Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

Tenascin C activates the toll­like receptor 4/NF­κB signaling pathway to promote the development of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a globally prevalent gynecological disorder among women of childbearing age. The present study aimed to investigate the role of tenascin C (TNC) in PCOS and its potential mechanisms. Fasting blood glucose and serum insulin, the homeostasis model assessment of insulin resistance and the serum hormone levels were determined in PCOS rats. In addition, H&E staining was used for assessing pathology. In addition, the effects of TNC on oxidative stress and inflammation response in PCOS rat and cell models was assessed. Furthermore, the roles of TNC on KGN cell proliferation and apoptosis were determined employing EdU assay and flow cytometry. TLR4/NF­κB pathway­related proteins were measured using western blotting, immunofluorescence and immunohistochemistry. It was found that the mRNA and protein expression was upregulated in PCOS rats and in KGN cells induced by dihydrotestosterone (DHT). Knockdown of TNC relieved the pathological characteristics and the endocrine abnormalities of PCOS rats. Knockdown of TNC inhibited ovarian cell apoptosis, oxidative stress and inflammation in PCOS rats. Knockdown of TNC reversed the DHT­induced reduction in cell proliferation and increase in apoptosis in KGN cells. Furthermore, knockdown of TNC alleviated oxidative stress and inflammatory responses induced by DHT in KGN cells. Additionally, knockdown of TNC inhibited the toll­like receptor 4 (TLR4)/NF­κB signaling pathway in PCOS rats and DHT­treated KGN cells. In conclusion, knockdown of TNC could ameliorate PCOS in both rats and a cell model by inhibiting cell apoptosis, oxidative stress and inflammation via the suppression of the TLR4/NF­κB signaling pathway.

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.

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.

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.

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.

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.

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.

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.

[Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits].

Objective: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. Methods: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. Results: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Conclusion: Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Caveolin-1-dependent tenascin C inclusion in extracellular vesicles is required to promote breast cancer cell malignancy.

Background: Elevated expression of CAV1 in breast cancer increases tumor progression. Extracellular vesicles (EVs) from CAV1-expressing MDA-MB-231 breast cancer cells contain Tenascin C (TNC), but the relevance of TNC remained to be defined. Methods: EVs were characterized by nanotracking analysis, microscopy and western blotting. The uptake of EVs by cells was studied using flow cytometry. The effects of EVs on breast cancer cells were tested in migration, invasion, colony formation and in vivo assays. Results: EVs were taken up by cells; however, only those containing TNC promoted invasiveness. In vivo, EVs lacking TNC ceased to promote tumor growth. Conclusion: CAV1 and TNC contained in breast cancer cell-derived EVs were identified as proteins that favor progression of breast cancer.

Caveolin-1 (CAV1) is a protein that in breast cancer increases with disease progression. Extracellular vesicles (EVs) from breast cancer cells with CAV1 also contain Tenascin C (TNC) protein, but the importance of TNC remained to be defined. EVs were identified by size, microscopy and protein analysis. The effects of EVs on breast cancer cells were studied using cells and experiments in animals. CAV1 expression promotes TNC inclusion into EVs, which increased the aggressiveness of recipient breast cancer cells. In animals, only EVs with TNC increased features associated with cancer spread, while EVs lacking TNC reduced tumor growth.

Tenascin-C-enriched regeneration-specific extracellular matrix guarantees superior muscle regeneration in Ambystoma mexicanum.

Severe muscle injury causes distress and difficulty in humans. Studying the high regenerative ability of the axolotls may provide hints for the development of an effective treatment for severe injuries to muscle tissue. Here, we examined the regenerative process in response to a muscle injury in axolotls. We found that axolotls are capable of complete regeneration in response to a partial muscle resection called volumetric muscle loss (VML), which mammals cannot perfectly regenerate. We investigated the mechanisms underlying this high regenerative capacity in response to VML, focusing on the migration of muscle satellite cells and the extracellular matrix (ECM) formed during VML injury. Axolotls form tenascin-C (TN-C)-enriched ECM after VML injury. This TN-C-enriched ECM promotes the satellite cell migration. We confirmed the importance of TN-C in successful axolotl muscle regeneration by creating TN-C mutant animals. Our results suggest that the maintenance of a TN-C-enriched ECM environment after muscle injury promotes the release of muscle satellite cells and supports eventually high muscle regenerative capacity. In the future, better muscle regeneration may be achieved in mammals through the maintenance of TN-C expression.

Chemokine Binding to Tenascin-C Influences Chemokine-Induced Immune Cell Migration.

Tenascin-C (TNC) is a complex glycoprotein of the extracellular matrix (ECM) involved in a plethora of (patho-)physiological processes, such as oncogenesis and inflammation. Since chemokines play an essential role in both disease processes, we have investigated here the binding of TNC to some of the key chemokines, namely CCL2, CCL26, CXCL8, CXCL10, and CXCL12. Thereby, a differential chemokine-TNC binding pattern was observed, with CCL26 exhibiting the highest and CCL2 the lowest affinity for TNC. Heparan sulfate (HS), another member of the ECM, proved to be a similarly high-affinity ligand of TNC, with a Kd value of 730 nM. Chemokines use glycosa-minoglycans such as HS as co-receptors to induce immune cell migration. Therefore, we assumed an influence of TNC on immune cell chemotaxis due to co-localization within the ECM. CCL26- and CCL2-induced mobilization experiments of eosinophils and monocytes, respectively, were thus performed in the presence and the absence of TNC. Pre-incubation of the immune cells with TNC resulted in a 3.5-fold increase of CCL26-induced eosinophil chemotaxis, whereas a 1.3-fold de-crease in chemotaxis was observed when monocytes were pre-incubated with CCL2. As both chemokines have similar HS binding but different TNC binding affinities, we speculate that TNC acts as an attenuator in monocyte and as an amplifier in eosinophil mobilization by impeding CCL2 from binding to HS on the one hand, and by reinforcing CCL26 to bind to HS on the other hand.

Tenascin-C-EGFR activation induces functional human satellite cell proliferation and promotes wound-healing of skeletal muscles via oleanic acid.

Human satellite cells (HuSCs) have been deemed to be the potential cure to treat muscular atrophy diseases such as Duchenne muscular dystrophy. However, the clinical trials of HuSCs were restricted to the inadequacy of donors because of that freshly isolated HuSCs quickly lost the Pax7 expression and myogenesis capacity in vivo after a few days of culture. Here we found that oleanic acid, a kind of triterpenoid endowed with diverse biological functions with treatment potential, could efficiently promote HuSCs proliferation. The HuSCs cultured in the medium supplement with oleanic acid could maintain a high expression level of Pax7 and retain the ability to differentiate into myotubes as well as facilitate muscle regeneration in injured muscles of recipient mice. We further revealed that Tenascin-C acts as the core mechanism to activate the EGFR signaling pathway followed by HuSCs proliferation. Taken together, our data provide an efficient method to expand functional HuSCs and a novel mechanism that controls HuSCs proliferation, which sheds light on the HuSCs-based therapy to treat muscle diseases.

Presence of anti-modified protein antibodies in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Studies of autoimmunity and anti-citrullinated protein antibodies (ACPA) in idiopathic pulmonary fibrosis (IPF) have been confined to investigations of anti-cyclic citrullinated peptide (anti-CCP) antibodies which utilize synthetic peptides as surrogate markers for in vivo citrullinated antigens. We studied immune activation by analysing the prevalence of in vivo anti-modified protein antibodies (AMPA) in IPF. METHODS: We included patients with incident and prevalent IPF (N = 120), sex and smoking-matched healthy controls (HC) (N = 120) and patients with RA (N = 104). Serum (median time: 11 months [Q1-Q3: 1-28 months] from diagnosis) was analysed for presence of antibodies towards native and posttranslational modified (citrullinated [Cit, N = 25]; acetylated [Acet, N = 4] and homocitrullinated [Carb, N = 1]) peptides derived from tenascin (TNC, N = 9), fibrinogen (Fib, N = 11), filaggrin (Fil, N = 5), histone (N = 8), cathelicidin (LL37, N = 4) and vimentin (N = 5) using a custom-made peptide microarray. RESULTS: AMPA were more frequent and in increased levels in IPF than in HC (44% vs. 27%, p < 0.01), but less than in RA (44% vs. 79%, p < 0.01). We specifically observed AMPA in IPF towards certain citrullinated, acetylated and carbamylated peptides versus HC: tenascin (Cit(2033) -TNC2025-2040 ; Cit(2197) -TNC2177-2200 ; Cit(2198) -TNC2177-2200 ), fibrinogen (Cit(38,42) -Fibα36-50 ; Cit(72) -Fibß60-74 ) and filaggrin (Acet-Fil307-324 , Carb-Fil307-324 ). No differences in survival (p = 0.13) or disease progression (p = 0.19) between individuals with or without AMPA was observed in IPF. However, patients with incident IPF had better survival if AMPA were present (p = 0.009). CONCLUSION: A significant proportion of IPF patients present with specific AMPA in serum. Our results suggest autoimmunity as a possible characteristic for a subgroup of IPF that may affect disease outcome.

Tenascin-C in Tissue Repair after Myocardial Infarction in Humans.

Adverse ventricular remodeling after myocardial infarction (MI) is progressive ventricular dilatation associated with heart failure for weeks or months and is currently regarded as the most critical sequela of MI. It is explained by inadequate tissue repair due to dysregulated inflammation during the acute stage; however, its pathophysiology remains unclear. Tenascin-C (TNC), an original member of the matricellular protein family, is highly up-regulated in the acute stage after MI, and a high peak in its serum level predicts an increased risk of adverse ventricular remodeling in the chronic stage. Experimental TNC-deficient or -overexpressing mouse models have suggested the diverse functions of TNC, particularly its pro-inflammatory effects on macrophages. The present study investigated the roles of TNC during human myocardial repair. We initially categorized the healing process into four phases: inflammatory, granulation, fibrogenic, and scar phases. We then immunohistochemically examined human autopsy samples at the different stages after MI and performed detailed mapping of TNC in human myocardial repair with a focus on lymphangiogenesis, the role of which has recently been attracting increasing attention as a mechanism to resolve inflammation. The direct effects of TNC on human lymphatic endothelial cells were also assessed by RNA sequencing. The results obtained support the potential roles of TNC in the regulation of macrophages, sprouting angiogenesis, the recruitment of myofibroblasts, and the early formation of collagen fibrils during the inflammatory phase to the early granulation phase of human MI. Lymphangiogenesis was observed after the expression of TNC was down-regulated. In vitro results revealed that TNC modestly down-regulated genes related to nuclear division, cell division, and cell migration in lymphatic endothelial cells, suggesting its inhibitory effects on lymphatic endothelial cells. The present results indicate that TNC induces prolonged over-inflammation by suppressing lymphangiogenesis, which may be one of the mechanisms underlying adverse post-infarct remodeling.

Protein-protein interactions between tenascin-R and RPTPζ/phosphacan are critical to maintain the architecture of perineuronal nets.

Neural plasticity, the ability to alter the structure and function of neural circuits, varies throughout the age of an individual. The end of the hyperplastic period in the central nervous system coincides with the appearance of honeycomb-like structures called perineuronal nets (PNNs) that surround a subset of neurons. PNNs are a condensed form of neural extracellular matrix that include the glycosaminoglycan hyaluronan and extracellular matrix proteins such as aggrecan and tenascin-R (TNR). PNNs are key regulators of developmental neural plasticity and cognitive functions, yet our current understanding of the molecular interactions that help assemble them remains limited. Disruption of Ptprz1, the gene encoding the receptor protein tyrosine phosphatase RPTPζ, altered the appearance of nets from a reticulated structure to puncta on the surface of cortical neuron bodies in adult mice. The structural alterations mirror those found in Tnr-/- mice, and TNR is absent from the net structures that form in dissociated cultures of Ptprz1-/- cortical neurons. These findings raised the possibility that TNR and RPTPζ cooperate to promote the assembly of PNNs. Here, we show that TNR associates with the RPTPζ ectodomain and provide a structural basis for these interactions. Furthermore, we show that RPTPζ forms an identical complex with tenascin-C, a homolog of TNR that also regulates neural plasticity. Finally, we demonstrate that mutating residues at the RPTPζ-TNR interface impairs the formation of PNNs in dissociated neuronal cultures. Overall, this work sets the stage for analyzing the roles of protein-protein interactions that underpin the formation of nets.

Interaction of adipose-derived stem cells with active and dormant breast cancer cells.

BACKGROUND: Although autologous fat grafting is considered a successful method for the management of contour deformities, the fat graft could potentially induce cancer reappearance by fueling dormant breast cancer cells. Our aim was to characterize the role of adipose-derived stem cells on active and dormant breast cancer cell growth. METHODS: Cobalt chloride was used to induce dormancy in MCF-7 cancer cells. Proliferation of active and dormant cancer cells was determined in the presence of adipose-derived stem cells. A proteome array was used to detect cancer-related protein expression in the cell-conditioned medium. The migration of cancer cells was measured in response to conditioned medium from the adipose-derived stem cells. RESULTS: The adipose-derived stem cells showed variable effects on active MCF-7 cells growth and inhibited MCF-7 proliferation after the withdrawal of cobalt chloride. Of the 84 different proteins measured in the conditioned medium, only tenascin-C was differentially expressed in the co-cultures. MCF-7 cells alone did not express tenascin-C, whereas co-cultures between MCF-7 and adipose-derived stem cells expressed more tenascin-C versus adipose-derived stem cells alone. The conditioned medium from co-cultures significantly increased the migration of the cancer cells. CONCLUSIONS: Adipose-derived stem cells themselves neither increased the growth or migration of cancer cells, suggesting that autologous fat grafting may be oncologically safe if reconstruction is postponed until there is no evidence of active disease. However, interactions between adipose-derived stem cells and MCF-7 cancer cells could potentially lead to the production of factors, which further promote cancer cell migration.

Tenascin-C Activation of Lung Fibroblasts in a 3D Synthetic Lung Extracellular Matrix Mimic.

The lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung-metastatic breast cancer alters these cell-ECM interactions, promoting fibroblast activation. There is a need for bio-instructive ECM models that match the ECM composition and biomechanics of the lung to study these cell-matrix interactions in vitro. Here, a synthetic, bioactive hydrogel is synthesized that mimics the native lung modulus and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin-binding and matrix metalloproteinase (MMP)-mediated degradation in the lung, which enables quiescent culture of human lung fibroblasts (HLFs). Stimulation with transforming growth factor ß1 (TGF-ß1), metastatic breast cancer conditioned media (CM), or tenascin-C-derived integrin-binding peptide activated hydrogel-encapsulated HLFs demonstrates multiple environmental methods to activate HLFs in a lung ECM-mimicking hydrogel. This lung hydrogel platform is a tunable, synthetic approach to studying the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2.

Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.