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.

Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome.

Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Ehlers-Danlos Syndrome: Molecular and Clinical Characterization of TNXA/TNXB Chimeras in Congenital Adrenal Hyperplasia.

CONTEXT: The syndrome CAH-X is due to a contiguous gene deletion of CYP21A2 and TNXB resulting in TNXA/TNXB chimeras. OBJECTIVE: To analyze TNXB gene status and to clinically evaluate the Ehlers-Danlos syndrome phenotype in a large cohort of Argentine congenital adrenal hyperplasia (CAH) patients to assess the prevalence of this condition in our population. METHODS: TNXB gene analysis was performed in 66 nonrelated CAH patients that were carriers of the CYP21A2 gene deletion. A molecular strategy based on multiplex ligation-dependent probe amplification and Sanger sequencing analysis was developed allowing for the detection of different, previously described TNXA/TNXB chimeras, named CH1, CH2, and CH3. The main outcome measures were TNXB status of CAH patients that were carriers of the CYP21A2 deletion in the homozygous or heterozygous state. RESULTS: TNXA/TNXB CH1 was found in 41%, CH2 in 29%, and CH3 in 1% of nonrelated alleles carrying the CYP21A2 deletion. Thus, overall 71% of alleles were found to carry a contiguous gene deletion. Sixty-seven percent of patients analyzed had a monoallelic form and 6% a biallelic form. All patients with the biallelic form had severe skin hyperextensibility and generalized joint hypermobility. CONCLUSION: Based on the high frequency of TNXB alterations found in CYP21A2 deletion carrier alleles, we recommend evaluating TNXB status in these patients, and assessing connective tissue dysplasia, including cardiologic alterations in positive cases. The number of patients undergoing cardiological evaluation should be expanded to determine the incidence of structural and functional abnormalities in this cohort.

Higher Gene Expression of Healing Factors in Anterior Cruciate Ligament Remnant in Acute Anterior Cruciate Ligament Tear.

BACKGROUND: Anterior cruciate ligament (ACL) reconstruction with remnant preservation has been described and related to potential advantages. Literature is lacking regarding gene expression of potential factors related to ligament healing in the ACL remnant and its relation to time from injury. HYPOTHESIS: The mRNA expression of ligament healing factors in the ACL remnant would be higher in acute tears (<3 months from injury) than in intermediate (3-12 months) and chronic (>12 months) injuries. STUDY DESIGN: Controlled laboratory study. METHODS: Gene expression of 21 genes related to ligament healing factors was analyzed in 46 ACL remnants biopsied during surgical reconstruction with quantitative real-time polymerase chain reaction technique. Specimens were divided into 3 groups according to time from injury: acute (<3 months from injury; n = 19), intermediate (3-12 months; n = 12), and chronic (>12 months; n = 15). Histological and immunohistochemical evaluation was performed by analysis of hematoxylin and eosin, CD-34, and S-100 staining. RESULTS: Expression of COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL12A1, LOX, PLOD1, and TNC genes in ACL remnant was greater in acute compared with chronic injuries. COL1A1, COL5A1, COL12A1, and TNC genes were also expressed more in the acute group compared with the intermediate group. Furthermore, expression of the genes COL1A1 and COL5A2 was significantly higher in female than in male patients. No difference in the number of blood vessels and mechanoreceptors among groups was observed in the microscopic evaluation. CONCLUSION: The present study demonstrates that expression of COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL12A1, LOX, PLOD1, and TNC genes in ACL remnant is greater in acute (<3 months from injury) compared with chronic (>12 months) injuries. Furthermore, COL1A1, COL5A1, COL12A1, and TNC genes were expressed more in the acute group compared with the intermediate group (3-12 months from injury). CLINICAL RELEVANCE: ACL reconstructions with remnant preservation should be performed in patients with acute injuries, as in these cases the ACL remnant may present the greatest healing potential.

Differential expression of extracellular matrix genes in glenohumeral capsule of shoulder instability patients.

Anterior shoulder instability is a common orthopedic problem. After a traumatic shoulder dislocation, patients present a plastic deformation of the capsule. The shoulder instability biology remains poorly understood. We evaluated the expression of genes that encode the cartilage oligomeric matrix protein (COMP), fibronectin 1 (FN1), tenascin C (TNC) and tenascin XB (TNXB) in the glenohumeral capsule of anterior shoulder instability patients and controls. Moreover, we investigated the associations between gene expression and clinical parameters. The gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction in the antero-inferior (macroscopically injured region), antero-superior and posterior regions of the capsule of 29 patients with shoulder instability and 8 controls. COMP expression was reduced and FN1 and TNC expression was increased in the antero-inferior capsule region of cases compared to controls (p < 0.05). TNC expression was increased in the posterior capsule portion of shoulder instability patients (p = 0.022). COMP expression was reduced in the antero-inferior region compared to the posterior region of shoulder instability patients (p = 0.007). In the antero-inferior region, FN1 expression was increased in the capsule of patients with more than one year of symptoms (p = 0.003) and with recurrent dislocations (p = 0.004) compared with controls. FN1 and TNXB expression was correlated with the duration of symptoms in the posterior region (p < 0.05). Thus, COMP, FN1, TNC and TNXB expression was altered across the capsule of shoulder instability patients. Dislocation episodes modify FN1, TNC and TNXB expression in the injured tissue. COMP altered expression may be associated with capsule integrity after shoulder dislocation, particularly in the macroscopically injured portion.

Multiple sclerosis risk markers in HLA-DRA, HLA-C, and IFNG genes are associated with sex-specific childhood leukemia risk.

Previous epidemiologic studies showed four times increased risk of acute lymphoblastic leukemia (ALL) in children of women with multiple sclerosis (MS). MS shows a risk association with Human leukocyte antigens (HLA)-DRA single nucleotide polymorphism (SNP) rs3135388, which is a proxy marker for DRB1*1501. We examined the relevance of rs3135388 in childhood ALL risk along with two other HLA-DRA SNPs in two case-control groups: 114 cases and 388 controls from South Wales (UK) and 100 Mexican Mestizo cases and 253 controls. We first confirmed the correlation between rs3135388 and DRB1*1501 in HLA-typed reference cell lines. We noted a female-specific risk association in childhood ALL (pooled odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.5-4.5, Mantel-Haenszel P = 0.0009) similar to the stronger association of DRB1*1501 in females with MS. Examination of an HLA-C 5' flanking region SNP rs9264942, known to correlate with HLA-C expression, showed a protective association in girls (OR = 0.4, 95% CI = 0.2-0.7, Mantel-Haenszel P = 0.0003) similar to the protective HLA-Cw*05 association in MS. In a reference cell line panel, HLA-Cw5 homozygous samples (n = 8) were also homozygous for the minor allele of the SNP. Likewise, the male-specific protective association of interferon-gamma (IFNG) SNP rs2069727 in MS was replicated with the same sex specificity in childhood ALL (OR = 0.6, 95% CI = 0.4-1.0, Mantel-Haenszel P = 0.03). Two other SNPs in superkiller viralicidic activity 2-like and tenascin XB that are markers for systemic lupus erythematosus susceptibility showed female-specific associations but due to linkage disequilibrium with HLA-DRB1*15. Our observations supported the epidemiologic link between MS and childhood ALL and added the sex effect to this connection. It appears that only girls born to mothers with MS may have an increased risk of ALL. Investigating the mechanism of these sex-specific associations may help understand the pathogenesis of MS and ALL.

Expression of extracellular matrix proteins in human dental pulp stem cells depends on the donor tooth conditions.

INTRODUCTION: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. METHODS: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. CONCLUSIONS: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption).

Regional and cellular distribution of the extracellular matrix protein tenascin-C in the chick forebrain and its role in neonatal learning.

The juvenile brain's pronounced synaptic plasticity in response to early experience and learning events is related to the fact that the genetically pre-programmed molecular machinery mediating neuronal development and synapse formation, is activated throughout postnatal brain development and thereby can be recruited for learning and long-term memory formation. In situ hybridization and immunocytochemistry experiments revealed that tenascin-C, one candidate molecule which we suspect to be involved in neonatal learning, is expressed in the forebrain of domestic chicks around the sensitive period during which auditory filial imprinting takes place. The involvement of tenascin-C in this juvenile learning task was tested by injections of monoclonal antibodies directed to distinct domains of the tenascin-C molecule into the avian prefrontal cortex analog, the medio-rostral nidopallium/mesopallium (formerly termed medio-rostral neostriatum/hyperstriatum ventrale), a forebrain area which has been shown to be critically involved in auditory filial imprinting. Injections of monoclonal antibody Tn 68, which is directed against a cell-binding domain of the tenascin-C molecule, strongly reduced the imprinting rate, as opposed to injections of the monoclonal antibody Tn 578, which binds to a domain involved in neurite outgrowth. Double labeling immunohistochemistry revealed that tenascin-C is associated with neurons which express the Ca(2+)-binding protein parvalbumin, and displays a staining pattern highly reminiscent of perineuronal nets of the extracellular matrix. These results indicate that a distinct domain of tenascin-C is functionally involved in the juvenile learning process of filial imprinting and further suggest a critical role of a specific neuronal subpopulation.