BACKGROUND: Cervical squamous cell carcinoma (CESC), the most common subtype of cervical cancer, is primarily caused by the high-risk human papillomavirus (HPV) infection and genetic susceptibility. Single-cell RNA sequencing (scRNA-seq) has been widely used in CESC research to uncover the diversity of cell types and states within tumor tissues, enabling a detailed study of the tumor microenvironment (TME). This technology allows precise mapping of HPV infection in cervical tissues, providing valuable insights into the initiation and progression of HPV-mediated malignant transformation. METHODS: We performed the scRNA-seq to characterize gene expression in tumor tissues and paired adjacent para-cancerous tissues from four patients with early-stage CESC using the 10× Genomics platform. The HPV infection and its subtypes were identified using the scRNA data and viral sequence mapping, and trajectory analyses were performed using HPV+ or HPV- cells. Interactions between different types of keratinized cells and their interactions with other cell types were identified, and pathways and specificity markers were screened for proliferating keratinized cells. The Cancer Genome Atlas (TCGA) dataset was used to verify the prognostic correlation between tumor-specific PI3+S100A7+ keratinocyte infiltration and CESC, and the localization relationship between PI3+S100A7+ keratinocytes and macrophages was verified by immunofluorescence staining. RESULTS: Various types of keratinocytes and fibroblasts were the two cell types with the most significant differences in percentage between the tumor tissue samples and paired adjacent non-cancerous tissue samples in the early stages of CESC. We found that PI3+S100A7+ keratinocytes were associated with early HPV-positive CESC, and PI3+S100A7+ keratinocytes were more abundant in tumors than in adjacent normal tissues in the TCGA-CESC dataset. Analysis of clinical information revealed that the infiltration of PI3+S100A7+ keratinocytes was notably higher in tumors with poor prognosis than in those with good prognosis. Additionally, multiplex immunofluorescence analysis showed a specific increase in PI3+S100A7+ expression within tumor tissues, with PI3+S100A7+ keratinocytes and CD163+ macrophages being spatially very close to each other. In the analysis of cell-cell interactions, macrophages exhibited strong crosstalk with PI3+S100A7+ proliferating keratinocytes in HPV-positive CESC tumors, mediated by tumor necrosis factor (TNF), CCL2, CXCL8, and IL10, highlighting the dynamic and tumor-specific enhancement of macrophage-keratinocyte interactions, which are associated with poor prognosis and immune modulation. Using CIBERSORTx, we discovered that patients with high infiltration of both PI3+S100A7+ proliferating keratinocytes and macrophages had the shortest overall survival. In the analysis of cell-cell interactions, PI3+S100A7+ proliferating keratinocytes and macrophages were found to be involved in highly active pathways that promote differentiation and structure formation, including cytokine receptor interactions, the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, and TNF signaling pathway regulation. Further subtyping of fibroblast populations identified four subtypes. The C1 group, characterized by its predominance in tumor tissues, is a subtype enriched with cancer-associated fibroblasts (CAFs), whereas the C3 group is primarily enriched in adjacent non-cancerous tissues and consists of undifferentiated cells. Moreover, the distinct molecular and cellular differences between HPV16- and HPV66-associated tumors were demonstrated, emphasizing the unique tumor-promoting mechanisms and microenvironmental influences driven by each HPV subtype. CONCLUSIONS We discovered a heterogeneous population of keratinocytes between tumor and adjacent non-cancerous tissues caused by HPV infection and identified macrophages and specific CAFs that play a crucial role during the early stage in promoting the inflammatory response and remodeling the cancer-promoting TME. Our findings provide new insights into the transcriptional landscape of early-stage CESC to understand the mechanism of HPV-mediated malignant transformation in cervical cancer.
Humans ; Female ; Papillomavirus Infections/genetics* ; Uterine Cervical Neoplasms/genetics* ; Carcinoma, Squamous Cell/pathology* ; Keratinocytes/metabolism* ; Single-Cell Analysis/methods* ; Tumor Microenvironment/genetics*

OBJECTIVE: To determine the role of Tripterygium wilfordii multiglycoside (TGW) in the treatment of psoriatic dermatitis from a cellular immunological perspective. METHODS: Mouse models of psoriatic dermatitis were established by imiquimod (IMQ). Twelve male BALB/c mice were assigned to IMQ or IMQ₊TGW groups according to a random number table. Histopathological changes in vivo were assessed by hematoxylin and eosin staining. Ratios of immune cells and cytokines in mice, as well as PAM212 cell proliferation in vitro were assessed by flow cytometry. Pro-inflammatory cytokine expression was determined using reverse transcription quantitative polymerase chain reaction. RESULTS: TGW significantly ameliorated the severity of IMQ-induced psoriasis-like mouse skin lesions and restrained the activation of CD45⁺ cells, neutrophils and T lymphocytes (all P<0.01). Moreover, TGW significantly attenuated keratinocytes (KCs) proliferation and downregulated the mRNA levels of inflammatory cytokines including interleukin (IL)-17A, IL-23, tumor necrosis factor α, and chemokine (C-X-C motif) ligand 1 (P<0.01 or P<0.05). Furthermore, it reduced the number of γ δ T17 cells in skin lesion of mice and draining lymph nodes (P<0.01). CONCLUSIONS TGW improved psoriasis-like inflammation by inhibiting KCs proliferation, as well as the associated immune cells and cytokine expression. It inhibited IL-17 secretion from γ δ T cells, which improved the immune-inflammatory microenvironment of psoriasis.
Male ; Animals ; Mice ; Tripterygium ; Psoriasis/drug therapy* ; Keratinocytes ; Skin Diseases/metabolism* ; Cytokines/metabolism* ; Imiquimod/metabolism* ; Dermatitis/pathology* ; Disease Models, Animal ; Mice, Inbred BALB C ; Skin/metabolism*

Cutaneous neurogenic inflammation (CNI) is inflammation that is induced (or enhanced) in the skin by the release of neuropeptides from sensory nerve endings. Clinical manifestations are mainly sensory and vascular disorders such as pruritus and erythema. Transient receptor potential vanilloid 1 and ankyrin 1 (TRPV1 and TRPA1, respectively) are non-selective cation channels known to specifically participate in pain and CNI. Both TRPV1 and TRPA1 are co-expressed in a large subset of sensory nerves, where they integrate numerous noxious stimuli. It is now clear that the expression of both channels also extends far beyond the sensory nerves in the skin, occuring also in keratinocytes, mast cells, dendritic cells, and endothelial cells. In these non-neuronal cells, TRPV1 and TRPA1 also act as nociceptive sensors and potentiate the inflammatory process. This review discusses the role of TRPV1 and TRPA1 in the modulation of inflammatory genes that leads to or maintains CNI in sensory neurons and non-neuronal skin cells. In addition, this review provides a summary of current research on the intracellular sensitization pathways of both TRP channels by other endogenous inflammatory mediators that promote the self-maintenance of CNI.
Animals ; Chronic Disease ; Dendritic Cells ; metabolism ; pathology ; Dermatitis ; metabolism ; pathology ; Gene Expression Regulation ; Humans ; Inflammation ; metabolism ; pathology ; Keratinocytes ; metabolism ; pathology ; Mast Cells ; metabolism ; pathology ; Sensory Receptor Cells ; metabolism ; pathology ; TRPA1 Cation Channel ; biosynthesis ; TRPV Cation Channels ; biosynthesis

Skin wound healing is a complex event, and interrupted wound healing process could lead to scar formation. The aim of this study was to examine the morphological changes of scar tissue. Pathological staining (HE staining, Masson's trichrome staining, methenamine silver staining) was used to evaluate the morphological changes of regenerating epidermis in normal skin and scar tissue, and immunofluorescence staining to detect the expression of collagen IV, a component of basement membrane (BM), and the expression of integrinβ4, a receptor for BM laminins. Additionally, the expression of CK14, CK5, and CK10 was measured to evaluate the proliferation and differentiation of keratinocytes in normal skin and scar tissue. The results showed that the structure of the skin was histologically changed in scar tissue. Collagen IV, expressed under the epidermis of normal skin, was reduced distinctly in scar tissue. Integrinβ4, expressed in the basal layer of normal skin, was found absent in the basal layer of scar tissue. Additionally, it was found that keratinocytes in scarring epidermis were more proliferative than in normal skin. These results indicate that during the skin wound healing, altered formation of BM may affect the proliferation of keratinocytes, reepithelial and tissue remodeling, and then result in scar formation. Thus, remodeling BM structure during wound repair may be beneficial for improving healing in cutaneous wounds during clinical practice.
Adolescent ; Adult ; Cicatrix ; metabolism ; pathology ; Collagen Type IV ; metabolism ; Female ; Humans ; Integrin beta4 ; metabolism ; Keratinocytes ; cytology ; metabolism ; pathology ; Male ; Skin ; cytology ; metabolism ; pathology

OBJECTIVE: In order to investigate the interaction between the cytokines and keratinocyte and determine the role of cytokines in hyperproliferative of chronic otitis media with cholesteatoma, we observe the expression of matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and its receptor (KGFR) in middle ear cholesteatoma. METHOD: We examined the expression of MMP9, VEGF, KGF, KGFR and Ki-67 by immunohistochemistry in 50 specimens from chronic otitis media with cholesteatoma and 15 specimens from the normal skin of external auditory meatus. Ki-67 as an evaluation of cholesteatoma proliferation markers were used to detect the keratinocyte proliferative activity. RESULT: (1) The expression of VEGF and MMP9 in cholesteatoma specimens was higher than normal skin, and the difference was statistically significant (t = 4.914, P < 0.01; t = 3.284, P < 0.01). (2) The expression of KGF and KGFR in middle ear tissues was higher than normal skin, and the difference was statistically significant (t = 4.814, P < 0.01; t = 3.104, P < 0.01); The expression of KGF and KGFR increased, and the expression of Ki-67 also correspondly increased in the cholesteatoma. (3) In the tissue MMP9 and VEGF were positive. Mean optical density increased as well. KGF expression also increased accordingly. CONCLUSION MMP9, VEGF, KGF and KGFR proteins played an important role in hyperproliferation of cholesteatoma tissues. VEGF, MMP9 and KGF had a synergistic effect in hyperproliferation of cholesteatoma tissues.
Cholesteatoma, Middle Ear ; pathology ; Cytokines ; metabolism ; Ear Canal ; metabolism ; Ear, Middle ; metabolism ; Fibroblast Growth Factor 7 ; metabolism ; Humans ; Immunohistochemistry ; Keratinocytes ; cytology ; Ki-67 Antigen ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Otitis Media ; pathology ; Receptor, Fibroblast Growth Factor, Type 2 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.
Adult ; Cell Proliferation ; Chemokine CXCL12 ; genetics ; Epidermal Cells ; Epidermis ; pathology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; metabolism ; Gene Expression Regulation ; Humans ; Keratinocytes ; cytology ; pathology ; Signal Transduction ; Skin Diseases ; genetics ; pathology

PURPOSE: Recent findings of increased cathelicidin protein and its proteolytic fragments in rosacea suggest a pathogenic role for cathelicidin in this disease. The relationship between cathelicidin and protease-activated receptor 2 (PAR-2) is therefore of interest, as PAR-2, expressed principally in keratinocytes, regulates pro-inflammatory cytokine expression in the skin. The purpose of this study was to determine the relationship between expression of PAR-2 and cathelicidin in rosacea and to test the effect of direct PAR-2 activation on cathelicidin expression in keratinocytes. MATERIALS AND METHODS: Samples from 40 patients with clinicopathologic diagnosis of rosacea and facial skin tissue samples from 20 patients with no specific findings or milium without inflammation were retrieved. Intensities of immunohistochemical staining for PAR-2 and cathelicidin were compared between normal and rosacea-affected skin tissues. Additionally, correlations between PAR-2 and cathelicidin staining intensities within rosacea patients were analyzed. In cultured keratinocytes, changes in PAR-2, cathelicidin, and vascular endothelial growth factor (VEGF) mRNA and protein were analyzed after treatment with PAR-2 activating peptide (AP). RESULTS: Cathelicidin expression was significantly higher in rosacea skin tissues than in normal tissues (p<0.001), while PAR-2 expression was not significantly higher in rosacea tissues than in normal skin tissues. A positive correlation between PAR-2 and cathelicidin within rosacea samples was observed (R=0.330, p=0.037). After treatment of PAR-2 AP, both mRNA and protein levels for PAR-2, cathelicidin, and VEGF significantly increased in cultured keratinocytes, compared with PAR-2 control peptide treatment. CONCLUSION: PAR-2 may participate in the pathogenesis of rosacea through activation of cathelicidin LL-37, a mediator of innate immune responses in the skin.
Adult ; Aged ; Antimicrobial Cationic Peptides/*metabolism ; Cytokines/metabolism ; Female ; Humans ; Immunity, Innate ; Inflammation/metabolism ; Keratinocytes/*metabolism ; Male ; Middle Aged ; Receptor, PAR-2/*metabolism ; Rosacea/*pathology ; Skin/pathology ; Vascular Endothelial Growth Factor A/*metabolism

In covering wounds, efforts should include utilization of the safest and least invasive methods with goals of achieving optimal functional and cosmetic outcome. The recent development of advanced wound healing technology has triggered the use of cells to improve wound healing conditions. The purpose of this review is to provide information on clinically available cell-based treatment options for healing of acute and chronic wounds. Compared with a variety of conventional methods, such as skin grafts and local flaps, the cell therapy technique is simple, less time-consuming, and reduces the surgical burden for patients in the repair of acute wounds. Cell therapy has also been developed for chronic wound healing. By transplanting cells with an excellent wound healing capacity profile to chronic wounds, in which wound healing cannot be achieved successfully, attempts are made to convert the wound bed into the environment where maximum wound healing can be achieved. Fibroblasts, keratinocytes, adipose-derived stromal vascular fraction cells, bone marrow stem cells, and platelets have been used for wound healing in clinical practice. Some formulations are commercially available. To establish the cell therapy as a standard treatment, however, further research is needed.
Blood Platelets/metabolism ; Cell- and Tissue-Based Therapy ; Diabetes Mellitus, Type 2/complications/pathology ; Fibroblasts/cytology/transplantation ; Humans ; Keratinocytes/cytology/transplantation ; Stromal Cells/cytology/transplantation ; Tissue Engineering ; Ulcer/etiology/therapy ; *Wound Healing

OBJECTIVETo investigate the effects of aminoguanidine cream on the proliferation of keratinocytes (KC), content of advanced glycosylation end products (AGE) and oxidative stress in skin tissue of rats with diabetes.

METHODSStearic acid, liquid paraffin, vaseline, lanolin, isopropyl myristate fat, glycerol, 50 g/L alcohol paraben, aminoguanidine hydrochloride etc. were mixed in certain proportion to make aminoguanidine cream, and cream without aminoguanidine was used as matrix. The dorsal skin of normal rats were harvested and treated by aminoguanidine cream with dose of 5, 10 g/L, or 5 g/L together with 10 g/L azone. The transdermal effect was respectively measured at post treatment hour 2, 4, 7, 10, 12, 24. Thirty SD rats were divided into normal control (NC, n = 6), diabetes (D, n = 8), aminoguanidine cream-interfered (AI, n = 8), matrix cream-interfered groups (MI, n = 8) according to the random number table. Diabetes was reproduced by intraperitoneal injection of STZ (65 mg/kg) in rats of D, AI, and MI groups, and rats in NC group were injected with 0.05 mmol/L citrate buffer as control. One week later, dorsal skin of rats in AI and MI groups were respectively treated with 10 g/L aminoguanidine cream and matrix cream by external use for 4 weeks. AGE content was determined with fluorescence detection from skin collagen extract. KC cell cycle was detected by flow cytometry. Skin tissue specimens were obtained for determination of levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and total antioxidant capacity. Data were processed with t test.

RESULTSTransdermal effect of aminoguanidine cream with dose of 10 g/L was better than that with 5 g/L or 5 g/L + 10 g/L azone cream. One rat was not induced successfully in MI group. Four weeks after model reproduction, 4 rats died in D group and 1 rat died in AI group. The AGE content in D group was obviously higher than that in NC group [(36.8 +/- 2.6), (24.6 +/- 2.7) U per milligram hydroxyproline, respectively, t = 7.2, P < 0.01], and that in AI group [(28.6 +/- 3.7) U per milligram hydroxyproline] was also lower as compared with that in D group (t = -3.9, P < 0.05). There was no significant difference in AGE content between MI [(32.2 +/- 5.2) U per milligram hydroxyproline] and D groups (t = 1.6, P > 0.05). The percentage of KC in S phase was obviously lower in D group than in NC group [(5.3 +/- 0.6)%, (7.6 +/- 0.9)%, respectively, t = 4.50, P < 0.01], while that in MI group [(9.2 +/- 1.5)%] was higher as compared with that in D group ( t = 4.90, P < 0.01). It was more higher in AI group than in D group on KC percentage in S and G2/M phase (with t value respectively 6.80, 3.17, P values all below 0.01). The oxidative stress indexes of skin tissue in D group were all higher than those in NC group, in which levels of MPO and SOD showed statistical difference (with t value respectively 4.4, 3.7, P values all below 0.05). The oxidative stress indexes were all lower in AI group than in D group, especially in SOD level (t = -1.4, P < 0.05). Levels of MAD, MPO in MI group were significantly lower than those in D group (with t value respectively 2.6, 2.9, P values all below 0.05).

CONCLUSIONSAminoguanidine cream can promote KC proliferation and appropriately reduce oxidative stress through inhibiting AGE formation to a certain extent in skin tissue of rats with diabetes. Signal use of matrix cream can also reduce oxidative stress in skin tissue of rats with diabetes.

Administration, Cutaneous ; Animals ; Cell Proliferation ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Glycation End Products, Advanced ; metabolism ; Guanidines ; administration & dosage ; pharmacology ; Keratinocytes ; drug effects ; Male ; Ointments ; administration & dosage ; pharmacology ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; metabolism ; pathology

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.
Animals ; Cells, Cultured ; Chemokine CCL2/metabolism ; Complement C1r/metabolism ; Corneal Transplantation/*immunology/pathology ; Endothelium, Corneal/*metabolism/pathology ; *Gene Expression Profiling ; Graft Rejection/*immunology/pathology ; HLA-A Antigens/metabolism ; Humans ; Keratinocytes/*metabolism ; Oligonucleotide Array Sequence Analysis ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Transplantation, Heterologous ; Up-Regulation