Molecular histopathology of matrix proteins through autofluorescence super-resolution microscopy.

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training.

Association of podoplanin expression to histopathological grades of oral epithelial dysplasia and oral squamous cell carcinoma.

Objective: To determine the expression of podoplanin, and to correlate it with histopathological grades in oral epithelial dysplasia and oral squamous cell carcinoma cases. METHODS: The retrospective, analytical, cross-sectional study was conducted at the City Laboratory, Peshawar, Pakistan, and comprised specimen block data of histologically diagnosed cases of oral benign lesions, dysplastic lesions and oral squamous cell carcinoma from January 2017 to August 2021. Two sections (4um) were cut from each specimen block for Haematoxylin and Eosin staining and immunohistochemistry. The slides were re-evaluated by two pathologists for confirmation of the diagnosis, and podoplanin marker was applied to cases selected using immunohistochemistry. Data was analysed using SPSS 22. RESULTS: Of the 80 cases identified, 68(85%) were analysed. There were 20(29.4%) benign cases; 11(55%) females and 9(45%) males with mean age 39.90±16.23 years, 20(29.4%) oral dysplastic cases; 14(70%) males and 6(30%) females with mean age 57.75±12.02 years, and 28(41.2%) oral squamous cell carcinoma cases; 17(61%) males and 11(39%) females with mean age 50.55±14.80 years. Podoplanin expression in oral epithelial dysplasia cases was significant (p=0.028), while it was not significant in the other 2 groups (p>0.05). CONCLUSIONS: Podoplanin when used along with histopathological evaluation could aid as an adjuvant technique in the diagnosis and grading of oral epithelial dysplasia.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

The transcription factor BMI1 increases hypoxic signaling in oral cavity epithelia.

The tongue epithelium is maintained by a proliferative basal layer. This layer contains long-lived stem cells (SCs), which produce progeny cells that move up to the surface as they differentiate. B-lymphoma Mo-MLV insertion region 1 (BMI1), a protein in mammalian Polycomb Repressive Complex 1 (PRC1) and a biomarker of oral squamous cell carcinoma, is expressed in almost all basal epithelial SCs of the tongue, and single, Bmi1-labelled SCs give rise to cells in all epithelial layers. We previously developed a transgenic mouse model (KrTB) containing a doxycycline- (dox) controlled, Tet-responsive element system to selectively overexpress Bmi1 in the tongue basal epithelial SCs. Here, we used this model to assess BMI1 actions in tongue epithelia. Genome-wide transcriptomics revealed increased levels of transcripts involved in the cellular response to hypoxia in Bmi1-overexpressing (KrTB+DOX) oral epithelia even though these mice were not subjected to hypoxia conditions. Ectopic Bmi1 expression in tongue epithelia increased the levels of hypoxia inducible factor-1 alpha (HIF1α) and HIF1α targets linked to metabolic reprogramming during hypoxia. We used chromatin immunoprecipitation (ChIP) to demonstrate that Bmi1 associates with the promoters of HIF1A and HIF1A-activator RELA (p65) in tongue epithelia. We also detected increased SC proliferation and oxidative stress in Bmi1-overexpressing tongue epithelia. Finally, using a human oral keratinocyte line (OKF6-TERT1R), we showed that ectopic BMI1 overexpression decreases the oxygen consumption rate while increasing the extracellular acidification rate, indicative of elevated glycolysis. Thus, our data demonstrate that high BMI1 expression drives hypoxic signaling, including metabolic reprogramming, in normal oral cavity epithelia.

DPSCs regulate epithelial-T cell interactions in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion characterized by fibrous tissue deposition, the incidence of which correlates positively with the frequency of betel nut chewing. Prolonged betel nut chewing can damage the integrity of the oral mucosal epithelium, leading to chronic inflammation and local immunological derangement. However, currently, the underlying cellular events driving fibrogenesis and dysfunction are incompletely understood, such that OSF has few treatment options with limited therapeutic effectiveness. Dental pulp stem cells (DPSCs) have been recognized for their anti-inflammatory and anti-fibrosis capabilities, making them promising candidates to treat a range of immune, inflammatory, and fibrotic diseases. However, the application of DPSCs in OSF is inconclusive. Therefore, this study aimed to explore the pathogenic mechanism of OSF and, based on this, to explore new treatment options. METHODS: A human cell atlas of oral mucosal tissues was compiled using single-cell RNA sequencing to delve into the underlying mechanisms. Epithelial cells were reclustered to observe the heterogeneity of OSF epithelial cells and their communication with immune cells. The results were validated in vitro, in clinicopathological sections, and in animal models. In vivo, the therapeutic effect and mechanism of DPSCs were characterized by histological staining, immunohistochemical staining, scanning electron microscopy, and atomic force microscopy. RESULTS: A unique epithelial cell population, Epi1.2, with proinflammatory and profibrotic functions, was predominantly found in OSF. Epi1.2 cells also induced the fibrotic process in fibroblasts by interacting with T cells through receptor-ligand crosstalk between macrophage migration inhibitory factor (MIF)-CD74 and C-X-C motif chemokine receptor 4 (CXCR4). Furthermore, we developed OSF animal models and simulated the clinical local injection process in the rat buccal mucosa using DPSCs to assess their therapeutic impact and mechanism. In the OSF rat model, DPSCs demonstrated superior therapeutic effects compared with the positive control (glucocorticoids), including reducing collagen deposition and promoting blood vessel regeneration. DPSCs mediated immune homeostasis primarily by regulating the numbers of KRT19 + MIF + epithelial cells and via epithelial-stromal crosstalk. CONCLUSIONS: Given the current ambiguity surrounding the cause of OSF and the limited treatment options available, our study reveals that epithelial cells and their crosstalk with T cells play an important role in the mechanism of OSF and suggests the therapeutic promise of DPSCs.

Evaluation of stromal myofibroblasts in oral submucous fibrosis and its malignant transformation: An immunohistochemical study.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion, with oral squamous cell carcinoma (OSCC) being the most prevalent malignancy affecting the oral mucosa. The malignant transformation of OSF into OSCC is estimated to occur in 7-13% of cases. Myofibroblasts (MFs) play pivotal roles in both physiological and pathological processes, such as wound healing and tumorigenesis, respectively. This study aimed to explore the involvement of MFs in the progression of OSF and its malignant transformation. MATERIALS AND METHODS: In total, 94 formalin-fixed paraffin-embedded tissue blocks were collected, including normal oral mucosa (NOM; n = 10), early-moderate OSF (EMOSF; n = 29), advanced OSF (AOSF; n = 29), paracancerous OSF (POSF; n = 21), and OSCC (n = 5) samples. Alpha-smooth muscle actin was used for the immunohistochemical identification of MFs. RESULTS: NOM exhibited infrequent expression of MFs. A higher staining index of MFs was found in AOSF, followed by EMOSF and NOM. Additionally, a significant increase in the staining index of MFs was found from EMOSF to POSF and OSCC. The staining index of MFs in NOM, EMOSF, AOSF, POSF, and OSCC was 0.14 ± 0.2, 1.69 ± 1.4, 2.47 ± 1.2, 3.57 ± 2.6, and 8.86 ± 1.4, respectively. All results were statistically significant (P < 0.05). CONCLUSIONS: The expression of MFs exhibited a gradual increase as the disease progressed from mild to malignant transformation, indicating the contributory role of MFs in the fibrogenesis and potential tumorigenesis associated with OSF.

Expression of CLLD7 and CHC1L Proteins in Oral Epithelial Dysplasia in a Group of Thai Patients.

OBJECTIVES: Previous study showed aberrant CLLD7 and CHC1L protein expression in oral squamous cell carcinoma (OSCC) compared to normal oral mucosa (NOM). This study aimed to evaluate the expression of these proteins in oral epithelial dysplasia (OED). MATERIALS AND METHODS: Forty specimens of OED and 11 NOM were used. The expression of CLLD7 and CHC1L was determined by immunohistochemistry. In each case, at least 1000 cells were counted. Presence of nuclear, cytoplasmic, and/or membrane staining of CLLD7 and CHC1L were considered positive. Percentages of total positive cells and positive cells at different locations were recorded. SPSS version 18 was used to compare variation between groups with statistical significance at p<0.05. RESULTS: No significant differences in the percentages of total positive cells of CLLD7 and CHC1L were found between NOM and all grades of OED. Nevertheless, there were significant differences in subcellular staining of these two proteins. In CLLD7, the nuclear staining of the moderate and the severe OED groups was significantly lower than that of the NOM group (p<0.05). The percentages of membrane staining of CHC1L in moderate and severe OED were significantly higher than that of NOM (p<0.001). In addition, the nuclear staining of CHC1L in each grade of OED was significantly lower than that of NOM (p<0.05). CONCLUSION: The subcellular mislocalization of CLLD7 and CHC1L in OED suggests that the expression of these potential tumor suppressor proteins might be dysregulated during the dysplastic process. The distinct membrane staining of CHC1L observed in OED but not in NOM is a useful characteristic that can be used to separate OED from NOM. Thus, CHC1L may be a good marker to assist in the diagnosis of OED.

Rattus norvegicus Glutathione Transferase Omega 1 Localization in Oral Tissues and Interactions with Food Phytochemicals.

Glutathione transferases are xenobiotic-metabolizing enzymes with both glutathione-conjugation and ligandin roles. GSTs are present in chemosensory tissues and fluids of the nasal/oral cavities where they protect tissues from exogenous compounds, including food molecules. In the present study, we explored the presence of the omega-class glutathione transferase (GSTO1) in the rat oral cavity. Using immunohistochemistry, GSTO1 expression was found in taste bud cells of the tongue epithelium and buccal cells of the oral epithelium. Buccal and lingual extracts exhibited thiol-transferase activity (4.9 ± 0.1 and 1.8 ± 0.1 µM/s/mg, respectively). A slight reduction from 4.9 ± 0.1 to 4.2 ± 0.1 µM/s/mg (p < 0.05; Student's t test) was observed in the buccal extract with 100 µM GSTO1-IN-1, a specific inhibitor of GSTO1. RnGSTO1 exhibited the usual activities of omega GSTs, i.e., thiol-transferase (catalytic efficiency of 8.9 × 104 M-1·s-1), and phenacyl-glutathione reductase (catalytic efficiency of 8.9 × 105 M-1·s-1) activities, similar to human GSTO1. RnGSTO1 interacts with food phytochemicals, including bitter compounds such as luteolin (Ki = 3.3 ± 1.9 µM). Crystal structure analysis suggests that luteolin most probably binds to RnGSTO1 ligandin site. Our results suggest that GSTO1 could interact with food phytochemicals in the oral cavity.

Aberrantly downregulated FENDRR by arecoline elevates ROS and myofibroblast activation via mitigating the miR-214/MFN2 axis.

Long non-coding RNA FENDRR possesses both anti-fibrotic and anti-cancer properties, but its significance in the development of premalignant oral submucous fibrosis (OSF) remains unclear. Here, we showed that FENDRR was downregulated in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs), and overexpression of FENDRR mitigated various myofibroblasts hallmarks, and vice versa. In the course of investigating the mechanism underlying the implication of FENDRR in myofibroblast transdifferentiation, we found that FENDRR can directly bind to miR-214 and exhibit its suppressive effect on myofibroblast activation via titrating miR-214. Moreover, we showed that mitofusin 2 (MFN2), a protein that is crucial to the fusion of mitochondria, was a direct target of miR-214. Our data suggested that FENDRR was positively correlated with MFN2 and MFN2 was required for the inhibitory property of FENDRR pertaining to myofibroblast phenotypes. Additionally, our results showed that the FENDRR/miR-214 axis participated in the arecoline-induced reactive oxygen species (ROS) accumulation and myofibroblast transdifferentiation. Building on these results, we concluded that the aberrant downregulation of FENDRR in OSF may be associated with chronic exposure to arecoline, leading to upregulation of ROS and myofibroblast activation via the miR-214-mediated suppression of MFN2.

Multifunctional hyaluronic acid/gelatin methacryloyl core-shell microneedle for comprehensively treating oral mucosal ulcers.

Oral ulceration is the most common oral mucosal disease. Oral mucosal ulcers are extremely painful, may interfere with eating and speaking, and potentially complicate systemic symptoms in severe cases. The humid and highly dynamic environment of the oral cavity makes local drug administration for treating oral mucosal ulcers challenging. To overcome these challenges, we designed and prepared a novel dissolving microneedle (MN) patch containing multiple drugs in a core-shell to promote oral ulcer healing. The MNs contained a methacrylate gelatin shell layer of basic fibroblast growth factor (bFGF), a hyaluronic acid (HA) core loaded with dexamethasone (DXMS), and zeolite imidazoline framework-8 (ZIF-8) encapsulated in the HA-based backplane. Progressive degradation of gelatin methacryloyl (GelMA) from the tip of the MN patch in the oral mucosa resulted in sustained bFGF release at the lesion site, significantly promoting cell migration, proliferation, and angiogenesis. Moreover, the rapid release of HA and, subsequently, DXMS inhibited inflammation, and the remaining MN backing after the tip dissolved behaved as a dressing, releasing ZIF-8 for its antimicrobial effects. This novel, multifunctional, transmucosal core-shell MN patch exhibited excellent anti-inflammatory, antimicrobial, and pro-healing effects in vivo and in vitro, suggesting that it can promote oral ulcer healing.

Antifibrotic effect of silymarin on arecoline-induced fibrosis in primary human buccal fibroblasts: an in silico and in vitro analysis.

BACKGROUND: This study aimed to assess silymarin's anticancer and antifibrotic potential through in silico analysis and investigate its impact on in vitro arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS & RESULTS: The study utilized iGEMDOCK for molecular docking, evaluating nine bioflavonoids, and identified silymarin and baicalein as the top two compounds with the highest target affinity, followed by subsequent validation through a 100ns Molecular Dynamic Simulation demonstrating silymarin's stable behavior with Transforming Growth Factor Beta. HBF cell lines were developed from tissue samples obtained from patients undergoing third molar extraction. Arecoline, a known etiological factor in oral submucous fibrosis (OSMF), was employed to induce fibrogenesis in these HBFs. The inhibitory concentration (IC50) of arecoline was determined using the MTT assay, revealing dose-dependent cytotoxicity of HBFs to arecoline, with notable cytotoxicity observed at concentrations exceeding 50µM. Subsequently, the cytotoxicity of silymarin was assessed at 24 and 72 h, spanning concentrations from 5µM to 200µM, and an IC50 value of 143µM was determined. Real-time polymerase chain reaction (qPCR) was used to analyze the significant downregulation of key markers including collagen, epithelial-mesenchymal transition (EMT), stem cell, hypoxia, angiogenesis and stress markers in silymarin-treated arecoline-induced primary buccal fibroblast cells. CONCLUSION: Silymarin effectively inhibited fibroblast proliferation and downregulated genes associated with cancer progression and EMT pathway, both of which are implicated in malignant transformation. To our knowledge, this study represents the first exploration of silymarin's potential as a novel therapeutic agent in an in vitro model of OSMF.

Expression of FAP in Oral Leukoplakia and Oral Squamous Cell Carcinoma.

OBJECTIVE: This study aimed to investigate the potential of fibroblast activation protein (FAP) as a biomarker in the progression of oral leukoplakia (OLK) carcinogenesis. This was achieved by evaluating FAP expression at different levels of the organisation, namely oral normal mucosa (NM), OLK, and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Altogether, 88 paraffin-embedded tissue samples were examined, including 55 cases of OLK, 13 cases of OSCC, and 20 cases of NM (control group). An exhaustive investigation was performed to examine FAP expression in NM, OLK, and OSCC tissues via immunohistochemistry (IHC). The relationship between FAP expression and clinical pathologic characteristics was analysed. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB) also proved the expression of FAP in NM, OLK, and OSCC cells. Aberrant FAP expression in OLK and OSCC was explored using in vitro experiments. RESULTS: Immunohistochemical results showed that high FAP expression was significantly correlated with histopathologic grade (P = .038) but not correlated with age, sex, or region (P = .953, .622, and .108, respectively). The expression level of FAP in NM tissues (0.15 ± 0.01) was minimal, whereas it was observed in OLK (0.28 ± 0.04) and OSCC (0.39 ± 0.02) tissues with a noticeable increase in expression levels (P < .001). The expression level of FAP in OLK with severe abnormal hyperplasia (S-OLK) tissues (0.33 ± 0.04) was significantly higher than in OLK with mild abnormal hyperplasia (MI-OLK, 0.26 ± 0.02) and OLK with moderate abnormal hyperplasia (MO-OLK, 0.28 ± 0.03) tissues (P < .001 and P = .039, respectively). The results of RT-PCR illustrated that the relative expression of FAP mRNA in OLK cells (2.63 ± 0.62) was higher than in NM cells (0.87 ± 0.14), but lower than in OSCC cells (5.63 ± 1.06; P = .027 and .012, respectively). FAP expression was minimal in NM cells (0.78 ± 0.06), modest in OLK cells (1.04 ± 0.06), and significantly elevated in OSCC cells (1.61 ± 0.09) based on the results of WB (P < .001). CONCLUSIONS: Significant variations in FAP expression were observed in NM, OLK, and OSCC tissues and cells. These findings revealed that FAP may be a reliable biomarker for the early diagnosis and evaluation of OLK carcinogenesis.

The administration of oral mucosal mesenchymal-derived stem cells improves hepatic inflammation, oxidative stress, and histopathology following traumatic brain injury.

BACKGROUND: The inflammatory mediators produced after traumatic brain injury (TBI) are reaching peripheral organs causing organ and tissue damage, including the liver. Our study assessed the effect of intravenous (i.v.) infusion of oral mesenchymal stem cells (OMSCs) on TBI-induced liver damage by measuring liver inflammatory factors and liver oxidative stress. METHODS: Twenty-eight adult male Wistar rats were divided into four groups: 1) sham control; 2) TBI alone (TBI); 3) TBI vehicle (Veh)-control; and 4) TBI with OMSC treatment (SC). OMSCs were obtained from oral mucosa biopsies. OMSCs were administered and administered i.v. at 1 and 24 h after TBI. Within 48 h after TBI, multiple parameters were analyzed, including inflammation, oxidative stress, and histopathological changes. RESULTS: In comparison to sham controls, the TBI alone showed in liver significantly increased levels of interleukin-1ß (IL-1ß; P < 0.001), interleukin-6 (IL-6; P < 0.001), malondialdehyde (MDA; P < 0.001), and protein carbonyl (PC; P < 0.001). At the same time the TBI alone decreased the liver levels of superoxide dismutase (SOD; P < 0.001), total antioxidant capacity (TAC; P < 0.001), catalase (CAT; P < 0.001), and interleukin-10 (IL-10; P < 0.001). In comparison to the TBI alone group, the therapeutic group treated with i.v. infusion of OMSCs demonstrated significantly reduced changes of IL-1ß (P < 0.001), IL-6 (P < 0.01), MDA (P < 0.01), PC (P < 0.05), SOD (P < 0.001), TAC (P < 0.01), CAT (P < 0.01), and IL-10 (P < 0.01). Histopathological evaluation showed in TBI alone group that the total score of liver tissue injury included extensive hydropic degeneration, lobular necrosis, inflammation as well as central vein congestion with subendothelial hemorrhage increased compared the sham group (P < 0.001). Administration of OMSC showed significantly smaller increase in the injury score compared to the TBI alone group (P < 0.001). CONCLUSION: Therapy with i.v. OMSCs administration after TBI reduces liver injury, as measured by inflammation and oxidative stress. The use of OMSCs can be considered for treatment of liver injury caused by TBI.

Boosting systemic absorption of peptides with a bioinspired buccal-stretching patch.

Biopharmaceuticals, including proteins and peptides, have revolutionized the treatment of a wide range of diseases, from diabetes and cardiovascular disorders to virus infections and cancer. Despite their efficacy, most of these macromolecular drugs require parenteral administration because of their high molecular weight and relative instability. Over the past 40 years, only a few oral peptide drugs have entered clinical trials, even when formulated with substantial amounts of permeation enhancers. To overcome the epithelial barrier, devices that inject drugs directly into the gastrointestinal mucosa have been proposed recently. However, the robustness and safety of those complex systems are yet to be assessed. In this study, we introduced an innovative technology to boost drug absorption by synergistically combining noninvasive stretching of the buccal mucosa with permeation enhancers. Inspired by the unique structural features of octopus suckers, a self-applicable suction patch was engineered, enabling strong adhesion to and effective mechanical deformation of the mucosal tissue. In dogs, this suction patch achieved bioavailability up to two orders of magnitude higher than those of the commercial tablet formulation of desmopressin, a peptide drug known for its poor oral absorption. Moreover, systemic exposure comparable to that of the approved oral semaglutide tablet was achieved without further optimization. Last, a first-in-human study involving 40 healthy participants confirmed the dosage form's acceptability, thereby supporting the clinical translatability of this simple yet effective platform technology.

Fibrotic Matrix Induces Mesenchymal Transformation of Epithelial Cells in Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is a potentially malignant disorder of the oral mucosa; however, whether and how the fibrotic matrix of OSF is involved in the malignant transformation of epithelial cells remains unknown. Herein, oral mucosa tissue from patients with OSF, OSF rat models, and their controls were used to observe the extracellular matrix changes and epithelial-mesenchymal transformation (EMT) in fibrotic lesions. Compared with controls, oral mucous tissues from patients with OSF showed an increased number of myofibroblasts, a decreased number of blood vessels, and increased type I and type III collagen levels. In addition, the oral mucous tissues from humans and OSF rats showed increased stiffness, accompanied by increased EMT activities of epithelial cells. The EMT activities of stiff construct-cultured epithelial cells were increased significantly by exogenous piezo-type mechanosensitive ion channel component 1 (Piezo1) activation, and decreased by yes-associated protein (YAP) inhibition. During ex vivo implantation, oral mucosal epithelial cells of the stiff group showed increased EMT activities and increased levels of Piezo1 and YAP compared with those in the sham and soft groups. These results indicate that increased stiffness of the fibrotic matrix in OSF led to increased proliferation and EMT of mucosal epithelial cells, in which the Piezo1-YAP signal transduction is important.

The Specifics of Neovascularization of Wound Defects in the Oral Mucosa during Its Regeneration under a Piezoelectric Polymer Membrane.

We studied restoration of microvessels in the oral mucosa wound defects under a polymer piezoelectric membrane (group 2) and without it (group 1). The control group included animals with intact mucosa. On day 3, the expression of the vascular endothelial growth factor (VEGF) increased in all experimental groups, while the expression of CD34 increased only in group 2, which attested to intensive neoangiogenesis. On day 7, we observed a decrease in VEGF expression and an increase in CD34 expression that was more pronounced in group 2, which reflected the beginning of blood vessels maturation. More rapid formation and maturation of blood vessels in group 2 was confirmed by electron microscopy: on day 7, endothelial cells with mature organelles and signs of active transcapillary exchange were seen. On day 12, the immature blood vessels still predominated in group 1, while in group 2, the expression of angiogenesis markers decreased though remained above the control, which created prerequisites for the complete restoration of wound area vascularization in group 2. In group 1, the expression of VEGF and CD34 was significantly below the control, which attested to the development of poorly vascularized scar tissue.

Cytokines secreted by inflamed oral mucosa: implications for oral cancer progression.

The oral mucosa has an essential role in protecting against physical, microbial, and chemical harm. Compromise of this barrier triggers a wound healing response. Key events in this response such as immune infiltration, re-epithelialization, and stroma remodeling are coordinated by cytokines that promote cellular migration, invasion, and proliferation. Cytokine-mediated cellular invasion and migration are also essential features in cancer dissemination. Therefore, exploration of cytokines that regulate each stage of oral wound healing will provide insights about cytokines that are exploited by oral squamous cell carcinoma (SCC) to promote tumor development and progression. This will aid in identifying potential therapeutic targets to constrain SCC recurrence and increase patient survival. In this review, we discuss cytokines that overlap in oral wounds and SCC, emphasizing how these cytokines promote cancer progression.

MiR-424/TGIF2-Mediated Pro-Fibrogenic Responses in Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) has been recognized as a potentially malignant disorder and is characterized by inflammation and the deposition of collagen. Among various regulators of fibrogenesis, microRNAs (miR) have received great attention but the detailed mechanisms underlying the miR-mediated modulations remain largely unknown. Here, we showed that miR-424 was aberrantly overexpressed in OSF tissues, and then we assessed its functional role in the maintenance of myofibroblast characteristics. Our results demonstrated that the suppression of miR-424 markedly reduced various myofibroblast activities (such as collagen contractility and migration ability) and downregulated the expression of fibrosis markers. Moreover, we showed that miR-424 exerted this pro-fibrosis property via direct binding to TGIF2, an endogenous repressor of the TGF-ß signaling. In addition, our findings indicated that overexpression of miR-424 activated the TGF-ß/Smad pathway, leading to enhanced myofibroblast activities. Altogether, our data revealed how miR-424 contributed to myofibroblast transdifferentiation, and targeting the miR-424/TGIF2 axis may be a viable direction for achieving satisfactory results from OSF treatment.

Molecular pathways of oral submucous fibrosis and its progression to malignancy.

OBJECTIVE: The review aims to comprehend various factors engaged in the alteration of molecular events resulting in Oral submucous fibrosis (OSMF) and its malignant transformation. DESIGN: Literature pertinent to pathways involved in OSMF were explored in databases such as PubMed, Scopus and Google Scholar. The relevant literature was reviewed and critically appraised in this narrative review. RESULTS: Areca nut components influence myriad of cellular molecules such as cytokines, growth factors, myofibroblasts, non-coding RNAs and alter their expression. These aberrantly expressed molecules drive the progression of OSMF from localized inflammation to fibrosis of buccal mucosa. The oral tissue suffers from oxidative stress, hypoxia, autophagy, aberration of cell cycle and DNA damage. Apoptosis of epithelial layer results in its atrophy facilitating deeper penetration of areca nut elements. With the advance of disease, epithelial-mesenchymal transition eventuates and promotes dysplasia. The jeopardized expression of various cellular molecules, suppressed apoptosis, along with increased genetic alterations and neovascularization favors the malignant transformation. CONCLUSION: OSMF is a progressive disorder with complex mechanism of pathogenesis initiated by inflammation of oral mucosa. Continuous habit of areca nut chewing and the resulting insult to the tissues prevents healing process and is destined to debilitating disease which affects the quality of life with a higher probability of progression to malignancy.

XIST/let-7i/HMGA1 axis maintains myofibroblasts activities in oral submucous fibrosis.

Long non-coding RNA XIST promotes the development of various types of head and neck cancers, but its role in the progression of precancerous oral submucous fibrosis (OSF) has not been determined yet. As such, we aimed to examine whether XIST implicates in the regulation of myofibroblast activation. Our results showed that the expression of XIST was upregulated in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs), and the silencing of XIST downregulated several myofibroblasts features. We demonstrated that elevation of let-7i after inhibition of XIST may lead to reduced myofibroblast activation. On the contrary, overexpression of high mobility group AT-Hook 1 (HMGA1) following the suppression of let-7i may result in enhanced myofibroblast activities. Moreover, we showed that the suppressive effect of silencing of XIST on myofibroblasts hallmarks was reversed by let-7i inhibition or HMGA1 overexpression, suggesting the pro-fibrotic property of XIST was mediated by downregulation of let-7i and upregulation of HMGA1. These findings revealed that myofibroblast activation of fBMFs may attribute to the alteration of the XIST/let-7i/HMGA1 axis. Therapeutic approaches to target this axis may serve as a promising direction to ameliorate the malignant progression of OSF.