6: Cyclin‐dependent kinase 6; TM4SF1: transmembrane‐4‐l‐six‐family‐1; SATB1: special AT‐rich sequence binding protein; GAB1: growth factor receptor binding protein1; IGFBP5: Insulin‐like growth factor binding protein‐ 5; NR2F2: nuclear receptor subfamily 2, group F, member 2; THBS1: Platelet reactive protein; ZEB2: Zinc finger E‐box Binding homeobox 2; FOXF1: Forkhead box protein F1; PHLPP2: PH domain leucine‐rich repeat protein phosphatase 2; PPARβ: peroxisome proliferator activated receptors‐β; PAI‐1: Fibrinogen activator inhibitor 1; FAK: Focal Adhesion Kinase; SIRT7: Silent Mating Type Information Regulation 2 Homologue 7; ARGLU1: arginine and glutamate‐rich 1 ; TRHDE‐AS1: Thyrotropin‐releasing hormone degrading enzyme antisense RNA 1; KLF4: Krüppel‐like factor4; DCN: Core proteoglycan gene; uPA: Urokinase‐type fibrinogen activator.

6.2. DNA methylation

DNA methylation is the chemical modification process of transferring methyl groups to cytosines via cytosine DNA methyltransferase (DNMT), most commonly the CpG (cytosine linked to guanine via a phosphate group, cytosine‐phosphate‐guanine, 5′‐CpG‐3′) site. ⁷¹ Loss of scar‐free healing capacity is followed by extensive changes in DNA methylation expression profiles. Based on a genome‐wide scan of CpG sites in KD, a total of 100 000 differentially methylated CpG sites were identified, of which 20 695 were hypomethylated and 79 305 were hypermethylated. ⁷² DNA methylation is involved in the integration process and various aspects of KD formation, including cell proliferation, invasion, mFB activation, COL deposition, and pigmentation. ⁷³ DNA methylation is increased in the CpG island of the CDC2L1 gene promoter region in KD tissues. It significantly suppresses the CDK11p58 protein expression in KD and inhibits FB apoptosis. ⁷⁴ DNA methylation is the most common epigenetic modification that plays an essential role in pathological KD pathogenesis, and DNA methylation is the ability to be a potential therapy that can reverse destructive epigenetic modifications.

6.3. Histone modification

Post‐translational histone modifications, such as acetylation, ubiquitination, and phosphorylation, are epigenetic mechanisms that regulate chromatin structure and gene expression by modulating the degree of chromatin compression. ⁷⁵ Histone acetylation is the most commonly studied histone modification in KD. ⁷⁶ Histone acetylation is a dynamic process regulated by the interaction of histone acetyltransferase (HAT) and histone deacetylase (HDAC). ⁶⁹ In KD, the overproduction of HDAC2 and the ability of TSA and CUDC‐907 to inhibit HDAC promotes the acetylation of histone H3, which inhibits the proliferation, invasion and ECM deposition of KFBs. ⁵⁹ , ⁷⁷ , ⁷⁸ The pro‐fibrotic transcriptional pattern in FBs is regulated by histone modifications. Additionally, regulatory histone modifications, such as CUDC‐907 and TSA may reverse the fibrosis of KD.

7. EFFECT OF AUTOPHAGY OF FBS IN PATHOLOGICAL SCAR FORMATION

Autophagy is a lysosome‐dependent catabolic mechanism that transports abnormal cellular contents to lysosomes for degradation to maintain cellular homeostasis. Under physiological conditions, autophagy facilitates cellular stability in response to adverse environments. Under pathological conditions, excessive autophagy causes “autophagic cell death”, which is considered as type II programmed cell death. ⁷⁹ An increasing number of studies have shown that autophagy is closely related to the maintenance, activity, differentiation, and apoptosis of cutaneous FBs during wound repair. Shi et al. found that HF exhibited higher levels of autophagy than normal FBs. ⁸⁰ Gingival tissue heals faster than skin and is less prone to scar formation. Recent studies have shown that gingival tissue wound healing does not involve autophagy, which results in reduced mFB differentiation and decreased COL deposition. ⁸¹ The transcription factor EB (TFEB) is a key regulator of lysosomal biosynthesis and autophagy. Zhou et al. found that TFEB‐mediated autophagy can maintain FB survival and function by degrading misfolded or unfolded proteins Alternatively, during secretory autophagy, misfolded or unfolded COLI can be secreted into the extracellular environment via autolysosomes. This misfolded or unfolded COLI is likely to be misassembled and irregularly aligned COL fibres in the HTS. ³⁷ Downregulation of FB autophagy induces apoptosis, which degrades excess intracellular fibronectin, ameliorates excessive ECM deposition, and inhibits fibrosis progression. ⁷⁹ Deng et al. found that inhibition of autophagy with oxymorphone resulted in smaller scar formation, reduced epidermal and dermal thickness, and significant downregulation of pro‐fibrotic factors in wound repair. ⁸² However, the mechanisms of autophagy regulation of FB apoptosis and fibrosis appear to be complex. Several studies have shown that the presence of autophagic death can induce FB apoptosis and alleviate scar formation during wound healing. Rapamycin, an mTOR inhibitor, enhances cellular autophagy and downregulates the pro‐inflammatory and mFB differentiation of the Notch1‐NLRP3 inflammasome signalling pathway in KD. ⁵⁷ Another study showed that autophagy can inhibit the blockade of the NLRP3 inflammasome signalling pathway and TGF‐β to alleviate tubulointerstitial fibrosis. ⁸³ Whether the upregulation of autophagy promotes FB proliferation or leads to further autophagic death, and how it affects other signalling pathways in FB, needs to be further elucidated in future studies. Regulation of autophagy is expected to be a new target for the treatment of skin scarring and skin fibrosis.

8. CONCLUSIONS

The pathogenesis of pathological scarring is still not fully understood. Inflammatory responses, mechanical stress, hypoxic conditions, and genetics all play a role in its development. Each factor causes FB abnormalities to various degrees. The abnormal trauma repair process affects the proliferation and function of FB due to the trauma microenvironment, signalling pathways, epigenetics, and autophagy. However, these factors are not independent of each other. Excessive hypoxia causes persistent chronic inflammation, which promotes abnormal proliferation of FBs and endothelial cells, as well as TGF‐β expression, leading to abnormal blood vessel formation and COL deposition, thereby further aggravating hypoxia. Because these factors interact and promote each other, the treatment of KD is complex, and in‐depth studies are needed to clarify the mechanism of FB abnormalities in pathological scarring, clarify the extent of the role of various factors, and achieve precise treatment of pathological scarring.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Ma Y, Liu Z, Miao L, et al. Mechanisms underlying pathological scarring by fibroblasts during wound healing. Int Wound J. 2023;20(6):2190‐2206. doi: 10.1111/iwj.14097

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this scoping review article, as no new data were created or analysed in this study.