Oral Submucous Fibrosis as an Overhealing Wound: Implications in Malignant Transformation

Mohit Sharma, Smitha S. Shetty and Raghu Radhakrishnan
1 Department of Oral Pathology, Sudha Rustagi College of Dental Sciences & Research, Greater Faridabad – 121002, Haryana (India);
2 Department of Oral Pathology, Faculty of Dentistry, Melaka Manipal Medical College, Manipal University, Manipal – 576104, India;
3 Department of Oral Pathology, Manipal College of Dental Sciences, Manipal University, Manipal – 576104, India

Oral submucous fibrosis is an oral potentially malignant disorder with high incidence of malignant transformation and rising global prevalence. However, the genesis of oral sub- mucous fibrosis is still unclear despite superfluity of literature. In the background of ineffective treat- ment, it is necessary to decode its onset and progression before designing customized treatment regi- mens.
The objective of this article is to decipher the pathogenesis of oral submucous fibrosis in order to identify novel drug targets.
A thorough literature review based on oral submucous fibrosis being an overhealing wound was conducted; several related patents were identified and herewith reviewed. Necessary pathways were elaborated and deliberated in the manuscript in the form of schemas, keeping our hypothesis in mind. Several novel molecular targets were identified and discussed in detail.
Several patents demonstrating inhibition of fibrosis via chemokine ligand mimetics, anticon- nexon antibodies, stem cell therapy, fibronectin blocking peptides, HIF inhibitors, recombinant erythropoietin, xanthine oxidase inhibitors, long non-coding RNAs, targeting inflammation, increasing TH-1/TH-2 cytokine ratio, t-box protein 4, chromium containing compositions, Iron-based nanocom- posites, Lactate Dehydrogenase-5 inhibitors, Carbonic Anhdrase-9 inhibitors, proton pump inhibitors, liposomal encapsulated glutathione, monocarboxylate-4 inhibitors, autophagy inhibitors, Submucosal anti-IL-6 antibodies, fibrin degradation products for monitoring of malignancy and fibrosis, small molecule antagonists like vorapaxar, tiplaxtinin, and TM-5275, TGF-β signalling inhibitors were iden- tified as future therapeutic avenues.
Considering, oral submucous fibrosis as an overhealing wound explains both pathogenesis and malignant transformation. Certainly, abnormalities in coagulation and fibrinolytic system are a common denominator in the profibrotic milieu and associated malignancy.

A steady increase in the availability of Betel Quid (BQ) along with a concomitant increase in the incidence of Oral Submucous Fibrosis (OSF) grants it the status of a future global epidemic [1]. In the backdrop of ineffective treatment [2], it is necessary to decode its onset and progression, before designing a tailored treatment algorithm. The mechanismof OSF genesis is still vague, although ample literature is available. Likening OSF to a normal wound healing is the premise of this review (Fig. 1). Based on our literature re- view, we suggest OSF as an overhealing wound due to chronic injury to the oral mucosa. Indeed, the proximity of gutkha or areca nut with oral mucosa and subsequent devel- opment of OSF in that region endorses such an inference. However, considering OSF as an overhealing wound solely due to chronic physical injury to oral mucosa fails to explain the occurrence of OSF in multiple sites in unilateral chewers. Nevertheless, the concept of salivary pooling addresses this discrepancy [3].
OSF probably represents an overhealing wound because of chronic physical, chemical, and mechanical injury to the oral mucosa [4-6]. Accordingly, the stages of progression of OSF correspond to the stages of maturation of granulation tissue [7]. Moreover, immunolocalization patterns of ma- tricellular proteins perlecan and fibronectin implicate them in the maturation of this granulation tissue into fibrosis [7]. Additionally, Monocyte Chemoattractant Protein-1 (MCP- 1)/Chemokine (C-C motif) Ligand 2 (CCL-2) based recruit- ment of myofibroblast to the site of epithelial injury in OSF, lends further credibility to this hypothesis [6]. Furthermore, upregulated CCL-2 consequent to chronic epithelial micro- trauma neutralizes epithelially derived antifibrotic prosta- glandin E-2 (PGE-2) via C-C chemokine receptor type 2 (CCR2) or cluster of differentiation 192 (CD192), promoting fibrosis [8]. Certainly, the use of CCR2 antagonists for inhi- bition of fibrosis could be envisaged (US8841313) [9]. An orally active antifibrotic agent blocking the binding of MCP- 1/CCL2 to CCR2 and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES)/Chemokine (C-C motif) ligand 5 (CCL-5), Macrophage Inflammatory Protein- 1a (MIP-1a), and Macrophage Inflammatory Protein-1β (MIP-1β) to C-C chemokine receptor type 5 (CCR5) could also be expected (EP3191100) [10]. Such a mechanism also explains the frequent juxtaposition of atrophic epithelium with subjacent connective tissue fibrosis [11]. Epithelial in- jury might lead to fibrosis of subjacent connective tissue viarecruitment of the myofibroblast to these sites. The subse- quent fibrosis mediated vascular rarefaction (vessel com- pression or obliteration) [12] denies nutrients and oxygen to mucosa leading to its atrophy. In line with such a conclusion, the role of other chemokines as additional recruiting agents for myofibroblasts and inflammatory cells, and their subse- quent extrapolation to the fibrotic process of OSF merits further exploration [13].

The purpose of fibrosis is to shield an injured tissue from relentless trauma and allow the substitution of lost and dam- aged cells [14]. In simple terms, it is an attempt of the body to encapsulate injured tissue (US8975237) [15]. The damage to the plasma membrane leads to release of intracellular ATP, through gap junctions (made up of two hexameric con- nexons that form a channel between adjacent plasma mem- branes) [15, 16]. This extracellular ATP serves as a chemo- tactic signal for inflammatory cell recruitment in the area for the purpose of repair and fibrosis [16]. Indeed, anticonnexon antibodies do retard fibrosis (US8975237) [15]. The persis- tent physical and chemical trauma to oral mucosa owing to habitual BQ chewing is liable for the genesis of OSF. The role of physical, chemical, mechanical trauma and matrix stiffness in the genesis of OSF is discussed in sections 2.1, 2.2, 2.3, 2.4, 2.5.1, 2.5.2, 2.6.1 & 2.6.2.

2.1. Role of Physical Trauma
The abrasive character of areca nut (AN) inflicts local- ized microtrauma (LMT) to the oral mucosa [17]. The con- stant LMT and consequent incessant remodeling in the ECM of injured mucosa transforms mechanical microenvironment via disruption of the protective nature of the cross-linked ECM, which in its native state stress shields the resident fi- broblasts [18-20]. Additionally, loss of rete ridges due to atrophy decreases the surface area of epithelial connective tissue interface, concentrating forces on fibroblast leading to their augmented differentiation into myofibroblasts (Fig. 2, Bx-1). As a result, the fibroblasts acquire contractile stress fibers composed of cytoplasmic actins. Eventually, these proto-myofibroblasts differentiate into contractile and secre- tory myofibroblasts with a de novo expression of a-Smooth Muscle Actin (a-SMA) [18]. LMT also increases the permeability of oral mucosa by inducing breaks and assists the ingress of BQ derived alkaloids and flavonoids into subepithelial connective tissue (Fig. 2) [6, 17]. Hence, the LMT may incessantly act as a trigger for myofibroblast dif- ferentiation in OSF. As masticatory mucosa has more rete ridges per unit area as compared to lining-mucosa, it is rela- tively resistant to OSF and seldom OSF affects masticatory mucosa [21]. Indeed, consistent with this inference gingival fibroblast do not readily adopt a full-blown myofibroblast phenotype, in response to TGF-β [21, 22].
The fine particulate character of Pan Masala and Gutkha (PMG) and high probability of adhesion to traumatized mu- cosa imparts severe mechanical irritation and injury to the oral mucosa [6], underpinning the earlier onset of OSF in PMG chewers when compared to AN chewers (Fig. 2) [23]. Based on our literature survey, any abrasive substance is capable of causing OSF if chewed chronically. Misi is a sub- stance used by the female villagers in some parts of India. It is a “black coloured powder containing various chemical substances like washing soda, borax, powdered alum, char- coal of myrobatan and fullers earth in varying proportions” [24]. Indeed, its composition seems to endow it with the po- tential to cause OSF according to our suggestion, which in- deed was the case in this rare and only study on this agent [24].
BQ chewing also has a drying effect on the oral mucosa [25] mediated through the desiccating action of its constitu- ent arecoline [26]. This further augments the permeability of the BQ chewers mucosa via epithelial shrinkage [26] leading to enlargement of intercellular spaces and gaps in basement membrane (Fig. 2) [27]. The mechanical irritation to the oral mucosa by the coarse fibers of areca nut also assists in perco- lation of these ingredients (Fig. 2) [5].

2.2. Stem Cell Activity in Fibrosis
Oral mucosa depends on basal stem cell layer for self- renewal. The increased permeability of the epithelium allows the stromal access to BQ associated carcinogens and irritants [28] leading to stem cell hypoplasia with ensuing loss of rete ridges (Fig. 2). The integrity of basal stem cell layer is essen- tial for the epithelial homeostasis and its disruption initiates fibrosis [29, 30] (Fig. 2).
All these studies testify a definite impairment in the self- renewing capacity of the basal stem cell layer in OSF with atrophic epithelium. Actually, stem cell therapy could treat several types of fibrosis in the body (WO2018003997, WO2016054155, US20100143312) [31-34]. Even regenera-tion of normal tissue from fibrotic tissue has already been contemplated through stem cell therapy (US20160312223) [35]. Unquestionably, stem cell therapy in OSF has shown promising results [36, 37]. Thus, restoration of renewal abil- ity of basal stem cell layer via stem cell therapy should be the fundamental and obligatory component of OSF therapy and will take care of both fibrosis and the developing malig- nancy [37-40].
Considering the fact, that oral mucosa is severely atro- phic in OSF the development of hyperplastic Oral Potentially Malignant Disorders (OPMDs) like leukoplakia and erythro- plakia deserve serious thought, as it signifies the switchover from fibrosis to malignancy [41].

2.3. Role of Chemical Trauma
BQ constituents released during chewing cause chemical trauma to the oral mucosa [5]. Slaked lime or aqueous cal- cium hydroxide, one of the BQ constituents, due to its strong alkaline nature (pH=11) can cause chemical irritation to the oral mucosa [6, 42]. Also, the orientation of fibrotic bands produced on exposure of mucosa to an alkali like sodium hydroxide (pH=13.5-15), is indistinguishable from the ones seen in OSF [43]. Undeniably, collagen fiber orientation in OSF is similar to other desmoplastic states like scleroderma, keloid, and hypertrophic scars. All of these show collagen fiber orientation parallel to the epithelium, in contrast to loose and randomly arranged collagen fibers in normal epi- thelium [44, 45].
With every chew the in-vivo salivary pH in BQ chewers rises rapidly to 10; however, an alkaline pH of 8–8.2 is suffi- cient for fibroblast toxicity and death [42]. A phenomenon akin to natural selection operates and leads to destruction and replacement of most of the original oral mucosal fibroblasts by phenotypically altered fibroblasts in OSF [42, 46]. The original fibroblasts have a phenotypic similarity to fetal fi- broblasts and are anti-fibrotic. The OSF fibroblasts, how- ever, undergo phenotypic alterations [42, 46] thus producing lower amounts of matrix metalloproteinase’s (MMPs) (col- lagenolytic) and augmented quantity of tissue inhibitors of matrix metalloproteinase’s (TIMP) (anti-collagenolytic) [42]. Indeed, OSF fibroblast shows 150% boost in collagen pro- duction as compared to controls [47]. OSF fibroblast also shows a reduction in collagen phagocytic activity compared to normal oral mucosal fibroblasts. Thus, OSF fibroblasts are inherently profibrotic [42, 47].
However, with respect to stromal MMP and TIMP con- tent, both increase with fibrosis. This paradox is explained through a decrease in MMP/TIMP activity ratio, leading to net ECM accumulation. Chronic injury activated plasmin cleaves inactive stromal MMP-1, 2, 3 and 9 into active forms (Fig. 2, Bx-2). Certainly, Rajendran et al., 2006 have shown increased stromal MMP-1, MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 in OSF stroma, however, in gelatin zymography these MMPs were inactive forms of en- zymes (Fig. 2, Bx-2) [48]. The MMPs are highly dependent on microenvironmental pH [42, 49] for their optimum func- tion and they show a drastically reduced activity at pH=9 followed by irreversible inactivation at pH=10 [42]. The alkaline pH causes the sequestration of ionic calcium, retarding the activity of MMPs, since they are highly reliant on ionic calcium. An overall three–five fold reduction in collagenase activity occurs in OSF when compared to the normal oral mucosa (Fig. 2, Bx-2) [50]. Thus, even though both stromal MMP and TIMP are increasing, there is inhibi- tion of MMP activity while TIMP is not inhibited resulting in the accumulation of ECM with declining MMP/TIMP activity ratio (Fig. 2, Bx-2).

2.4. Is OSF Stroma A – “Fertile Soil for Malignancy”?
Fortunately, for our hypothesis, regarding OSF as an overhealing wound explains fibrosis as well as malignant transformation (Fig. 2). Trauma augmented epithelial perme- ability [28] coupled with the reduced vascularity impedes the removal of carcinogens from stroma leading to rebound car- cinogenic exposure of the overlying epithelium (Fig. 2). The high incidence of OPMDs arising in a background of OSF is an affirmation of such an inference (Fig. 2) [28, 51]. Cor- roborating this inference is the report of 23% more OED and 13% more OSCC arising in the background of OSF (OSCC- OSF) due to trauma [28, 52, 53]. Moreover, a malpositioned third molar, particularly within the maxilla, augments the risk of OSCC-OSF through persistent impingement of OSF mucosa against coronoid process [28, 41].
Chourasia et al., (2015) reported the propensity of OSCC-OSF to be 25.71%, while Zachariah et al. (1966) re- ported it to be 40%; however, Shiau et al., (1979) reported it to be just 23% [41]. It appears that malignant potential of OSF has been severely under reported in the literature [41]. They reported cheek as the most common site while tongue was second most common site [41]. Chaturvedi et al., (2013) have however reported anterior 2/3rd of the tongue as the most common site for OSCC-OSF [54]. They have attributed the loss of protective barrier of tongue due to papillary atro- phy for such propensity [54]. Based on our clinical experi- ence, we have often observed atrophy of tongue papillae on the lateral border of tongue in OSF patients. Gadbail et al., (2017) have reported sharp edges and severe attrition in pos- terior teeth in BQ chewing individuals suffering from OSF [55]. The fibrous and hard consistency of AN might be im- plicated for such an observation. They also reported lateral border of tongue as the second most common site for the origin of OSCC-OSF, while buccal mucosa was the most common site [55]. They implicated amalgamation of en- hanced ingress of BQ carcinogens and chronic trauma from the sharp teeth, as a reason for enhanced malignancy in this region [55]. They concluded that carcinogenic process in OSCC-OSF occurs accelerated pace when compared to con- ventional OSCC [55].
Incidentally, the surgically treated cases of OSF develop rebound fibrosis [56, 57] as well as malignancy [56, 58, 59]. The incidence of OSCC-OSF post surgery has been around 23 % [58]. The malignant conversion of the surgically trans- posed flap into a prior area of OSF is an omen of corrupted stroma (Fig. 2) [59-61]. Such an implication assumes signifi- cance considering the fact that volumetric proportion ofstroma vs. epithelium is highest in oral cavity when com- pared to elsewhere in the body [60].
Several studies show that OSCC-OSF is a clinically dis- tinct disease with better prognosis and better grade of differ- entiation (Well Differentiated OSCC-OSF (WD-OSCC- OSF)) when compared to conventional OSCC [41, 54-56, 58], therefore exhibit lesser incidence of metastasis. Despite all this evidence [41, 54-56, 58], we still disagree with these studies as all of these had a very short follow-up time i.e. within 5 years. Such a short follow-up is insufficient to ac- count, quantify, and compare the biological aggressiveness of OSCC-OSF with conventional OSCC. Even the conven- tional OSCC requires 20 or more years for biological evolu- tion, thus it seems to be imprudent to consider five years as adequate follow-up time, and this could well be the reason that most of these investigators reported WD-OSCC-OSF. Another reason for increased reporting of WD-OSCC-OSF could be their early discovery due to the symptoms associ- ated with OSF. A longer follow-up will definitely, detect poorer grades and a consequent worsened prognosis. Indeed, there are reports of Moderately Differentiated OSCC-OSF and Poorly Differentiated OSCC-OSF [54, 62]. Indeed, HIF shows the highest expression in OSCC-OSF when compared to OSCC or OSF alone [63]. The higher expression of HIF in OSCC-OSF, when compared to OSCC or OSF alone may be due to the biological convergence of pathways associated with fibrosis and malignancy, which may synergistically enhance HIF expression; this translates more angiogenesis and aggressiveness to cancer. Certainly, OSCC-OSF is more invasive and shows higher metastasis and recurrence rate when compared to conventional OSCC [63].
Thus, it is safe to conclude that unremitting physical, chemical and mechanical abuse of oral mucosa associated with areca nut chewing in the background of reduced vascu- larity would turn it into a “Fertile Soil for Malignancy”. In other words, OSF stroma serves as a good model of stromal field cancerization hitherto a lesser well-known entity [59- 61, 64, 65].

2.5. Role of Mechanical Stress
Based on the characteristic histological alteration, an ob- vious question one would ask is, why an inherently nonk- eratinized buccal mucosa displays keratinization in areas of fibrosis? A straightforward explanation could be a defensive response of buccal mucosa to continuous physical and chemical onslaught. Such an inference is validated by the appearance of Loricin (absent in nonkeratinized mucosa, like buccal mucosa), a component of the cornified cell envelope in the OSF affected buccal mucosa [25, 38]. Loricin is inci- dentally upregulated by mechanical stress and Ca++ [25, 38]. The mechanical stress is a direct result of BQ chewing while the slaked lime component of BQ supplies Ca++ ions (Fig. 2, Bx-3) [25]. Persuasively, in high-throughput oligonucleotide microarray loricin is most amplified gene within the list of 661 upregulated genes in OSF patients [38]. Moreover, as K- 19 expression is incompatible with keratinization of NOM, its diminution in nonkeratinized mucosa is consistent with induction of keratinization [40]. A recent quantitative pro- teomics analysis identified Filamin-A (FLNA) as one of the two most consistently upregulated biomarkers ofsquamous cell carcinoma arising in the background of OSF. FLNA is known to shield cells from shear stress and its upregulation indicates the protective response of oral mucosa towards areca nut chewing induced mechanical shear stresses [66].
2.5.1. Loss of Caveolin-1 (Cav-1) is the Linchpin of Fibro- sis
Cav-1 loss from fibroblast mediates fibrosis of several organs [67]; its subsequent reintroduction by various means mitigates the fibrosis via inhibition of TGF-β signalling at various levels [67]. Low membrane expression of Phosphate and Tensin Homologue deleted on Chromosome 10 (PTEN) often correlates with low membrane expression of Cav-1 [68]. Furthermore, Cav-1 overexpression restores PTEN lev- els, inhibits AKT phosphorylation and fibroblast prolifera- tion, signifying the role of Cav-1 as a determinant of mem- brane PTEN levels [69]. Indeed, PTEN protein contains a Cav-1 binding consensus sequence, which facilitates its co- localization with Cav-1 in the plasma membrane [69]. On fibroblasts interaction with polymerized type I collagen, Cav-1 forms a complex with PTEN and β1 integrin on the plasma membrane positioning PTEN in a precise spatial lo- cation to inhibit PI3K/AKT signal generated through β1 in- tegrin-matrix interaction [69], promoting fibroblast apopto- sis. However, loss of Cav-1 leads to reduced membrane ac- cumulation of PTEN-Cav-1-β1 integrin complex. This re- stricts the ability of PTEN to inactivate AKT phosphoryla- tion [69], leading to fibroblast persistence thereby facilitating fibrosis.
Additionally, Cav-1 functions as an inhibitor of TGF-β Type I Receptor (Activin Like Kinase -5 (ALK-5)) signalling via enhancement of its internalization from cell membrane [67, 70]. Undeniably, the inflammatory environ- ment in OSF through inflammatory cytokines like IL-6 might shift caveolin receptors to non-raft fractions, diminish- ing Cav-1 mediated TGF-β receptor internalization thereby enhancing TGF-β signaling [67]. Additionally, Cav-1 loss induces oxidative stress [70], which may then activate latent TGF-β into its active form [71]. Indeed, Cav-1 loss mediated enhanced TGF-β signalling augments transcription TGF-β target genes like CTGF [72]. CTGF then coerce metabolic reprogramming via HIF-1 activation, driving autophagy, Reverse Warburg Effect (RWE), and senescence in fibro- blasts [71]. This autophagy has been shown as a requisite factor for myofibroblast differentiation during healing of oral mucosa [21]. Consistent with this observation, a novel link between autophagy and OSF has been recently demonstrated [73]. Definitely, in human and murine fibroblasts and macrophages deficient in the key elements of the autophagic machinery, the TGF-β1 secretion is completely abolished [74].
Higher Cav-1 levels in normal fibroblasts, but lower lev- els in fibrotic fibroblasts, is consistent with this antifibrotic role [75]. Certainly, the introduction of caveolin-1 scaffold- ing domain (CSD peptide) has been shown to inhibit colla- gen and tenascin-C expression in normal fibroblasts and fi- brotic fibroblasts (US8058227) [75]. Moreover, this CSD peptide inhibits a-SMA expression in fibrotic fibroblasts, but not in normal fibroblasts [75]. This antifibrotic activity ofCSD peptide stems through inhibition of PI-3K-Akt, MEK- ERK and JNK signalling [75]. Essentially, Cav-1 loss is mandatory to invoke constitutive myofibroblastic phenotype [70].
2.5.2. Cav-1 Loss Mediates Fibrosis Via Augmented Re- sponse To Mechanical Stress
The Caveolae buffer the mechanical stress imparted on cell through mechanical disassembly [67, 76]. Cav-1 is the member of Caveolin family, indispensable for the Caveolae formation [67]. Obviously, Cav-1 deficient fibroblasts have a proclivity for myofibroblast conversion on mechanical stress exposure [67]. Additionally, Cav-1 is downregulated by TGF-β in a SMAD independent manner via p38-MAPK, triggering increased differentiation of myofibroblast under mechanical stresses (Fig. 2, Bx-1) [77]. Since TGF-β signalling is highly upregulated in OSF, it is safe to assume that Cav-1 is down regulated in OSF, and implies predisposi- tion of affected tissue to mechanical stress; to best of our knowledge Cav-1 downregulation has not yet been reported in OSF (Fig. 2, Bx-1). The continual mechanical stress on mucosa leads to upregulation of profibrotic mediators like CTGF, TGF-β, and TGM2 via mechanosensitive proteins like Yes-Associated Protein (YAP) and transcriptional co- activator with PDZ-binding motif (TAZ) (Fig. 3) [78]. How- ever, mechanical stress on its own leads to a partial myofi- broblastic phenotype and full differentiation requires simul- taneous presence of fibronectin splice variant Extra Domain- A (ED-A), mechanical stress and TGF-β [79].

2.6. Role of Augmented Matrix Stiffness
Increasing Matrix stiffness can itself contribute to fibro- sis besides being its byproduct [80]. The enhanced matrix cross-linking due to activity of enzymes such as TGM-2 and LOX leads to stiffening of the matrix, which might further promote the differentiation of myofibroblast [81]. Matrix cross-linking foster the inflammatory response beyond the threshold level, independent of TGF-β, to stimulate further ECM secretion from fibroblasts and advance the fibrosis [80]. The practice of constantly chewing areca nut can on its own mediate OSF through constant mechanical stress. Cer- tainly, a Stretch Responsive Element (SRE) comprising of nucleotide sequence GAGACC in the CTGF gene promoter has now been, verified. The activation of SRE via Cyclic Mechanical Stretch (CMS) through constant chewing of areca nut may independently upregulate CTGF production[82] (Fig. 3). Even TGF-β1 seems to have a stretch respon-sive element, activated by CMS in the same manner (Fig. 3) [83, 84]. Both CTGF and TGF-β1 may then augment each other, through a mutualistic amplifying circuit (Fig. 3) [85] and thus synergize in stretch-mediated fibrosis. Enhanced matrix cross-linking promotes aberrant angiogenesis (charac- terized by leaky and tortuous blood vessels), leading to di- minished oxygen supply to the area (hypoxia) [86]. Pecu- liarly, this effect is independent of matrix density, as a sub- sequent increase in matrix density decreases angiogenesis; further adding to local hypoxia (Fig. 3) [86].
TGF-β1 mediated hyaluronan synthesis and retention sta- bilize focal adhesion involved in cell attachment and this expedites myofibroblast phenotype [87]. Evidently, thera-peutic disruption of hyaluronan leads to loss of focal adhe- sion, loss of mechanical tension and myofibroblast apoptosis (Fig. 3, Bx-1) [87]. Increased retention of hyaluronan in ECM expedites retention of inflammatory cells, thereby promoting fibrosis (Fig. 3) [87]. Hyaluronan rich ECM helps in retaining and recruiting the eosinophils in the area, ex- plaining the ubiquitous presence of eosinophils in almost every stage of OSF (Fig. 3, Bx-2). Indeed, recent studies have shown the effective treatment of OSF through intrale- sional injection of Hyaluronidase [88, 89]. Fibrosis, once initiated is a self-perpetuating process and may eventually involve the surrounding normal mucosa (Fig. 3).
2.6.1. Matrix Stiffness as a Rheostat Controlling TGF-β Function
Besides fibrosis, an augmented tissue stiffness might also be central in the malignant progression of OSF [90]. Cer- tainly, increase in matrix stiffness beyond a certain level inhibits TGF-β mediated epithelial apoptosis which never- theless promotes epithelial mesenchymal transition (EMT), the key to metastasis [91]. Conversely, decreased matrix stiffness boosts TGF-β mediated epithelial apoptosis while inhibiting EMT [91].

Normal wound healing involves the deposition of colla- gen; however, it is excessive in fibrosis. Hemostasis or co-agulation is a paramount step in healing and is carried out by platelet aggregation and formation of a fibrin clot, which then matures into a Provisional Fibrin Matrix (PFM) [92].
PFM is composed of several polymeric plasma proteins like fibrin, fibrinogen, fibronectin, vitronectin, hyaluronic acid, and heparin sulfate [92, 93]. Fibroblasts activated dur- ing normal healing facilitate reepithelialization via accretion of PFM [93], thereby restoring tissue barrier function [94]. These fibroblasts differentiate into myofibroblasts, character- ized by the presence of a-smooth muscle actin. Even though fibroblasts are sufficient to achieve wound closure, it is the myofibroblasts, which repair both normal and abnormal tis- sue by their enhanced contractile ability (Fig. 1, Bx-1, Bx-2, Bx-3 & Bx-4) [81].
The normal wound repair concludes through the dissolu- tion of PFM via enhanced fibrinolytic activity [85, 93], myo- fibroblast apoptosis [95, 96], myofibroblasts de- differentiation [97, 98], fibronectin phagocytosis, decrease in inflammatory cytokines and decrease in profibrotic cytoki- nes, TGF-β and CTGF (Fig. 1, Bx-2) [93, 95, 99]. However, the persistence of PFM due to deficient fibrinolytic activity, myofibroblast persistence, and reduced fibronectin phagocy- tosis leads to fibrosis [85, 100]. Indeed, fibronectin functions as a primary matrix for the organization of collagenous con- nective tissue during the tissue repair process [100, 101]. Accordingly, the persistence of fibronectin due to deficient phagocytosis by fibroblast leads to fibrosis (Fig. 1, Bx-3 &Bx-4) [102]. A topical application of fibronectin blocking peptides by preventing fibronectin polymerization could ef- fectively treat fibrosis while avoiding systemic side effects (US9364516) [100].

3.1. Role of Inflammation
Subsequent to tissue injury, an inflammatory stimulus is essential to commence tissue repair [46, 103]. Three specific types of cell – macrophages, T helper (TH) cells, and myofi- broblasts, control both inflammation and fibrosis via several common mechanisms (US20160030509) [103, 104]. The CD+ T Cells are sub-classified into TH-1 or TH-2 based on cytokines repertoire, the former characterized by INF-y, IL- 12, IL-2 and latter by IL-4, 5, 6, 8, 10, 13 [105, 106]. A de-creased TH-1/ TH-2 cytokine ratio is consistent with the evolution of both fibrosis and malignancy [105-108]. Indeed, via the use of BCG vaccine a shift in ratio towards TH-1 cy- tokines is antifibrotic (CN101138633) [105]. Additionally, administration of IL-12 is antifibrotic through the promotion of TH-1 immune response (US8247370) [109]. It is possible that inflammatory cytokines like Il-6 can independently of TGF-β promote OSF by JAK-STAT-3, MAPK-ERK, and PI3K-Akt pathways [107, 110, 111]. Incidentally, IL-6 also serves as an autocrine growth factor for fibroblasts (WO2017053963) [112]. IL-13 mediates fibrosis through activation and production of TGF-β and also promotes fibro- sis independently of TGF-β (WO2017053963) [112]. Inter- leukin-β1 (IL-β1) and Tumor necrosis factor-a (TNF-a) are important profibrotic mediators, that amplify IL-6 expression (WO2017053963) [112]. Indeed, a localized fibrotic disorderas OSF could be treated through a TNF receptor 2 (TNFR2) antagonists (EP3265107) [113].
Tissue injury can induce IL-11 expression; moreover, profibrotic agents like TGF-β1, ET-1 or PDGF can kindle IL-11 production by oral fibroblasts (US20170174759) [114]. IL-11 can also upregulate its own production in an autocrine manner (US20170174759) [114]. An agent capable of forming a complex with IL-11 reducing its ability to bind to an IL-11 receptor might have antifibrotic effects (US20170174759) [114].

Undoubtedly, myofibroblasts usually undergo apoptosis once wound is fully covered with epithelium [21], due to the reestablishment of cell-specific contacts and cellular relief from stress [110]. Certainly, properly repaired tissue recu- perates its original mechanical properties, once again stress- shielding resident fibroblasts and inhibiting their further myofibroblast conversion [110]. This mechanism is experi- mentally validated in murine models of fibrosis, where splinting of granulation tissue leads to myofibroblast persis- tence, whereas its release causes myofibroblast apoptosis [110]. Although the factors that mediate fibroblast activation and differentiation are well characterized, the factors mediat- ing the persistence of fibroblast in fibrotic tissue is not well known [99].
Myofibroblast persistence in OSF may be due to persis- tent physical and chemical injury to oral mucosa by habitualBQ chewing that leads to relentless failed reepithelization attempts thwarting tissue barrier function [6, 94, 95, 99]. The normal repair is TGF-β dependent and demonstrates only transient CTGF expression (Fig. 1, Bx-1 & Bx-2) [115]. Fi- brosis is, however, TGF-β independent and demonstrates persistent CTGF expression (Fig. 1, Bx-3 & Bx-4) [115, 116]. CTGF chronically elevated at the sites of persistent microtrauma in OSF could be a reason for persistent fibro- blast activation (Fig. 1, Bx-3 & Bx-4) [85]. Not surprisingly, only the early stages of OSF demonstrate elevated expres- sion of TGF-β1 in the submucosa, but subsequently, there is no difference in its expression levels with respect to normal tissues, yet fibrosis ensues [116]. Elevated, CTGF expression despite declining TGF-β1, because of positive feedback from several pathways, may perhaps be the only plausible expla- nation [85, 116]. Yet another mechanism for persistence of myofibroblasts in fibrosing tissues could be an epigenetic modification in gene function (Fig. 1, Bx-3) [14, 99], which might be mediated through chronic inflammation via chronic persistent physical and chemical injury to the oral mucosa.
The myofibroblast isolated from fibrotic tissue, neither undergoes apoptosis nor reverts back to fibroblast even when placed in a non-fibrotic environment [99]. This indicates that the phenotype changes in myofibroblast isolated from fi- brotic tissues are permanent and inheritable. Undeniably, OSF tissues do demonstrate the persistence of myofibro- blasts [5, 6, 95]. Moreover, these myofibroblasts serve as a marker for gauging the severity of OSF [5].

Hypoxia is a negative balance between O2 supply and demand. Normally this deficit is compensated by an increase in blood O2 utilization during acute (perfusion-limited) hy- poxia and through rise in the local blood flow during chronic (diffusion-limited) hypoxia. However, in pathological states, these compensatory mechanisms are not operative and hy- poxia ensues. Essentially, all wounds are hypoxic and all healing outcomes are directly proportional to oxygen avail- ability [117, 118]. Thus, considering OSF as an overhealing wound, it would not be inappropriate to contemplate hypoxia as the key protagonist of OSF.
Astoundingly, dilated blood vessels and neo- angiogenesis seem to deny the presence of hypoxia in OSF stroma [26, 119]. However, in most of the tissues of the body, including oral mucosa barring pulmonary vasculature where hypoxia causes vasoconstriction, the global response to hypoxia is vasodilatation [120-122]. By dilatation of blood vessels, the tissue allows greater perfusion alleviating hypoxia, albeit incomplete. Thus, using vasodilatation as an argument to deny the presence of hypoxia in OSF stroma is untenable [123, 124]. Likewise, the occurrence of angio- genesis is not an argument against hypoxia either; hypoxia being the key stimulus for angiogenesis [125-127]. Indeed, hypoxia-induced angiogenesis in OSF is exemplified by the presence of CD105+ blood vessels in the stroma [128, 129]. Moreover, several studies have shown that hypoxia is essen- tial for the maintenance of fibrosis [130, 131]. Incidentally, hypoxia worsens with progression of fibrosis due to a progressive increase in diffusion distances of oxygen fromblood vessels to the cells [132]. Additionally, hypoxia upregulates various profibrotic mediators like PAI-1, PDGF, TGF-β, CTGF, b-FGF, TGF-a, LOX, TIMP-1, ET-1, andVEGF [130-133]. Thus, hypoxia and fibrosis form a vicious cycle potentiating each other. Developing malignancy has again been utilized to deny the existence of hypoxia in OSF; however, the development of malignancy and continuance of fibrosis in a hypoxic environment can be explained by stro- mal compensatory mechanism discussed in section 5.4 (Fig. 4).
However, hypoxia can initiate fibrosis only with the help of TGF-β, as evidenced by the lack of fibronectin in hypoxia in the absence of TGF-β [134, 135]. Incidentally, TGF-β induced collagen I dwindles upon inhibition of HIF-1a [136]. Thus, hypoxia may play a role in the progression of the OSF once the disease process is initiated by areca quid [134, 137]. Remarkably, the incidence of OED in OSF was proportionate to the thickness of fibrosis in OSF [138]. Us- ing this evidence the authors concluded that inhibition of fibrosis through any means might inhibit the development of OSCC in the background of OSF via reduction of hypoxia [138]. Actually, fibrotic focus itself serves as a surrogate marker of hypoxia (WO2012166700) [139]. All these facts implicate hypoxia as an obligatory factor in the pathogenesis and malignant transformation OSF [12, 130-133, 140]. Hence, inhibiting HIF will inhibit both fibrosis and malig- nancy in OSF (US20170157112) [141].

5.1. Local State of Hypoxia in OSF
The stromal hypoxia in OSF has been previously con- templated [142]. However, the existence of the local state of hypoxia in OSF stroma was first conclusively demonstrated by Tsai et al. in 2015 [137]. They showed higher expression of HIF-1a in OSF fibroblasts as compared to normal buccal mucosal fibroblasts [137]. Arecoline was shown to be re- sponsible for the augmentation of PAI-1 expression [137, 143, 144] in hypoxia rather than normoxia [137]. Moreover, PAI-1 protein expression was abolished via HIF-1a inhibitor under hypoxic conditions [137]. The authors concluded that the transition from steady state of ECM metabolism to ECM accumulation is partly driven by augmentation of PAI-1 through hypoxia [137]. PAI-1 has a hypoxia response ele- ment (HRE) in its promoter conforming to such a role [145]. Several studies have shown that elevated PAI-1 level pro- motes fibrosis of oral mucosa [137, 143, 146].
PAI-1 expression promotes fibrosis by constraining plasmin formation via inhibition of plasminogen activators (tPA and uPA) and it thereby facilitates fibrin accumulation [85, 93, 147]. Among all the serine protease inhibitors (SERPINS), PAI-1 has the utmost efficacy in inhibiting plasminogen activators [147]. See the reference for the de- tailed mechanism of PAI-1 in OSF [148].

5.2. Mechanism for Development of Local Hypoxia in OSF
There may be several mechanisms for the development of local state of hypoxia in OSF. Indeed, OSF stroma demon- strates a change in vascular status from normal to dilated, constricted, and obliterated blood vessels with the progression of disease (Fig. 4, Bx-1) [12, 149]. The devel-opment of fibrosis in OSF leads to the organization of colla- gen fibers into sheets parallel to the surface [44, 150]. This, in turn, causes constriction and obliteration of blood vessels [12, 138, 142, 150-152], which then reduces the perfusion of tissues; leading to tissue hypoxia (Fig. 4, Bx-1) [151, 153, 154]. The congestion and fibrosis of blood vessels also take place (Fig. 4, Bx-1) [12, 150]. Undeniably, special stains can detect these changes in blood vessels in OSF. The Mallory and Van-Gieson’s stain can easily detect congested and con- stricted blood vessels, respectively [12].
It is important to note that fibrosis may not always affect the length or number of blood vessels, but may simply re- duce tissue perfusion through blood vessel constriction (Fig. 4, Bx-1) [12, 151]. Furthermore, despite various compensa- tory mechanisms like angiogenesis and vasodilatation, the stroma of OSF might be still hypoxic, due to the presence of other vascular abnormalities like endarteritis obliterans and endothelial dysfunction which may further intensify the pre- existing hypoxia [133]. Endarteritis obliterans might be countered via the use of recombinant erythropoietin, an an- giogenesis inducer (US20090280094) [155] while endothe- lial dysfunction might be tackled through xanthine oxidase inhibitors (US20170217948) [156], administration of multi- ple long non-coding RNA (lnc-RNA) (EP3054012) [157], or through Chromium containing compositions in amalgama- tion with Phyllanthus emblica extract and Shilajit [158].
Additionally, several studies have demonstrated that anemia can render tissue hypoxic through reduced O2 deliv- ery [95, 159-161]. Apparently, several studies have de- scribed the presence of chronic iron deficiency anemia in OSF patients [161-163]. A novel formula of iron-based nanocomposites for treatment of iron deficiency anemia is available (US20160022733) [164]. We have also previously discussed various mechanisms of stromal hypoxia in OSF, and on this basis, hyperbaric oxygen therapy (HBOT) was recommended as a treatment of OSF [133].
Recently, it was shown that the cytotoxic action of arecoline facilitates endothelial cell apoptosis [165]. A de- crease in endothelial cell density (measured by the number of endothelial cell/µ2) with progressive grades of OSF, corrobo- rates the cytotoxic action of arecoline [166, 167]. Addition- ally, augmented PAI-1 expression inhibits endothelial P53 degradation via modulation of proteosome activity and thereby promote endothelial apoptosis [168]. Moreover, the arecoline upregulation in OSF in the background of elevated PAI-1 would mediate endothelial apoptosis of cells with a greater efficacy than it would alone [148, 165, 168]. Even, thrombospondin (THBS-1) released from activated platelets as a consequence of chronic injury to oral mucosa can medi- ate endothelial apoptosis [169]. These might be the supple- mentary mechanisms of hypoxia in OSF, besides the ones described before (Fig. 4, Bx-1).
Recently, it has been demonstrated that the vascularity of the mucosa overlying the fibrotic bands diminishes as evi- denced by a reduction in blood flow velocity by Color Dop- pler and Spectral Doppler, while in the region between the fibrotic bands it is normal [152]. Evidently, the increased stiffness of sub-epithelium hinders even distribution of blood vessels within the connective tissue, which retards nutrient supply to the epithelial layer leading to its atrophy [170].
Thus, a local state of hypoxia might be responsible for atro- phy and ulceration of the epithelium since such epithelium often overlies fibrotic areas (Fig. 2) [11, 142, 152]. These facts indicate that the epithelial atrophy is chiefly attributable to ischemia [11, 12, 47, 142, 152, 170], among other mecha- nisms (Fig. 2) [46, 171]. Finally, it can be safely concluded that stroma of OSF is hypoxic, especially in focal areas of fibrosis [12, 142].

5.3. Hypoxic and Other Mechanisms of PAI-1 Stabiliza- tion
PAI-1 plays a definitive role in OSF pathogenesis [148]. However, PAI-1 is inherently unstable and converts to its latent form readily [147]. Its structure is nevertheless stabi- lized by directly binding to vitronectin, fibrin, a1 acid glyco- protein, and phospholipids or indirectly by fibrin through vitronectin [147, 172]. a1 acid glycoprotein, in turn, may be upregulated by the inflammatory component associated with the fibrosis (Fig. 4, Bx-2) [147, 172].

5.4. Stromal Compensatory Mechanism in Hypoxia
Adaptations to hypoxic microenvironment include revas- cularization and a glycolytic switch, which correlate with augmented invasion and metastasis [63]. This adaptation is mediated through HIF-1a upregulation [63]. Moreover, HIF- 1a upregulation mediates OSF and its malignant conversion [63]. Naturally, extending these mechanisms to OSF would offer novel insights into the mechanisms of development of OSCC-OSF.
The occurrence of glycolysis in the epithelial cells even in the presence of oxygen is termed as aerobic glycolysis or Warburg effect [173]. The Reverse Warburg Effect (RWE), however, is the occurrence of this phenomenon in the stro- mal cells [70, 173, 174]. RWE might be essential for pro- gression and maintenance of myofibroblastic state and thereby promote fibrosis [139, 175]. Certainly, several stud- ies have shown that RWE is crucial for fibrosis [70, 139, 175-177]. This fact might be exploited to transport inhibitory agents like arsenic bound to the sugar moiety into cell inte- rior (US20150038694) [178]. Moreover, the hypoxic state of OSF stroma may force aerobic glycolysis on stromal cells like fibroblast (i.e. RWE) through HIF-1a (Fig. 4, Bx-3) [70, 173, 174]. Certainly, HIF-1a upregulates glycolysis and downregulates Oxidative Phosphorylation (OXPHOS) even in normoxia [173]. To facilitate glycolysis, HIF-1a also upregulates Glucose Transporter -1 (Glut-1), allowing in- creased glucose entry into the cell [173, 179]. HIF-1a is also known to upregulate LDH-5 resulting in accumulation of intracellular lactic acid (Fig. 4, Bx-3) [173]. The enhanced intracellular acidity, due to intracellular lactic acid however is either lethal or inhibitory for proliferation [173]. However, HIF-1a mediated upregulation of Monocarboxylate Trans- porter -4 (MCT-4) and carbonic anhydrase-9 (CA-9) results in the export of lactate and protons (H+) from the cell effec- tively countering the augmented intracellular acidity (Fig. 4, Bx-3) [173, 179]. As a consequence of these activities cellu- lar pH gradient is reversed, resulting in a more acidic ex- tracellular pH (pHe) and a more alkaline intracellular pH (pHi) (referred to as Reversed pH Gradient) (Fig. 4) [173]. Alkaline pHi further promotes glycolysis via upregulation ofglycolytic enzymes, making pHe further acidic (Fig. 4, Bx-3) [173]. The resulting acidic pHe can stabilize PAI-1 through protonation of imidazole groups in its His364 residues (Fig. 4, Bx-2) [172]. Enhanced acidic pHe also stabilizes HIF-1a via nuclear sequestration of its inhibitor Von Hippel-Lindau (VHL) protein [180]. The other inhibitor of HIF expression in normoxia is the Factor Inhibiting HIF (FIH) [181]. miR- 31 targets the 3′ untranslated region (UTR) of FIH and re- presses its expression [181]. miR-31 may in turn be upregu- lated in fibroblast as a consequence of Cav-1 loss (Fig. 4, Bx-4) [182]. This will lead to activation of HIF signalling even in normoxia, providing an auxiliary pathway of promot- ing RWE in OSF stroma (Fig. 4). This may provide a mechanism for continuance of RWE in OSF stroma even when adequate oxygenation is available due to enhanced angiogenesis. These mechanisms may amplify HIF-1a levels [145], which will then further promote RWE in OSF Stroma forming a positive feedback loop; a phenomenon christened as “Domino Effect” by Chaudhary et al., in (2015) [63]. Upregulated HIF-1a can also augment PAI-1 levels through the HRE in PAI-1 promoter (Fig. 4) [145]. Definitely, LDH- 5 inhibitors might be effective in inhibiting OSF and its as- sociated malignancy (US20150335674, US20170029531) [183, 184].
Indeed, the elevation of CA-9 in OSF and OSCC is an evidence for the operation of RWE in OSF stromal cells like fibroblast [185]. Based on this mechanism local delivery ofCA-9 inhibitor could be contemplated to counter OSF (US20110034448) [186]. Moreover, the use of such an agent might be considered as an alternative to stem cell therapy for treating fibrosis (US20100135980) [187]. Additionally, these CA-9 inhibitor might also have anti-metastatic activity against OSCC arising in background of OSF (US20140148400, WO2017004543) [188, 189]. Alterna-tively, the submucosal injection of a Proton Pump Inhibitor (PPI) could be considered. Indeed, PPIs seem to have multi- ple antifibrotic effects through augmentation of iNOS activ- ity, downregulation of TGF-β1 receptor, TGF-β2 receptor, MMPs, fibronectin and inflammatory cytokines like TNF-a and IL-β [190]. They also demonstrate antioxidant effect via downregulation of ROS and upregulation of HO-1 [190]. Notably, we have previously contemplated the effectiveness of PPI in inhibiting oral and other cancers [173].
Considering the above facts, there seems to be no reason to believe that RWE does not operate in the stroma of OSF. Rather, RWE might provide the starved epithelial cells with a steady supply of nutrients (i.e. lactate and/or pyruvate) even in the presence of fibrosis and diminished vascularity (Fig. 4, Bx-4). Thus, RWE may allow the development of epithelial malignancy in the background of continuing fibro- sis and hypoxia, as vascularity is not essential for tumor cell proliferation. Hence as discussed before, developing malig- nancy in the background of OSF, should not be utilized to deny the existence of stromal hypoxia in OSF [26]. Due toRWE, OSF stroma rather serves as a reservoir of nutrient and raw materials (i.e. pyruvate and lactate) to the developing cancer cells. Cav-1 loss in stromal fibroblast serves as a marker of myofibroblastic phenotype and RWE [70, 139], which is evident by upregulation of five myofibroblast markers and eight glycolytic enzymes [70]. Cav-1 loss medi- ates upregulation of miR-31 and miR-43c, coercing stromal mitophagy and autophagy respectively that then drives the RWE, fibrosis and malignancy (US20140322705) (Fig. 4, Bx-4) [182]. Indeed, use of liposomal encapsulated glu- tathione might be a novel therapy for both cancer and fibro- sis via inhibition RWE (US20150030668) [191]. Targeting the RWE through MCT-4 inhibitors thorough inhibition of this stromal epithelial coupling might co-inhibit epithelial malignancy and fibrosis (WO2016201426, WO2017091885) [192, 193]. Autophagy inhibitors might also serve as dual inhibitory agent against fibrosis and malignancy (WO2016168721, EP2616082) [194, 195].
The novelty of our schema is that it explains how the progressive loss of vascularity leads to augmented fibrosis and we have provided a mechanism of cellular sustenance in such an environment. Besides the arecanut induced perio- dontitis [17, 196-198] might exacerbate or promote preexist- ing OSF, by furnishing several molecular mediators like i.e., IL-1, Lipopolysaccharide (LPS), TNF-a, C-Reactive Protein (CRP) [197, 199-202], which then upregulate PAI-1 expres- sion (Fig. 4, Bx-5) [168]. These molecular factors have an easy access to OSF stroma due to the enhanced permeability of OSF epithelium. Additionally, reduced salivary flow rate and a consequent decrease in clearance of these molecular mediators might boost their permeation through diseased mucosa (Fig. 4, Bx-6). IL-6 is also another factor produced in periodontitis [197] which may exacerbate fibrosis by shifting TGF-β receptors to non-raft fractions [67], augmenting TGF-β signalling, which then promotes fibrosis by itself or through upregulation of PAI-1. Thus, periodontal therapy in OSF patients might also augment response to anti- fibrotic therapy and reduce the incidence of malignancy aris- ing in background of OSF [198, 202]. Thus, localized deliv- ery of human engineered, anti-IL-6 antibodies to the submu- cous area might inhibit OSF (US20160355818, US20160222104) [203, 204].

Acute injury leads to transient coagulation pathway acti- vation that too for the period of repair. However, chronic injury leads to sustained coagulation pathway activation or- chestrating fibrosis [205]. Undeniably, coagulation pathway abnormalities epitomize the profibrotic milieu of several organs, including oral cavity [205-208]. Thus, extrapolating the abnormalities in coagulation cascade to OSF could help in deciphering its pathogenesis. The epithelial and/or endo- thelial damage triggering the coagulation pathway is the ini- tial response in healing of wounds and fibrosis [209]. Indeed, augmented factor VIII and platelet aggregates within the vascular lumen in the fibrotic areas in OSF affected indi- viduals, uphold such a mechanism [210, 211]. Additionally, thrombosis of blood vessels as a consequence of platelet activation [211], poor microcirculation and hypercoagulabil-ity of blood are frequent findings in OSF tissues (Fig. 5) [57].
The coagulation pathway induction in OSF via chronic physical and chemical injury through BQ chewing leads to activation of extrinsic (Fig. 5, Bx-1), intrinsic (Fig. 5, Bx-2) and final common pathway of coagulation (Fig. 5, Bx-3). These lead to the activation of Thrombin (II-a) which then converts fibrinogen (I) into fibrin monomers (Ia). These fi- brin monomers polymerize to cross-linked fibrin via Fibrin Stabilizing Factor-a (Factor XIII-a), the latter being generated from Fibrin Stabilizing Factor (Factor XIII) via the action of thrombin (II-a) [212]. Thrombin (II-a) also stimu- lates platelets to secrete thrombospondin (THBS-1), which then activates TGF-β via proteolytic cleavage (Fig. 5). In- deed, a study has shown THBS-1 as the principal activator of TGF-β in 100% of the OSF cases [213]. Thrombin (II-a) also serves as a fibroblast chemoattractant, recruiting fibroblasts from the neighboring areas into injured sites [208].
Fibrinogen is inert in circulation and unable to bind fi- bronectin [214]. Only upon polymerization of fibrin into the clot, the cryptic sites are exposed facilitating fibronectin in- corporation [214]. Fibronectin incorporation in fibrin is pro- moted through blood clotting factor XIII-a [215]. Certainly, fibronectin contains fibrin-binding sites Fib-1 and Fib-2 to facilitate its incorporation in the fibrin matrix [215]. Moreo- ver, fibronectin promotes fibroblast spreading and ECM ad- hesion and is an absolute requirement for its migration into a clot [214]. Definitely, experimental deficiency of fibrin de- lays the fibrosis (Fig. 5) [93].

Arecoline has been shown to upregulate both tPA and PAI-1 and the latter to a far greater extent (Fig. 4, Bx-7) [148]. The resulting unfavorable stoichiometry promotes fibrosis by allowing the accumulation of fibrin and to a lesser extent plasmin (See the reference for further explana- tion) (Fig. 4 & Fig. 5) [148]. This differential upregulation of mutually antagonistic molecules PAI-1 and tPA results in increased fibrosis because of the fibrin degradation products (FDPs) resulting from plasmin activity, stabilize PAI-1 to a far greater extent than fibrin alone. (Fig. 4 & Fig. 5) [148]. FDPs especially the DD and D fragments, but not E fragment are tenfold potent than the parent molecule fibrinogen in inducing PAI-1 [216, 217]. Presence of FDPs in the blood of OSF patients is an evidence for such a mechanism [11, 218, 219]. Furthermore, only betel nut chewers with OSF show FDP in plasma while those without OSF show no FDP in the plasma [11].
Augmentation of fibrin deposition in matrix leads to en- hanced formation of PFM and hence the quantum of fibrosis. Enhanced deposition of fibrin also increases plasma FDP due to residual plasmin activity [85]. In fact, the plasma FDP could be utilized as an early proxy indicator of fibrin deposi- tion and thereby the amount of fibrosis. Consequently, by measuring FDPs it is possible to assess the stage of OSF progression [11, 218]. Theses FDPs could also be utilized for detection and monitoring of OSCC arising in the background of OSF (EP2414843) [220-223].

The empirical evidence that surgical modalities to treat OSF have been unsatisfactory and even attended by rebound fibrosis (Our Clinical Experience); as, in essence, we are again creating a wound in already altered mucosa, provide further support to our hypothesis. Since abnormalities in coagulation cascade epitomize the profibrotic milieu of OSF; targeting coagulation pathway is a novel and viable thera- peutic option. However, general inhibition of the coagulation cascade might not be a right strategy for the treatment of fibrosis [224, 225]. Targeting a specific component of the coagulation cascade responsible for fibrosis might be a valu- able and novel therapy for OSF. Since Protease-Activated Receptors (PARs) play a central role in influencing inflam- matory and fibrotic responses, their pharmacological target- ing might be a novel therapy for fibrosis [206, 226]. There exist four PARs (PAR-1, 2, 3, 4) [208]. Among these, PAR-1 chiefly mediates profibrotic effects of thrombin [208].
Likewise, factor X-a also mediates its profibrotic effects through PAR-1 (Fig. 5) [208, 227, 228]. Certainly, thrombin elevated at the sites of microtrauma to the oral mucosa upregulate PAR-1, which in turn promotes CTGF secretionfrom the fibroblast (Fig. 4) [206, 229, 230]. Thus, specific PAR-1 antagonist might hold promise for the treatment of OSF [231]. Certainly, small molecule antagonist to PAR-1 receptor vorapaxar and atopaxar might be contemplated [205]. A simplified preparation of vorapaxar achieving high blood concentration has been mooted (CN106309396) [232]. PAI-1 inhibition through small molecule inhibitors like Ti- plaxtinin has antifibrotic and an anticancer role (US20110105574, US20160158188) [233, 234]. Enhancingthe activity of plasminogen activators uPA and tPA through TM-5275 (US20160144001) [235] also holds promise in the treatment of OSF [236, 237]. TM-5275 also has a novel anti- inflammatory action via inhibition of macrophage migration in the area [238]. Since TGF-β signalling is central to the initiation of fibrosis, a glucosylceramide synthase inhibitor or a lactosylceramide synthase inhibitor can impede nuclear translocation of phosphorylated Smad, serving as a fibrosis inhibitor through inhibition of TGF-β signalling (US20180008585) [239].
Any treatment protocol for OSF should be limited to medical therapy for early OSF and surgery should be reserved for advanced cases.

The genesis of OSF is still a mystery although a plethora of literature is available. The persistent physical, chemical and mechanical trauma of susceptible oral mucosa due to habitual BQ chewing resulting in the overzealous mucosal healing explains the onset of OSF. A CCL-2 based mecha- nism; recruiting myofibroblast to sites of epithelial injury in OSF adds credence to this hypothesis. Considering, OSF as an overhealing wound explains the pathogenesis of OSF as well as its malignant transformation. Essentially all wounds are hypoxic and hypoxia seems to be mandatory for both malignancy and fibrosis. Not surprisingly, the degree ofepithelial dysplasia in OSF is proportional to the thickness of fibrosis. Logically, considering OSF as an overhealing wound also entails coagulation pathway abnormalities. Cer- tainly, abnormalities in the coagulation cascade and fibri- nolytic system are a common denominator in the profibrotic milieu as well as associated malignancy. Since PARs medi- ate the cellular effects of coagulation factors, they play a vital role in inflammatory and fibrotic responses. Hence, pharmacological targeting of PARs, chiefly PAR-1; might be a novel therapy for OSF. A summary of patents for the treatment of Tiplaxtinin OSF as a paradigm of the overhealing wound is listed in Table 1.