S100 calcium-binding protein A9 promotes skin regeneration through toll-like receptor 4 during tissue expansion.

Background: In plastic surgery, tissue expansion is widely used for repairing skin defects. However, low expansion efficiency and skin rupture caused by thin, expanded skin remain significant challenges in promoting skin regeneration during expansion. S100 calcium-binding protein A9 (S100A9) is essential in promoting wound healing; however, its effects on skin regeneration during tissue expansion remain unclear. The aim of the present study was to explore the role of S100A9 in skin regeneration, particularly collagen production to investigate its importance in skin regeneration during tissue expansion. Methods: The expression and distribution of S100A9 and its receptors-toll-like receptor 4 (TLR-4) and receptor for advanced glycation end products were studied in expanded skin. These characteristics were investigated in skin samples of rats and patients. Moreover, the expression of S100A9 was investigated in stretched keratinocytes in vitro. The effects of S100A9 on the proliferation and migration of skin fibroblasts were also observed. TAK-242 was used to inhibit the binding of S100A9 to TLR-4; the levels of collagen I (COL I), transforming growth factor beta (TGF-ß), TLR-4 and phospho-extracellular signal-related kinase 1/2 (p-ERK1/2) in fibroblasts were determined. Furthermore, fibroblasts were co-cultured with stretched S100A9-knockout keratinocytes by siRNA transfection and the levels of COL I, TGF-ß, TLR-4 and p-ERK1/2 in fibroblasts were investigated. Additionally, the area of expanded skin, thickness of the dermis, and synthesis of COL I, TGF-ß, TLR-4 and p-ERK1/2 were analysed to determine the effects of S100A9 on expanded skin. Results: Increased expression of S100A9 and TLR-4 was associated with decreased extracellular matrix (ECM) in the expanded dermis. Furthermore, S100A9 facilitated the proliferation and migration of human skin fibroblasts as well as the expression of COL I and TGF-ß in fibroblasts via the TLR-4/ERK1/2 pathway. We found that mechanical stretch-induced S100A9 expression and secretion of keratinocytes stimulated COL I, TGF-ß, TLR-4 and p-ERK1/2 expression in skin fibroblasts. Recombined S100A9 protein aided expanded skin regeneration and rescued dermal thinning in rats in vivo as well as increasing ECM deposition during expansion. Conclusions: These findings demonstrate that mechanical stretch promoted expanded skin regeneration by upregulating S100A9 expression. Our study laid the foundation for clinically improving tissue expansion using S100A9.

Identification and Validation of Hub Immune-Related Genes in Non-Alcoholic Fatty Liver Disease.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common progressive liver disease worldwide. It can cause liver cancer and possibly death. Abnormal immune infiltration is involved in the progression of NAFLD. The aim of this study was to identify and validate the hub immune-related genes in NAFLD. Methods: Microarray data were downloaded from Gene Expression Omnibus, and immune-related differentially expressed genes (IRDEGs) were obtained. A protein-protein interaction network was used to further screen. The diagnostic value of the IRDEGs was evaluated by receiver operating characteristic curves. Differences in immune infiltration levels were analyzed using single-sample gene set enrichment analysis. Hub IRDEGs were identified by correlation analysis with immune infiltration levels. Finally, molecular experiments were used to confirm the expression of the hub IRDEGs and explore their roles in NAFLD. Results: We obtained 18 IRDEGs. Five hub genes were further identified by protein-protein interaction network, receiver operating characteristic curves and correlation analysis: AQP9, BACH2, CD4, IL17RE and S100A9. Based on functional enrichment analysis, the hub genes were enriched primarily in many immune-related pathways. In NAFLD, AQP9, CD4, and IL17RE expression was significantly reduced, whereas BACH2 and S100A9 expression was elevated. PCR, oil red O staining and triglyceride detection revealed that the knock-down of BACH2 and S100A9 reduced lipid accumulation in NAFLD cells. Conclusion: This study provided insight into the profile of immune infiltration underlying NAFLD and identified AQP9, BACH2, CD4, IL17RE and S100A9 as ancillary diagnostic indicators of NAFLD. And BACH2 and S100A9 might be therapeutic targets for NAFLD.

Expression of S100A9 in adamantinomatous craniopharyngioma and its association with wet keratin formation.

Wet keratin is a hallmark of adamantinomatous craniopharyngioma (ACP), which is frequently infiltrated by inflammatory cells. S100 calcium-binding protein A9 (S100A9) has been confirmed to play a decisive role in the development of inflammation. However, the relationship between wet keratin (keratin nodules) and S100A9 in ACP is poorly understood. The objective of the present study was to explore the expression of S100A9 in ACP and its association with wet keratin formation. Immunohistochemistry and immunofluorescence were used to detect the expression of S100A9, ß-catenin and Ki67 in 46 cases of ACP. A total of three online databases were used to analyze S100A9 gene expression and protein data. The results revealed that S100A9 was primarily expressed in wet keratin and some intratumoral and peritumoral cells, and its expression in wet keratin was upregulated in the high inflammation group (P=1.800x10-3). In addition, S100A9 was correlated with the degree of inflammation (r=0.6; P=7.412x10-3) and the percentage of Ki67-positive cells (r=0.37; P=1.000x10-2). In addition, a significant correlation was noted between the area of wet keratin and the degree of inflammation (r=0.51; P=2.500x10-4). In conclusion, the present study showed that S100A9 was upregulated in ACP and may be closely associated with wet keratin formation and the infiltration of inflammatory cells in ACP.

S100A9 promotes glycolytic activity in HER2-positive breast cancer to induce immunosuppression in the tumour microenvironment.

Purpose: The purpose of this study was to investigate the correlation between S100 calcium binding protein A9 (S100A9), tumour glycolysis and tumour infiltrating lymphocytes (TIL) in human epidermal growth factor receptor 2 (HER2) - positive breast cancer (BRCA). Materials and methods: A total of 667 BRCA patients in Xiangya Hospital of Central South University were enrolled in this study. Haematoxylin and eosin (H&E) staining were used to count TIN in tissues. Human breast cancer cell lines (SK-BR-3 cells and BT474 cells) were transfected with S100A9 specific small interfering RNA (siRNA). The expressions of S100A9, glycolytic enzymes and lymphocyte markers were detected by immunohistochemistry (IHC) staining, Western blot and immunofluorescence. Lactate production, glucose consumption and the extracellular acidification rate (ECAR) were detected to assess glycolysis activity. Results: S100A9 was significantly overexpressed in HER2+ cases. The expressions of phosphoglycerol kinase 1 (PGK1), lactate dehydrogenase A (LDHA) and enolase α (ENO1) were significantly up-regulated in S100A9 dominant tissues. The expressions of PGK1, LDHA and ENO1 detected in S100A9 silenced cell lines were significantly down-regulated. Moreover, S100A9 silencing significantly altered lactate production, glucose uptake and ECAR levels in HER2+ cell lines. Co-expression of S100A9 and c-Myc was detected in HER2+ tissues. The absence of S100A9 greatly hindered ß-catenin expression in cell lines, which later induced the phosphorylation of c-Myc.The amount of TILs in cases with abundant S100A9 and LDHA was much greater than in cases with low S100A9 levels and poorer LDHA. TIL deficiency and elevated S100A9 intensity are factors affecting the survival rate of HER2+ BRCA cases. Conclusions: S100A9 overexpression upregulated the glycolysis activity of tumour cells through the c-Myc-related pathway, suppressing lymphocyte infiltration in the tumour stroma, affecting the efficacy of immune regulation and long-term survival of patients.

S100A9­containing serum exosomes obtained from patients with burn injuries promote myocardial cell pyroptosis through NLRP3.

S100 calcium-binding protein A9 (S100A9) is highly expressed in the serum exosomes of patients with burn injuries. The present study aimed to investigate the underlying mechanisms of burn injury-associated exosomes in the regulation of myocardial cell pyroptosis. Reverse transcription-quantitative PCR and western blotting were used to examine relative mRNA and protein levels. The morphology of exosomes was visualized using transmission electron microscopy. The expression levels of IL-1ß and IL-18 in cells were examined using ELISA kits. Finally, cell pyroptosis was examined using flow cytometry. When AC16 cells were treated with the serum exosomes obtained from patients with burn injuries, pyroptosis was significantly promoted and the expression levels of IL-1ß and IL-18 were increased. NLR family pyrin domain containing 3 (NLRP3), S100A9, caspase-1 and Gasdermin D (GSDMD)-N expression levels were also upregulated. However, these were significantly reversed by anti-S100A9 antibodies. Thereafter, CY-09, an NLRP3 inhibitor, was revealed to restore the increase in pyroptosis and IL-18, IL-1ß, caspase-1, NLRP3 and GSDMD-N expression levels caused by recombinant S100A9 to be similar to the control. These findings suggested that burn injury-associated exosomes containing S100A9 can affect AC16 cell pyroptosis through NLRP3.

Selecting Multitarget Peptides for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial disease with a complex pathogenesis. Developing multitarget drugs could be a powerful strategy to impact the progressive loss of cognitive functions in this disease. The purpose of this study is to select a multitarget lead peptide candidate among a series of peptide variants derived from the neutrophil granule protein cathepsin G. We screened eight peptide candidates using the following criteria: (1) Inhibition and reversion of amyloid beta (Aß) oligomers, quantified using an enzyme-linked immunosorbent assay (ELISA); (2) direct binding of peptide candidates to the human receptor for advanced glycation end-products (RAGE), the Toll-like receptor 4 (TLR4) and the S100 calcium-binding protein A9 (S100A9), quantified by ELISA; (3) protection against Aß oligomer-induced neuronal cell death, using trypan blue to measure cell death in a murine neuronal cell line; (4) inhibition of TLR4 activation by S100A9, using a human TLR4 reporter cell line. We selected a 27-mer lead peptide that fulfilled these four criteria. This lead peptide is a privileged structure that displays inherent multitarget activity. This peptide is expected to significantly impact cognitive decline in mouse models of Alzheimer's disease, by targeting both neuroinflammation and neurodegeneration.

S100A9 Derived from Chemoembolization-Induced Hypoxia Governs Mitochondrial Function in Hepatocellular Carcinoma Progression.

Transarterial chemoembolization (TACE) is the major treatment for advanced hepatocellular carcinoma (HCC), but it may cause hypoxic environment, leading to rapid progression after treatment. Here, using high-throughput sequencing on different models, S100 calcium binding protein A9 (S100A9) is identified as a key oncogene involved in post-TACE progression. Depletion or pharmacologic inhibition of S100A9 significantly dampens the growth and metastatic ability of HCC. Mechanistically, TACE induces S100A9 via hypoxia-inducible factor 1α (HIF1A)-mediated pathway. S100A9 acts as a scaffold recruiting ubiquitin specific peptidase 10 and phosphoglycerate mutase family member 5 (PGAM5) to form a tripolymer, causing the deubiquitination and stabilization of PGAM5, leading to mitochondrial fission and reactive oxygen species production, thereby promoting the growth and metastasis of HCC. Higher S100A9 level in HCC tissue or in serum predicts a worse outcome for HCC patients. Collectively, this study identifies S100A9 as a key driver for post-TACE HCC progression. Targeting S100A9 may be a promising therapeutic strategy for HCC patients.

Integrating serum proteomics and metabolomics to compare the common and distinct features between acute aggressive ischemic stroke (APIS) and acute non-aggressive ischemic stroke (ANPIS).

Understanding common and distinct pathophysiological features between acute progressive ischemic stroke (APIS) and acute non-progressive ischemic stroke (ANPIS) is a prerequisite to making clear the mechanism to determine the prognosis of acute ischemic stroke (AIS). Here, we recruited three independent sets of subjects, all of which included the APIS, ANPIS, and control groups. They were used for serum proteomic and metabolomic analyses, and validation of the critical pathophysiological processes and potential biomarkers of APIS, respectively. Results showed that there were both common and distinct metabolome and proteome between APIS and ANPIS. APIS and ANPIS shared basic processes of AIS in inflammation and oxidative stress response. Coagulation and lipid metabolism disorder, activation of the complement system, and inflammation may enhance with each other in the symptom worsening of APIS. The contents of serum amyloid A1 (SAA1) and S100 calcium-binding protein A9 (S100-A9) in the validation set confirmed the key pathophysiological processes indicated by omics data; they also jointly conferred a moderate value to distinguish APIS from ANPIS. Collectively, disturbance in coagulation and lipid metabolism, complement activation, and inflammation may be synergistically involved in symptom deterioration in APIS. SAA1 and S100-A9 serve as a potential biomarker panel to distinguish APIS from ANPIS. THE SIGNIFICANCE: In this study, we integrated serum proteomics and metabolomics to explore the similarities and differences in pathophysiological processes between APIS and ANPIS. The global metabolic networks have been constructed, and the crucial common pathophysiological processes and the key distinct pathophysiological features between APIS and ANPIS were investigated based on the differentially expressed proteins and metabolites (DEPs/DEMs). Furthermore, pivotal serum proteins (SAA1 and S100A9) were detected in a dependent set to validate the key pathophysiological characteristics, as well as to assess the possibility of them being used as a biomarker panel. Taken together, the multi-omics integration strategy used in this clinical study shows potential to comprehensively interpret and compare the pathophysiological processes of AIS in various conditions, as well as to screen a reliable new biomarker panel.

Effects of S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9) on matrix metalloproteinase (MMP) expression in nasopharyngeal carcinoma CNE-2 cells.

BACKGROUND: Studies have shown that S100A8 and S100A9 are highly expressed in a variety of tumors, including NPC, and are associated with tumor invasion and migration. MMPs are associated with the invasion and migration of tumor cells. To further investigate the mechanism by which S100A8 and S100A9 affect the invasion and migration of NPC cells, the present study examined the effects of S100A8 and S100A9 on MMPs in NPC CNE-2 cells. METHODS: Recombinant pEGFP-N1-S100A8 and recombinant pEGFP-N1-S100A9 overexpression vectors and S100A8 and S100A9 RNA interference (RNAi) vectors were constructed and transfected into NPC CNE-2 cells. The transfection efficiency in each group of cells was assessed, and the gene and protein expression of MMP7, MMP9 and MMP12 were determined. RESULTS: The transfection efficiency was approximately 60-70%. Compared with those in the control group, the expression levels of MMP7, MMP9 and MMP12 in the S100A8 and S100A9 overexpression groups was significantly higher (P<0.05), and the expression levels of MMP7, MMP9 and MMP12 in the S100A8-RNAi and S100A9-RNAi groups were significantly lower (P<0.05). The number of cells in S100A8 overexpression group and S100A9 overexpression group at 24, 48 and 72 h was higher than that in RNAi group, RNAi control group, overexpression control group and normal control group, with statistical significance; The cell doubling time in S100A8 and S100A9 overexpression group was significantly shorter than that in RNAi control group, overexpression control group and normal control group, with statistical significance. CONCLUSIONS: High S100A8 and S100A9 expression may promote the expression of MMP7, MMP9 and MMP12, which are related to the invasion and metastasis of NPC cells.

S100 Calcium Binding Protein A9 Represses Angiogenic Activity and Aggravates Osteonecrosis of the Femoral Head.

Ischemic damage aggravation of femoral head collapse is a prominent pathologic feature of osteonecrosis of the femoral head (ONFH). In this regard, S100 calcium binding protein A9 (S100A9) is known to deteriorate joint integrity, however, little is understood about which role S100A9 may play in ONFH. In this study, a proteomics analysis has revealed a decrease in the serum S100A9 level in patients with ONFH upon hyperbaric oxygen therapy. Serum S100A9 levels, along with serum vascular endothelial growth factor (VEGF), soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-6 (IL-6), and tartrate-resistant acid phosphatase 5b levels were increased in patients with ONFH, whereas serum osteocalcin levels were decreased as compared to healthy controls. Serum S100A9 levels were increased with the Ficat and Arlet stages of ONFH and correlated with the patients with a history of being on glucocorticoid medication and alcohol consumption. Osteonecrotic tissue showed hypovasculature histopathology together with weak immunostaining for vessel marker CD31 and von Willrbrand factor (vWF) as compared to femoral head fracture specimens. Thrombosed vessels, fibrotic tissue, osteocytes, and inflammatory cells displayed strong S100A9 immunoreactivity in osteonecrotic lesion. In vitro, ONFH serum and S100A9 inhibited the tube formation of vessel endothelial cells and vessel outgrowth of rat aortic rings, whereas the antibody blockade of S100A9 improved angiogenic activities. Taken together, increased S100A9 levels are relevant to the development of ONFH. S100A9 appears to provoke avascular damage, ultimately accelerating femoral head deterioration through reducing angiogenesis. This study provides insight into the molecular mechanism underlying the development of ONFH. Here, analysis also highlights that serum S100A9 is a sensitive biochemical indicator of ONFH.