Targeting myocardial inflammation: investigating the therapeutic potential of atrial natriuretic peptide in atrial fibrosis.

BACKGROUND: Atrial Fibrillation (AF), a prevalent arrhythmic condition, is intricately associated with atrial fibrosis, a major pathological contributor. Central to the development of atrial fibrosis is myocardial inflammation. This study focuses on Atrial Natriuretic Peptide (ANP) and its role in mitigating atrial fibrosis, aiming to elucidate the specific mechanisms by which ANP exerts its effects, with an emphasis on fibroblast dynamics. METHODS AND RESULTS: The study involved forty Sprague-Dawley rats, divided into four groups: control, Angiotensin II (Ang II), Ang II + ANP, and ANP only. The administration of 1 µg/kg/min Ang II was given to Ang II and Ang II + ANP groups, while both Ang II + ANP and ANP groups received 0.1 µg/kg/min ANP intravenously for a duration of 14 days. Cardiac fibroblasts were used for in vitro validation of the proposed mechanisms. The study observed that rats in the Ang II and Ang II + ANP groups showed an increase in blood pressure and a decrease in body weight, more pronounced in the Ang II group. Diastolic dysfunction, a characteristic of the Ang II group, was alleviated by ANP. Additionally, ANP significantly reduced Ang II-induced atrial fibrosis, myofibroblast proliferation, collagen overexpression, macrophage infiltration, and the elevated expression of Interleukin 6 (IL-6) and Tenascin-C (TN-C). Transcriptomic sequencing indicated enhanced PI3K/Akt signaling in the Ang II group. Furthermore, in vitro studies showed that ANP, along with the PI3K inhibitor LY294002, effectively reduced PI3K/Akt pathway activation and the expression of TN-C, collagen-I, and collagen-III, which were induced by Ang II. CONCLUSIONS: The study demonstrates ANP's potential in inhibiting myocardial inflammation and reducing atrial fibrosis. Notably, ANP's effect in countering atrial fibrosis seems to be mediated through the suppression of the Ang II-induced PI3K/Akt-Tenascin-C signaling pathway. These insights enhance our understanding of AF pathogenesis and position ANP as a potential therapeutic agent for treating atrial fibrosis.

Serum proteomic profiling of patients with compensated advanced chronic liver disease with and without clinically significant portal hypertension.

INTRODUCTION: Portal hypertension (PH) drives the progression of liver cirrhosis to decompensation and death. Hepatic venous pressure gradient (HVPG) measurement is the standard of PH quantification, and HVPG≥10 mmHg defines clinically significant PH (CSPH). We performed proteomics-based serum profiling to search for a proteomic signature of CSPH in patients with compensated advanced chronic liver disease (cACLD). MATERIALS AND METHODS: Consecutive patients with histologically confirmed cACLD and results of HVPG measurements were prospectively included. Serum samples were pooled according to the presence/absence of CSPH and analysed by liquid chromatography-mass spectrometry. Gene set enrichment analysis was performed, followed by comprehensive literature review for proteins identified with the most striking difference between the groups. RESULTS: We included 48 patients (30 with, and 18 without CSPH). Protein CD44, involved in the inflammatory response, vascular endothelial growth factor C (VEGF-C) and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), both involved in lymphangiogenesis were found solely in the CSPH group. Although identified in both groups, proteins involved in neutrophil extracellular traps (NET) formation, as well as tenascin C, autotaxin and nephronectin which mediate vascular contractility and lymphangiogenesis were more abundant in CSPH. DISCUSSION AND CONCLUSION: We propose that altered inflammatory response, including NET formation, vascular contractility and formation of new lymph vessels are key steps in PH development. Proteins such as CD44, VEGF-C, LYVE-1, tenascin C, Plasminogen activator inhibitor 1, Nephronectin, Bactericidal permeability-increasing protein, Autotaxin, Myeloperoxidase and a disintegrin and metalloproteinase with thrombospondin motifs-like protein 4 might be considered for further validation as potential therapeutic targets and candidate biomarkers of CSPH in cACLD.

Tenascin-X is increased with decreased expression of miR-378a-5p and miR-486-5p in mice fed a methionine-choline-deficient diet that induces hepatic fibrosis.

We previously reported that tenascin-X (Tnxb) aggravates hepatic fibrosis in mice fed a high-fat and high-cholesterol diet with high levels of phosphorus and calcium (HFCD). In this study, we investigated Tnxb expression in livers with fibrosis caused by administration of a methionine-chorine-deficient (MCD) diet in mice. Whole transcriptome analysis showed that Tnxb was one of the genes with increased expression in livers of MCD diet-fed mice compared with that in livers of normal diet (ND)-fed mice. In microarray and subsequent microRNA (miRNA) network analyses, miR-378a-5p and miR-486-5p were identified in livers of MCD diet-fed mice as downregulated miRNAs, which have their predicted target sites in the 3' untranslated region of Tnxb mRNA and might suppress the translation of Tnxb mRNA. RT-qPCR analyses of livers of MCD diet-fed mice compared with livers of ND-fed mice verified the upregulation of Tnxb and fibrosis-triggering genes and conversely the downregulation of miR-378a-5p and miR-486-5p. Overexpression of miR-378a-5p and miR-486-5p resulted in decreased level not only of the FLAG-tagged fibrinogen-like domain of Tnxb protein (FLAG-mTNX-FG) but also of endogenous Tnxb protein in murine cultured cells. These results indicate that expression of Tnxb is regulated by miR-378a-5p and miR-486-5p in hepatic fibrosis following MCD diet feeding.

Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons.

During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. The SMP core-matrisome was characterized and computationally predicted to form a basement membrane-like structure tailored for motor axon guidance, including basement membrane-associated ECM proteins, as collagen XV-B, one of the earliest core-matrisome gene transcribed in differentiating SMPs and the glycoprotein Tenascin C. To investigate how contact-mediated guidance cues are organized along the motor path to exert their function in vivo, we used microscopy-based methods to analyze and quantify motor axon navigation in tnc and col15a1b knock-out fish. We show that motor axon shape and growth rely on the timely expression of the attractive cue Collagen XV-B that locally provides axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. Our study offers evidence that not only the composition of ECM cues but their topology critically influences motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury as regenerating nerves follow their original path.

Association of Tenascin-C Gene Polymorphisms with Risk of Acute Coronary Syndrome in South Indian Population: A Case-Control Genetic Association Study.

Background: The extracellular matrix (ECM) glycoprotein changes are associated with the pathogenesis and complications of atherosclerosis, leading to acute coronary syndrome (ACS). Tenascin-C (TNC), an ECM protein, has been implemented in the pathogenesis, diagnosis, and prognosis of patients with cardiovascular disease. Aim: The study aimed to compare the genetic variants of the TNC gene (rs13321, rs2104772, and rs12347433) between South Indians with ACS and healthy participants. Materials and Methods: This case-control study recruited 150 ACS patients as cases and 150 healthy participants as controls. TNC genotyping was performed using TaqMan 5'-exonuclease allele discrimination assay. Serum TNC levels were measured by enzyme-linked immunosorbent assay. Results: Serum TNC levels were significantly higher in cases compared with controls. No significant difference was observed in allele and genotype frequencies of rs13321, rs2104772, and rs12347433 between cases and controls, which was confirmed by dominant, recessive, codominant, and homozygotic genetic models. The patients with heterozygous genotypes of rs13321, rs2104772, and rs12347433 had significantly lower serum TNC levels than patients with respective homozygous genotypes. Haplotype analyses revealed that the C-T-A haplotype in the block of rs13321-rs12347433-rs2104772 was associated with lower ACS risk (OR = 0.33, 95% CI: 0.15 - 0.75; p = 0.005). Also, the C-T-T and G-T-A haplotypes of the TNC gene were associated with higher and lower serum TNC levels, respectively. Conclusion: Our study demonstrated no genetic association between single nucleotide polymorphisms of the TNC gene and ACS risk; however, the C-T-A haplotype of the TNC gene might be associated with reduced ACS risk in South Indians.

Evaluating the efficacy of a long-read sequencing-based approach in the clinical diagnosis of neonatal congenital adrenocortical hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders predominantly characterized by impaired corticosteroid synthesis. Clinical phenotypes include hypoadrenocorticism, electrolyte disturbances, abnormal gonadal development, and short stature, of which severe hyponadrenocorticism and salt wasting can be life-threatening. Genetic analysis can help in the clinical diagnosis of CAH. However, the 21-OHD-causing gene CYP21A2 is arranged in tandem with the highly homologous CYP21A1P pseudogene, making it difficult to determine the exact genotypes using the traditional method of multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing or next-generation sequencing (NGS). We applied a long-read sequencing-based approach termed comprehensive analysis of CAH (CACAH) to 48 newborns with CAH that were diagnosed by clinical features and the traditional MLPA plus Sanger sequencing method for retrospective analysis, to evaluate its efficacy in the clinical diagnosis of neonatal CAH. Compared with the MLPA plus Sanger sequencing method, CACAH showed 100 % consistency in detecting SNV/indel variants located in exons and exon-intron boundary regions of CAH-related genes. It can directly determine the cis-trans relationship without the need to analyze parental genotypes, which reduces the time to diagnosis. Moreover, CACAH was able to distinguish different CYP21A1P/CYP21A2 and TNXA/TNXB chimeras, and detect additional variants (CYP21A2 variants c.-121C > T, c.*13G > A, c.*52C > T, c.*440C > T, c.*443 T > C, and TNXB variants c.12463 + 2 T > C, c.12204 + 5G > A). We also identified the TNXB variant c.11435_11524 + 30del alone instead of as a part of the TNXA/TNXB-CH-1 chimera in two newborns, which might be introduced by gene conversion. All of these characteristics enabled clinicians to better explain the phenotype of subjects and manage them more effectively. CACAH has a great advantage over the traditional MLPA and Sanger sequencing methods, showing substantial potential in the genetic diagnosis and screening of neonatal CAH.

Bioactive TNIIIA2 Sequence in Tenascin-C Is Responsible for Macrophage Foam Cell Transformation; Potential of FNIII14 Peptide Derived from Fibronectin in Suppression of Atherosclerotic Plaque Formation.

One of the extracellular matrix proteins, tenascin-C (TN-C), is known to be upregulated in age-related inflammatory diseases such as cancer and cardiovascular diseases. Expression of this molecule is frequently detected, especially in the macrophage-rich areas of atherosclerotic lesions; however, the role of TN-C in mechanisms underlying the progression of atherosclerosis remains obscure. Previously, we found a hidden bioactive sequence termed TNIIIA2 in the TN-C molecule and reported that the exposure of this sequence would be carried out through limited digestion of TN-C by inflammatory proteases. Thus, we hypothesized that some pro-atherosclerotic phenotypes might be elicited from macrophages when they were stimulated by TNIIIA2. In this study, TNIIIA2 showed the ability to accelerate intracellular lipid accumulation in macrophages. In this experimental condition, an elevation of phagocytic activity was observed, accompanied by a decrease in the expression of transporters responsible for lipid efflux. All these observations were mediated through the induction of excessive ß1-integrin activation, which is a characteristic property of the TNIIIA2 sequence. Finally, we demonstrated that the injection of a drug that targets TNIIIA2's bioactivity could rescue mice from atherosclerotic plaque expansion. From these observations, it was shown that TN-C works as a pro-atherosclerotic molecule through an internal TNIIIA2 sequence. The possible advantages of clinical strategies targeting TNIIIA2 are also indicated.

Tenascin-C promotes endochondral ossification and fracture healing through Hedgehog and Hippo signaling.

Fractures are frequent and severe musculoskeletal injuries. This study aimed to investigate the function of tenascin-C (TNC) in regulating chondrogenic during fracture healing and elucidate the underlying molecular mechanisms. A well-established femur fracture model in male C57BL/6J mice was used to transect the middle diaphysis of the femur. To identify the essential role of TNC, shTNC lentiviruses or TNC protein were administered in the animal model. Micro-CT analysis, histologic analysis, immunostaining assays, and gene expression analysis were employed to investigate the effect of TNC during fracture healing. An in vitro mesenchymal stem cell culture system was developed to investigate the role and molecular mechanism of TNC in regulating chondrogenesis. TNC expression was induced at the inflammatory phase and peaked at the cartilaginous callus phase during fracture healing. Knockdown of TNC expression in callus results in decreased callus formation and impaired fracture healing. Conversely, administration of exogenous TNC promoted chondrogenic differentiation, cartilage template formation and ultimately improved fracture healing. Both the Hedgehog and Hippo signaling pathways were found to be involved in the pro-chondrogenic function of TNC. Our observations demonstrate that TNC is a crucial factor responsible for endochondral ossification in fracture healing and provide a potential therapeutic strategy for promoting fracture healing.

Preclinical evaluation of an 18F-labeled Tenascin-C aptamer for PET imaging of atherosclerotic plaque in mouse models of atherosclerosis.

Tenascin-C is an extracellular matrix glycoprotein strongly expressed in coronary atherosclerotic plaque. Aptamers are single-stranded oligonucleotides that bind to specific target molecules with high affinity. This study hypothesized that tenascin-C expression at atherosclerotic plaque in vivo could be detected by tenascin-C specific aptamers using positron emission tomography (PET). This paper reports the radiosynthesis of a fluorine-18 (18F)-labeled tenascin-C aptamer for the biodistribution and PET imaging of the tenascin-C expression in apolipoprotein E-deficient (ApoE-/-) mice. The aortas ApoE-/- mice showed significantly increased positive areas of Oil red O staining than control C57BL/6 mice, and tenascin-C expression was detected in foam cells accumulated in the subendothelial lesions of ApoE-/- mice. The ex vivo biodistribution of the 18F-labeled tenascin-C aptamer showed significantly increased uptake at the aorta of ApoE-/- mice, and ex vivo autoradiography of aorta revealed the high accumulation of the 18F-labeled tenascin-C aptamer in the atherosclerotic lesions of ApoE-/- mice, which was consistent with the location of the atherosclerotic plaques detected by Oil red O staining. PET imaging of the 18F-labeled tenascin-C aptamer revealed a significantly higher mean standardized uptake in the aorta of the ApoE-/- mice than the control C57BL/6 mice. These data highlight the potential use of tenascin-C aptamer to diagnose atherosclerotic lesions in vivo.

The relationship of maternal age, BMI, serum Tenascin-C, and HOMA-IR values with gestational diabetes mellitus.

Objectives: To explore the relationship, if any, of gestational diabetes mellitus with maternal age, body mass index, serum tenascin-C and homeostatic model assessment for insulin resistance, and to see if these could act as predictive markers for gestational diabetes mellitus. METHODS: The case-control study was conducted from February to August 2022 at the outpatient department of gynaecology and obstetrics at the Civil Hospital, Karachi, and comprised pregnant females aged 18-40 years having gestational age 20-34 weeks. After noting down baseline characteristics and anthropometric measurements, the participants were subjected to oral glucose tolerance test on the basis of which they were divided into three groups; pregnant healthy controls in group 1, those with gestational diabetes mellitus on diet control in group 2, and those with gestational diabetes mellitus taking medicines for the condition in group 3. Fasting serum samples were used for further analysis using enzyme-linked immunosorbent assay kits. Data was analysed using SPSS 21. RESULTS: Of the 90 subjects, 30(33.3%) were in group 1 with mean age 26.0±4.9 years, 30(33.3%) were in group 2 with mean age 30.7±5.6 years, and 30(33.3%) were in group 3 with mean age 29.1±5.5 years. Age, gestational age, body mass index and homeostatic model assessment for insulin resistance values were significantly higher in groups 2 and 3 compared to group 1 (p<0.05), while serum Tenascin-C values were not significantly different (p>0.05). CONCLUSIONS: HOMA-IR values and BMI were more reliable in diagnosing GDM before its onset, and should be included in the screening test for GDM in early pregnancy.

Increased Elastase and Matrix Metalloproteinase Levels in the Pulmonary Arteries of Infants With Congenital Diaphragmatic Hernia.

BACKGROUND: Pulmonary vascular disease (PVD) complicated with pulmonary hypertension (PH) is a leading cause of mortality in congenital diaphragmatic hernia (CDH). Unfortunately, CDH patients are often resistant to PH therapy. Using the nitrogen CDH rat model, we previously demonstrated that CDH-associated PVD involves an induction of elastase and matrix metalloproteinase (MMP) activities, increased osteopontin and epidermal growth factor (EGF) levels, and enhanced smooth muscle cell (SMC) proliferation. Here, we aimed to determine whether the levels of the key members of this proteinase-induced pathway are also elevated in the pulmonary arteries (PAs) of CDH patients. METHODS: Neutrophil elastase (NE), matrix metalloproteinase-2 (MMP-2), epidermal growth factor (EGF), tenascin-C, and osteopontin levels were assessed by immunohistochemistry in the PAs from the lungs of 11 CDH patients and 5 normal age-matched controls. Markers of proliferation (proliferating cell nuclear antigen (PCNA)) and apoptosis (cleaved (active) caspase-3) were also used. RESULTS: While expressed by both control and CDH lungs, the levels of NE, MMP-2, EGF, as well as tenascin-C and osteopontin were significantly increased in the PAs from CDH patients. The percentage of PCNA-positive PA SMCs were also enhanced, while those positive for caspase-3 were slightly decreased. CONCLUSIONS: These results suggest that increased elastase and MMPs, together with elevated tenascin-C and osteopontin levels in an EGF-rich environment may contribute to the PVD in CDH infants. The next step of this study is to expand our analysis to a larger cohort, and determine the potential of targeting this pathway for the treatment of CDH-associated PVD and PH. TYPE OF STUDY: Therapeutic. LEVEL OF EVIDENCE: LEVEL III.

Extracellular Matrix-Trapped Bioinspired Lipoprotein Prolongs Tumor Retention to Potentiate Antitumor Immunity.

The immunomodulatory effects of many therapeutic agents are significantly challenged by their insufficient delivery efficiency and short retention time in tumors. Regarding the distinctively upregulated fibronectin (FN1) and tenascin C (TNC) in tumor stroma, herein a protease-activated FN1 and/or TNC binding peptide (FTF) is designed and an extracellular matrix (ECM)-trapped bioinspired lipoprotein (BL) (FTF-BL-CP) is proposed that can be preferentially captured by the TNC and/or FN1 for tumor retention, and then be responsively dissociated from the matrix to potentiate the antitumor immunity. The FTF-BL-CP treatment produces a 6.96-, 9.24-, 6.72-, 7.32-, and 6.73-fold increase of CD3+CD8+ T cells and their interferon-γ-, granzyme B-, perforin-, and Ki67-expressing subtypes versus the negative control, thereby profoundly eliciting the antitumor immunity. In orthotopic and lung metastatic breast cancer models, FTF-BL-CP produces notable therapeutic benefits of retarding tumor growth, extending survivals, and inhibiting lung metastasis. Therefore, this ECM-trapping strategy provides an encouraging possibility of prolonging tumor retention to potentiate the antitumor immunity for anticancer immunotherapy.

The role of TNC in atherosclerosis and drug development opportunities.

Tenascin C (TNC), a rich glycoprotein of the extracellular matrix, exhibits a pro-atherosclerosis or anti-atherosclerosis effect depending on its location. TNC, especially its C domain/isoform (TNC-C), is strongly overexpressed in atherosclerotic plaque active areas but virtually undetectable in most normal adult tissues, suggesting that TNC is a promising delivery vector target for atherosclerosis-targeted drugs. Many delivery vectors were investigated by recognizing TNC-C, including G11, G11-iRGD, TN11, PL1, and PL3. F16 and FNLM were also investigated by recognizing TNC-A1 and TNC, respectively. Notably, iRGD was undergoing clinical trials. PL1 not only recognizes TNC-C but also the extra domain-B (EDB) of fibronectin (FN), which is also a promising delivery vector for atherosclerosis-targeted drugs, and several conjugate agents are undergoing clinical trials. The F16-conjugate agent F16IL2 is undergoing clinical trials. Therefore, G11-iRGD, PL1, and F16 have great development value. Furthermore, ATN-RNA and IMA950 were investigated in clinical trials as therapeutic drugs and vaccines by targeting TNC, respectively. Therefore, targeting TNC could greatly improve the success rate of atherosclerosis-targeted drugs and/or specific drug development. This review discussed the role of TNC in atherosclerosis, atherosclerosis-targeted drug delivery vectors, and agent development to provide knowledge for drug development targeting TNC.

Tenascin-C expressing touch dome keratinocytes exhibit characteristics of all epidermal lineages.

The touch dome (TD) keratinocytes are specialized epidermal cells that intimately associate with the light touch sensing Merkel cells (MCs). The TD keratinocytes function as a niche for the MCs and can induce de novo hair follicles upon stimulation; however, how the TD keratinocytes are maintained during homeostasis remains unclear. scRNA-seq identified a specific TD keratinocyte marker, Tenascin-C (TNC). Lineage tracing of Tnc-expressing TD keratinocytes revealed that these cells maintain themselves as an autonomous epidermal compartment and give rise to MCs upon injury. Molecular characterization uncovered that, while the transcriptional and chromatin landscape of the TD keratinocytes is remarkably similar to that of the interfollicular epidermal keratinocytes, it also shares certain molecular signatures with the hair follicle keratinocytes. Our study highlights that the TD keratinocytes in the adult skin have molecular characteristics of keratinocytes of diverse epidermal lineages.

Assessment of Tenascin C levels in the serum of patients with bronchial asthma.

Asthma is a heterogeneous disease that affects a large proportion of the global population and is distinguished by airway hyperresponsiveness to direct and indirect stimulations. It is a multifactorial disease that is triggered by heredity and environmental causes. Tenascin C (TNC) is an extracellular matrix glycoprotein that promotes inflammatory cell migration from the interstitium to the airways. Stimulation of TNC is through cytokines from T helper 2 (Th2) cells, in addition, it proliferates within basement membranes of the airways in asthmatic patients. This study aimed to determine whether serum TNC can be used as a novel biomarker for asthma diagnosis and to evaluate the association between serum TNC measurement and asthma severity. This case-control study included 64 patients with mild to severe bronchial asthma, diagnosed according to GINA 2022, referred to the Allergy and Clinical Immunology outpatient clinic at Ain Shams University Hospital, and 64 normal subjects as controls. Serum TNC levels were measured by ELISA. Serum TNC levels were significantly higher among bronchial asthma patients than controls (p ˂0.001). The sensitivity of serum TNC measurement in the diagnosis of bronchial asthma was 93.75%, the specificity 60.94%, and the negative predictive value 90.7%. Besides, a significant relation was found between serum TNC levels and the severity of bronchial asthma (p=0.004), as elevated serum TNC levels were the highest among severe asthmatic patients. In conclusion, the results gained in this study revealed that serum TNC level could be proposed as a potential biomarker for the diagnosis of bronchial asthma and a potential predictor of disease severity.

Characteristics of n6-methyladenosine (m6A) regulators and role of FTO/TNC in scleroderma.

BACKGROUND: m6A regulators have important roles in a variety of autoimmune diseases, but their potential function in scleroderma, a refractory connective tissue disease, remains unclear. Tenascin C (TNC) is known to be a factor promoting collagen deposition in the development of scleroderma, but the regulatory relationship between TNC and m6A regulators is unknown. METHODS: We extracted GSE33463 data consisting of forty-one healthy controls and sixty-one patients with scleroderma, and we analyzed the expression levels of twenty-one m6A regulators as well as the associations between them. In addition, we obtained random forest (RF) and nomogram models to predict the likehood of scleroderma. Next, we categorized the m6Aclusters and geneclusters by consensus clustering, and we performed an immune cell infiltration analysis for each cluster. Finally, we injected adenoviruses into a bleomycin (BLM)-induced mouse model of scleroderma, which was used to overexpress FTO and TNC. We assess the extent of skin fibrosis in the mice samples using pathology stains and measuring their hydroxyproline content and collagen mRNA. RESULTS: We initially identified fourteen differentially expressed m6A regulators (WTAP, RBM15, CBLL1, FTO, ALKBH5, YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, RBMX, HNRNPC, IGFBP1 and IGFBP2). We found ALKBH5 to be positively associated with CBLL1 and RBM15, and FTO to be negatively associated with WTAP. In addition, we identified four m6A regulators (CBLL1, IGFBP1, YTHDF2 and IGFBP2) using a RF model, and we designed a nomogram model with those variables that proved reliable according to the calibration curve and clinical impact curve. We found that the m6Acluster A was correlated with Type 1 T helper cell infiltration and the genecluster A was correlated with regulatory T cell infiltration. Finally, we showed that FTO overexpression downregulated the m6A and mRNA levels of TNC, and alleviated skin fibrosis in the mouse model of scleroderma. Thus, our overexpression experiments provide preliminary evidence suggesting that TNC is an adverse factor in scleroderma. CONCLUSION: Our approach might be useful as a new and accurate scleroderma diagnosis method. Moreover, our results suggested that FTO/TNC might be a novel scleroderma therapeutic target.

Engrailed 2 serves as a master regulator of the super-enhancer in the TNC gene locus in non-small cell lung cancer.

Engrailed 2 (EN2) is a homeodomain-containing protein that is dysregulated in many types of cancer. However, the role of EN2 in non-small cell lung cancer (NSCLC) and the mechanism underlying its biological function are largely unclear. Here, we showed that EN2 played an oncogenic function in NSCLC and greatly enhanced the malignant phenotype of NSCLC cells. Meanwhile, EN2 was able to boost the expression of a well-studied oncogenic Tenascin-C (TNC) gene, which in turn activated the AKT signaling pathway. Interestingly, we found that EN2 directly bound to the super-enhancer (SE) region in the TNC locus. The histone marker H3K27ac was also enriched in the region, indicating the activation of the SE. Treatment of the cells with JQ1, an inhibitor of SE activity, abrogated the effect of EN2 on the expression of TNC and phosphorylation of AKT-Ser473. Collectively, our work unveils a novel mode of EN2 function, in which EN2 governs the SE in the TNC locus, consequently activating the oncogenic TNC-AKT axis in NSCLC.

Role of tenascin C in lesion formation in early peritoneal endometriosis.

OBJECTIVE: To identify cytokines or extracellular matrix components that contribute to adhesion to, and invasion of, the peritoneum, proximal to lesions in the early phase of endometriosis. DESIGN: Laboratory-based study. SETTING: University Hospital and Laboratory of Animal Science. PATIENTS AND ANIMALS: Five women with ovarian endometrioma, 138 wild-type (WT) C57BL/6N mice, and 48 Tenascin C (Tnc) knockout (TncKO) mice. INTERVENTIONS: To establish a murine endometriosis model, 20 pieces of minced uterine tissue fragments from each horn were administered intraperitoneally to syngeneic mice. Three days later, endometriotic lesions and peritoneal tissues were collected. Separately, we transfected human peritoneal mesothelial cells (HMrSV5) or human endometrial stromal cells (hESCs) with Tnc small interfering ribonucleic acid. MAIN OUTCOME MEASURES: We employed a polymerase chain reaction array to profile gene expression in the murine peritoneum, in both peritoneum distal to lesions and peritoneum surrounding lesions (PSL). The expression of upregulated genes in the PSL was verified in the peritoneal samples by real-time reverse transcription-polymerase chain reaction. TncKO mice were used to investigate the role of Tnc in the development of endometriosis. We evaluated the proliferative activity or inflammatory state of lesions by Ki67 or CD3 immunostaining. Intraperitoneal distribution of macrophages was assessed by fluorescence-activated cell sorting. Using Tnc small interfering ribonucleic acid, we examined the invasive capacity of hESCs in a coculture system with HMrSV5. RESULTS: Tnc gene expression was significantly higher in PSL than in peritoneum distal to lesions. The weight and number of TncKO lesions in TncKO hosts were lower than those of WT lesions in WT hosts. In contrast, the weight and number of nonattached TncKO lesions in TncKO hosts were higher than those of nonattached WT lesions in WT hosts. We observed decreased Ki67-positive cells or H-scores for CD3, a lower proportion of M1 macrophages, and a higher proportion of M2 macrophages in TncKO lesions in TncKO recipients. Silencing of Tnc expression in hESCs and HMrSV5 diminished the invasivity of hESCs. CONCLUSION: Tnc may be a crucial factor in the development of early peritoneal endometriosis.

Clinical advances in TNC delivery vectors and their conjugate agents.

Tenascin C (TNC), a glycoprotein that is abundant in the tumor extracellular matrix (ECM), is strongly overexpressed in tumor tissues but virtually undetectable in most normal tissues. Many TNC antibodies, peptides, aptamers, and nanobodies have been investigated as delivery vectors, including 20A1, α-A2, α-A3, α-IIIB, α-D, BC-2, BC-4 BC-8, 81C6, ch81C6, F16, FHK, Ft, Ft-NP, G11, G11-iRGD, GBI-10, 19H12, J1/TN1, J1/TN2, J1/TN3, J1/TN4, J1/TN5, NJT3, NJT4, NJT6, P12, PL1, PL3, R6N, SMART, ST2146, ST2485, TN11, TN12, TNFnA1A2-Fc, TNfnA1D-Fc, TNfnBD-Fc, TNFnCD-Fc, TNfnD6-Fc, TNfn78-Fc, TTA1, TTA1.1, and TTA1.2. In particular, BC-2, BC-4, 81C6, ch81C6, F16, FHK, G11, PL1, PL3, R6N, ST2146, TN11, and TN12 have been tested in human tissues. G11-iRGD and simultaneous multiple aptamers and arginine-glycine-aspartic acid (RGD) targeting (SMART) may be assessed in clinical trials because G11, iRGD and AS1411 (SMART components) are already in clinical trials. Many TNC-conjugate agents, including antibody-drug conjugates (ADCs), antibody fragment-drug conjugates (FDCs), immune-stimulating antibody conjugates (ISACs), and radionuclide-drug conjugates (RDCs), have been investigated in preclinical and clinical trials. RDCs investigated in clinical trials include 111In-DTPA-BC-2, 131I-BC-2, 131I-BC-4, 90Y-BC4, 131I81C6, 131I-ch81C6, 211At-ch81C6, F16124I, 131I-tenatumomab, ST2146biot, FDC 131I-F16S1PF(ab')2, and ISAC F16IL2. ADCs (including FHK-SSL-Nav, FHK-NB-DOX, Ft-NP-PTX, and F16*-MMAE) and ISACs (IL12-R6N and 125I-G11-IL2) may enter clinical trials because they contain components of marketed treatments or agents that were investigated in previous clinical studies. This comprehensive review presents historical perspectives on clinical advances in TNC-conjugate agents to provide timely information to facilitate tumor-targeting drug development using TNC.

Extracellular matrix complexity in biomarker studies: a novel assay detecting total serum tenascin-C reveals different distribution to isoform-specific assays.

Serum biomarkers are the gold standard in non-invasive disease diagnosis and have tremendous potential as prognostic and theranostic tools for patient stratification. Circulating levels of extracellular matrix molecules are gaining traction as an easily accessible means to assess tissue pathology. However, matrix molecules are large, multimodular proteins that are subject to a vast array of post-transcriptional and post-translational modifications. These modifications often occur in a tissue- and/or disease-specific manner, generating hundreds of different variants, each with distinct biological roles. Whilst this complexity can offer unique insight into disease processes, it also has the potential to confound biomarker studies. Tenascin-C is a pro-inflammatory matrix protein expressed at low levels in most healthy tissues but elevated in, and associated with the pathogenesis of, a wide range of autoimmune diseases, fibrosis, and cancer. Analysis of circulating tenascin-C has been widely explored as a disease biomarker. Hundreds of different tenascin-C isoforms can be generated by alternative splicing, and this protein is also modified by glycosylation and citrullination. Current enzyme-linked immunosorbent assays (ELISA) are used to measure serum tenascin-C using antibodies, recognising sites within domains that are alternatively spliced. These studies, therefore, report only levels of specific isoforms that contain these domains, and studies on the detection of total tenascin-C are lacking. As such, circulating tenascin-C levels may be underestimated and/or biologically relevant isoforms overlooked. We developed a highly specific and sensitive ELISA measuring total tenascin-C down to 0.78ng/ml, using antibodies that recognise sites in constitutively expressed domains. In cohorts of people with different inflammatory and musculoskeletal diseases, levels of splice-specific tenascin-C variants were lower than and distributed differently from total tenascin-C. Neither total nor splice-specific tenascin-C levels correlated with the presence of autoantibodies to citrullinated tenascin-C in rheumatoid arthritis (RA) patients. Elevated tenascin-C was not restricted to any one disease and levels were heterogeneous amongst patients with the same disease. These data confirm that its upregulation is not disease-specific, instead suggest that different molecular endotypes or disease stages exist in which pathology is associated with, or independent of, tenascin-C. This immunoassay provides a novel tool for the detection of total tenascin-C that is critical for further biomarker studies. Differences between the distribution of tenascin-C variants and total tenascin-C have implications for the interpretation of studies using isoform-targeted assays. These data highlight the importance of assay design for the detection of multimodular matrix molecules and reveal that there is still much to learn about the intriguingly complex biological roles of distinct matrix proteoforms.