Proteolysis of the pericellular matrix: Pinpointing the role and involvement of matrix metalloproteinases in early osteoarthritic remodeling.

The pericellular matrix (PCM) serves a critical role in signal transduction and mechanoprotection in chondrocytes. Osteoarthritis (OA) leads to a gradual deterioration of the cartilage, marked by a shift in the spatial arrangement of chondrocytes from initially isolated strands to large cell clusters in end-stage degeneration. These changes coincide with progressive enzymatic breakdown of the PCM. This study aims to assess the role and involvement of specific matrix metalloproteinases (MMPs) in PCM degradation during OA. We selected cartilage samples from 148 OA patients based on the predominant spatial chondrocyte patterns. The presence of various MMPs (-1,-2,-3,-7,-8,-9,-10,-12,-13) was identified by multiplexed immunoassays. For each pattern and identified MMP, the levels and activation states (pro-form vs. active form) were measured by zymograms and western blots. The localization of these MMPs was determined using immunohistochemical labeling. To verify these results, healthy cartilage was exposed to purified MMPs, and the consecutive structural integrity of the PCM was analyzed through immunolabeling and proximity ligation assay. Screening showed elevated levels of MMP-1,-2,-3,-7, and -13, with their expression profile showing a clear dependency of the degeneration stage. MMP-2 and -7 were localized in the PCM, whereas MMP-1,-7, and -13 were predominantly intracellular. We found that MMP-2 and -3 directly disrupt collagen type VI, and MMP-3 and -7 destroy perlecan. MMP-2, -3, and -7 emerge as central players in early PCM degradation in OA. With the disease's initial stages already displaying elevated peaks in MMP expression, this insight may guide early targeted therapies to halt abnormal PCM remodeling. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) causes a gradual deterioration of the articular cartilage, accompanied by a progressive breakdown of the pericellular matrix (PCM). The PCM's crucial function in protecting and transmitting signals within chondrocytes is impaired in OA. By studying 148 OA-patient cartilage samples, the involvement of matrix metalloproteinases (MMPs) in PCM breakdown was explored. Findings highlighted elevated levels of certain MMPs linked to different stages of degeneration. Notably, MMP-2, -3, and -7 were identified as potent contributors to early PCM degradation, disrupting key components like collagen type VI and perlecan. Understanding these MMPs' roles in initiating OA progression, especially in its early stages, provides insights into potential targets for interventions to preserve PCM integrity and potentially impeding OA advancement.

Optimized allele-specific silencing of the dominant-negative COL6A1 G293R substitution causing collagen VI-related dystrophy.

Collagen VI-related dystrophies (COL6-RDs) are a group of severe, congenital-onset muscular dystrophies for which there is no effective causative treatment. Dominant-negative mutations are common in COL6A1, COL6A2, and COL6A3 genes, encoding the collagen α1, α2, and α3 (VI) chains. They act by incorporating into the hierarchical assembly of the three α (VI) chains and consequently produce a dysfunctional collagen VI extracellular matrix, while haploinsufficiency for any of the COL6 genes is not associated with disease. Hence, allele-specific transcript inactivation is a valid therapeutic strategy, although selectively targeting a pathogenic single nucleotide variant is challenging. Here, we develop a small interfering RNA (siRNA) that robustly, and in an allele-specific manner, silences a common glycine substitution (G293R) caused by a single nucleotide change in COL6A1 gene. By intentionally introducing an additional mismatch into the siRNA design, we achieved enhanced specificity toward the mutant allele. Treatment of patient-derived fibroblasts effectively reduced the levels of mutant transcripts while maintaining unaltered wild-type transcript levels, rescuing the secretion and assembly of collagen VI matrix by reducing the dominant-negative effect of mutant chains. Our findings establish a promising treatment approach for patients with the recurrent dominantly negative acting G293R glycine substitution.

COL6A1 expression as a potential prognostic biomarker for risk stratification of T1 high grade bladder cancer: Unveiling the aggressive nature of a distinct non-muscle invasive subtype.

PURPOSE: T1 high grade (T1HG) bladder cancer (BC) is a type of non-muscle invasive BC (NMIBC) that is recognized as an aggressive subtype with a heightened propensity for progression. Current risk stratification methods for NMIBC rely on clinicopathological indicators; however, these approaches do not adequately capture the aggressive nature of T1HG BC. Thus, new, more accurate biomarkers for T1HG risk stratification are needed. Here, we enrolled three different patient cohorts and investigated expression of collagen type VI alpha 1 (COL6A1), a key component of the extracellular matrix, at different stages and grades of BC, with a specific focus on T1HG BC. MATERIALS AND METHODS: Samples from 298 BC patients were subjected to RNA sequencing and real-time polymerase chain reaction. RESULTS: We found that T1HG BC and muscle invasive BC (MIBC) exhibited comparable expression of COL6A1, which was significantly higher than that by other NMIBC subtypes. In particular, T1HG patients who later progressed to MIBC had considerably higher expression of COL6A1 than Ta, T1 low grade patients, and patients that did not progress, highlighting the aggressive nature and higher risk of progression associated with T1HG BC. Moreover, Cox and Kaplan-Meier survival analyses revealed a significant association between elevated expression of COL6A1 and poor progression-free survival of T1HG BC patients (multivariate Cox hazard ratio, 16.812; 95% confidence interval, 3.283-86.095; p=0.001 and p=0.0002 [log-rank test]). CONCLUSIONS: These findings suggest that COL6A1 may be a promising biomarker for risk stratification of T1HG BC, offering valuable insight into disease prognosis and guidance of personalized treatment decisions.

Retrospective clinical and genetic analysis of COL6-RD patients with a long-term follow-up at a single French center.

Collagen type VI-related dystrophies (COL6-RD) are rare diseases with a wide phenotypic spectrum ranging from severe Ullrich's congenital muscular dystrophy Ullrich congenital muscular dystrophy to much milder Bethlem myopathy Both dominant and recessive forms of COL6-RD are caused by pathogenic variants in three collagen VI genes (COL6A1, COL6A2 and COL6A3). The prognosis of these diseases is variable and difficult to predict during early disease stages, especially since the genotype-phenotype correlation is not always clear. For this reason, studies with long-term follow-up of patients with genetically confirmed COL6-RD are still needed. In this study, we present phenotypic and genetic data from 25 patients (22 families) diagnosed with COL6-RD and followed at a single French center, in both adult and pediatric neurology departments. We describe three novel pathogenic variants and identify COL6A2:c.1970-9G>A as the most frequent variant in our series (29%). We also observe an accelerated progression of the disease in a subgroup of patients. This large series of rare disease patients provides essential information on phenotypic variability of COL6-RD patients as well as on frequency of pathogenic COL6A gene variants in Southern France, thus contributing to the phenotypic and genetic description of Collagen type VI-related dystrophies.

New Clinical and Immunofluoresence Data of Collagen VI-Related Myopathy: A Single Center Cohort of 69 Patients.

Pathogenetic mechanism recognition and proof-of-concept clinical trials were performed in our patients affected by collagen VI-related myopathies. This study, which included 69 patients, aimed to identify innovative clinical data to better design future trials. Among the patients, 33 had Bethlem myopathy (BM), 24 had Ullrich congenital muscular dystrophy (UCMD), 7 had an intermediate phenotype (INTM), and five had myosclerosis myopathy (MM). We obtained data on muscle strength, the degree of contracture, immunofluorescence, and genetics. In our BM group, only one third had a knee extension strength greater than 50% of the predicted value, while only one in ten showed similar retention of elbow flexion. These findings should be considered when recruiting BM patients for future trials. All the MM patients had axial and limb contractures that limited both the flexion and extension ranges of motion, and a limitation in mouth opening. The immunofluorescence analysis of collagen VI in 55 biopsies from 37 patients confirmed the correlation between collagen VI defects and the severity of the clinical phenotype. However, biopsies from the same patient or from patients with the same mutation taken at different times showed a progressive increase in protein expression with age. The new finding of the time-dependent modulation of collagen VI expression should be considered in genetic correction trials.

Interleukin 36 receptor-inducible matrix metalloproteinase 13 mediates intestinal fibrosis.

Background: Fibrostenotic disease is a common complication in Crohn's disease (CD) patients hallmarked by transmural extracellular matrix (ECM) accumulation in the intestinal wall. The prevention and medical therapy of fibrostenotic CD is an unmet high clinical need. Although targeting IL36R signaling is a promising therapy option, downstream mediators of IL36 during inflammation and fibrosis have been incompletely understood. Candidate molecules include matrix metalloproteinases which mediate ECM turnover and are thereby potential targets for anti-fibrotic treatment. Here, we have focused on understanding the role of MMP13 during intestinal fibrosis. Methods: We performed bulk RNA sequencing of paired colon biopsies taken from non-stenotic and stenotic areas of patients with CD. Corresponding tissue samples from healthy controls and CD patients with stenosis were used for immunofluorescent (IF) staining. MMP13 gene expression was analyzed in cDNA of intestinal biopsies from healthy controls and in subpopulations of patients with CD in the IBDome cohort. In addition, gene regulation on RNA and protein level was studied in colon tissue and primary intestinal fibroblasts from mice upon IL36R activation or blockade. Finally, in vivo studies were performed with MMP13 deficient mice and littermate controls in an experimental model of intestinal fibrosis. Ex vivo tissue analysis included Masson's Trichrome and Sirius Red staining as well as evaluation of immune cells, fibroblasts and collagen VI by IF analysis. Results: Bulk RNA sequencing revealed high upregulation of MMP13 in colon biopsies from stenotic areas, as compared to non-stenotic regions of patients with CD. IF analysis confirmed higher levels of MMP13 in stenotic tissue sections of CD patients and demonstrated αSMA+ and Pdpn+ fibroblasts as a major source. Mechanistic experiments demonstrated that MMP13 expression was regulated by IL36R signaling. Finally, MMP13 deficient mice, as compared to littermate controls, developed less fibrosis in the chronic DSS model and showed reduced numbers of αSMA+ fibroblasts. These findings are consistent with a model suggesting a molecular axis involving IL36R activation in gut resident fibroblasts and MMP13 expression during the pathogenesis of intestinal fibrosis. Conclusion: Targeting IL36R-inducible MMP13 could evolve as a promising approach to interfere with the development and progression of intestinal fibrosis.

Alopecia in Patients with Collagen VI-Related Myopathies: A Novel/Unrecognized Scalp Phenotype.

Collagen VI-related myopathies are characterized by severe muscle involvement and skin involvement (keratosis pilaris and impaired healing with the development of abnormal scars, especially keloids). Scalp involvement and hair loss have not been reported among cutaneous changes associated with collagen VI mutations. The aim of this study is to describe the clinical, trichoscopic, and histological findings of the scalp changes in patients affected by COL VI mutations and to estimate their prevalence. Patients with Ullrich congenital muscular dystrophy were enrolled and underwent clinical and trichoscopic examinations and a scalp biopsy for histopathology. Five patients were enrolled, and all complained of hair loss and scalp itching. One patient showed yellow interfollicular scales with erythema and dilated, branched vessels, and the histological findings were suggestive of scalp psoriasis. Two patients presented with scarring alopecia patches on the vertex area, and they were histologically diagnosed with folliculitis decalvans. The last two patients presented with scaling and hair thinning, but they were both diagnosed with folliculitis and perifolliculitis. Ten more patients answered to a "scalp involvement questionnaire", and six of them confirmed to have or have had scalp disorders and/or itching. Scalp involvement can be associated with COL VI mutations and should be investigated.

Collagen VI in the Musculoskeletal System.

Collagen VI exerts several functions in the tissues in which it is expressed, including mechanical roles, cytoprotective functions with the inhibition of apoptosis and oxidative damage, and the promotion of tumor growth and progression by the regulation of cell differentiation and autophagic mechanisms. Mutations in the genes encoding collagen VI main chains, COL6A1, COL6A2 and COL6A3, are responsible for a spectrum of congenital muscular disorders, namely Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM), which show a variable combination of muscle wasting and weakness, joint contractures, distal laxity, and respiratory compromise. No effective therapeutic strategy is available so far for these diseases; moreover, the effects of collagen VI mutations on other tissues is poorly investigated. The aim of this review is to outline the role of collagen VI in the musculoskeletal system and to give an update about the tissue-specific functions revealed by studies on animal models and from patients' derived samples in order to fill the knowledge gap between scientists and the clinicians who daily manage patients affected by collagen VI-related myopathies.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

Circulating Levels of Endotrophin Are Prognostic for Long-Term Mortality after AKI.

Background: AKI involves a rapid decrease in kidney function that may be associated with structural damage. Early markers predicting AKI are emerging, but tools to assess patients' long-term health risks after AKI are still lacking. Endotrophin (ETP) is a bioactive molecule released during the formation of collagen type VI. We evaluated the potential of circulating ETP as a prognostic biomarker of adverse outcomes after AKI. Methods: We measured ETP in plasma samples collected 1 year after an episode of AKI, using the PRO-C6 ELISA in 801 patients (393 patients with AKI and 408 controls) from the prospective AKI Risk in Derby (ARID) study (ISRCTN25405995), who were then followed until year 3. Kidney disease progression was defined as ≥25% decline in eGFR combined with a decline in CKD stage. Results: ETP levels were significantly higher in the AKI group compared with controls (P<0.001). In the AKI group, ETP could discriminate patients with kidney disease progression at year 3 (AUC=0.67, P<0.01), whereas eGFR could not (AUC=0.51, P=0.57). In logistic regression including common risk factors, ETP was independently associated with kidney disease progression in patients with AKI (OR=1.10, P<0.01). ETP could discriminate survivors from nonsurvivors at year 3 (AUC=0.64, P<0.01). In a Cox proportional hazards regression for mortality after AKI that included common risk factors, only ETP (HR=1.05; P<0.001) and age (HR=1.06, P<0.01) were retained in the final model. Conclusions: Patients in the AKI group had higher levels of plasma ETP at year 1 as compared with those who had not had AKI. In the AKI group, ETP levels predict kidney disease progression and mortality. Because ETP is a profibrotic molecule, our findings may indicate that ETP identifies patients with active fibrogenesis after AKI, suggestive of long-term renal remodeling, which is associated with patient outcome.

A novel variant in the COL6A1 gene causing Ullrich congenital muscular dystrophy in a consanguineous family: a case report.

BACKGROUND: Collagen VI-related dystrophies are a subtype of congenital muscular dystrophy caused by pathogenic variants in COL6A1, COL6A2 or COL6A3 genes affecting skeletal muscles and connective tissue. The clinical phenotype ranges from the milder Bethlem myopathy to the severe Ullrich congenital muscular dystrophy (UCMD). Herein, we report the first consanguineous Sri Lankan family with two children affected with UCMD due to a novel variant in the COL6A1 gene. CASE PRESENTATION: Two sisters, aged 10-years and 7-years, presented with progressive, bilateral proximal muscle weakness. Both probands had delayed motor milestones and demonstrated difficulty in standing from a squatting position, climbing stairs and raising arms above the shoulders. Cognitive, language and social development were age appropriate. Examination showed proximal muscle weakness of the upper and lower extremities and hyperlaxity of the wrist and fingers in both with some variability in clinical severity noted between the two siblings. Serum creatine kinase levels were elevated, and electromyography showed low polyphasic motor unit potentials in the 10-year-old and myopathic features with short duration motor unit potentials with no polyphasia in the 7-year-old. Whole exome sequencing (WES) was performed and a novel, homozygous missense, likely pathogenic variant in exon 25 of COL6A1 gene [NM_001848: c.1667G > T;NP_001839.2:p.Gly556Val] was identified in both probands. This variant was validated by Sanger sequencing in proband 1 as well as proband 2, and the parents and an unaffected sibling were found to be heterozygote carriers for the same variant. CONCLUSIONS: The findings in this family add to the expanding number of COL6A1 variants identified and provides a better understanding of the genotype-phenotype correlations associated with UCMD.

Clinical and Molecular Spectrum Associated with COL6A3 c.7447A>G p.(Lys2483Glu) Variant: Elucidating its Role in Collagen VI-related Myopathies.

BACKGROUND: Dominant and recessive autosomal pathogenic variants in the three major genes (COL6A1-A2-A3) encoding the extracellular matrix protein collagen VI underlie a group of myopathies ranging from early-onset severe conditions (Ullrich congenital muscular dystrophy) to milder forms maintaining independent ambulation (Bethlem myopathy). Diagnosis is based on the combination of clinical presentation, muscle MRI, muscle biopsy, analysis of collagen VI secretion, and COL6A1-A2-A3 genetic analysis, the interpretation of which can be challenging. OBJECTIVE: To refine the phenotypical spectrum associated with the frequent COL6A3 missense variant c.7447A>G (p.Lys2483Glu). METHODS: We report the clinical and molecular findings in 16 patients: 12 patients carrying this variant in compound heterozygosity with another COL6A3 variant, and four homozygous patients. RESULTS: Patients carrying this variant in compound heterozygosity with a truncating COL6A3 variant exhibit a phenotype consistent with COL6-related myopathies (COL6-RM), with joint contractures, proximal weakness and skin abnormalities. All remain ambulant in adulthood and only three have mild respiratory involvement. Most show typical muscle MRI findings. In five patients, reduced collagen VI secretion was observed in skin fibroblasts cultures. All tested parents were unaffected heterozygous carriers. Conversely, two out of four homozygous patients did not present with the classical COL6-RM clinical and imaging findings. Collagen VI immunolabelling on cultured fibroblasts revealed rather normal secretion in one and reduced secretion in another. Muscle biopsy from one homozygous patient showed myofibrillar disorganization and rimmed vacuoles. CONCLUSIONS: In light of our results, we postulate that the COL6A3 variant c.7447A>G may act as a modulator of the clinical phenotype. Thus, in patients with a typical COL6-RM phenotype, a second variant must be thoroughly searched for, while for patients with atypical phenotypes further investigations should be conducted to exclude alternative causes. This works expands the clinical and molecular spectrum of COLVI-related myopathies.

COL6A6 inhibits the proliferation and metastasis of non-small cell lung cancer through the JAK signalling pathway.

BACKGROUND: Collagen type VI alpha 6 chain (COL6A6) plays a vital role in maintaining cell structural integrity and regulating cell function. It has been proven to be a tumor suppressor gene and molecular therapeutic target. However, the mechanism of COL6A6 in non-small cell lung cancer (NSCLC) has not been elucidated. The purpose of this study was to investigate the relationship between COL6A6 and NSCLC. METHODS: We analyzed the expression of COL6A6 in NSCLC using public databases and verified the findings in NSCLC tissues and cells. The protein expression of COL6A6 was evaluated by Western blot. The CCK8 and Transwell assays were used to assess the invasion ability of NSCLC cells after COL6A6 knockdown. At the same time, we discussed the role of the JAK signalling pathway that may be related to COL6A6. RESULTS: Bioinformatics analysis showed that COL6A6 expression was downregulated in NSCLC, and its high expression was associated with a better prognosis of NSCLC. In vitro, the expression of COL6A6 in NSCLC tissues was significantly lower than that in adjacent tissues. Furthermore, COL6A6 knockout accelerated the proliferation, invasion, and migration of NSCLC cells and activated the JAK signalling pathway. CONCLUSIONS: Our study illustrates that COL6A6 is a tumor suppressor gene in NSCLC and is involved in NSCLC tumorigenesis by regulating the JAK signalling pathway. Therefore, COL6A6 holds promise as a molecular therapeutic target for NSCLC.

Biopsy-Controlled Non-Invasive Quantification of Collagen Type VI in Kidney Transplant Recipients: A Post-Hoc Analysis of the MECANO Trial.

The PRO-C6 assay, a reflection of collagen type VI synthesis, has been proposed as a non-invasive early biomarker of kidney fibrosis. We aimed to investigate cross-sectional and longitudinal associations between plasma and urine PRO-C6 and proven histological changes after kidney transplantation. The current study is a post-hoc analysis of 94 participants of the MECANO trial, a 24-month prospective, multicenter, open-label, randomized, controlled trial aimed at comparing everolimus-based vs. cyclosporine-based immunosuppression. PRO-C6 was measured in plasma and urine samples collected 6 and 24 months post-transplantation. Fibrosis was evaluated in biopsies collected at the same time points by Banff interstitial fibrosis/tubular atrophy (IF/TA) scoring and collagen staining (Picro Sirius Red; PSR); inflammation was evaluated by the tubulo-interstitial inflammation score (ti-score). Linear regression analyses were performed. Six-month plasma PRO-C6 was cross-sectionally associated with IF/TA score (Std. ß = 0.34), and prospectively with 24-month IF/TA score and ti-score (Std. ß = 0.24 and 0.23, respectively) (p < 0.05 for all). No significant associations were found between urine PRO-C6 and any of the biopsy findings. Fibrotic changes and urine PRO-C6 behaved differentially over time according to immunosuppressive therapy. These results are a first step towards non-invasive fibrosis detection after kidney transplantation by means of collagen VI synthesis measurement, and further research is required.

COL6A3 promotes cellular malignancy of osteosarcoma by activating the PI3K/AKT pathway

SUMMARY OBJECTIVE In this study, we aimed to investigate the role of COL6A3 on cell motility and the PI3K/AKT signaling pathway in osteosarcoma. METHODS The relative expression of COL6A3 was achieved from a GEO dataset in osteosarcoma tissue. siRNA technology was applied to decrease the COL6A3 expression in cells, and cell counting kit-8 (CCK-8) assay and colony formation analysis were used to examine the cell proliferation potential. Knockdown COL6A3 made the proliferation and colony formation abilities worse than the COL6A3 without interference. Likewise, in contrast to the si-con group, cell invasion and migration were inhibited in the si-COL6A3 group. Moreover, the western blot results suggested that the PI3K/AKT signaling pathway was manipulated by measuring the protein expression of the PI3K/AKT pathway-related markers, due to the COL6A3 inhibition. CONCLUSION COL6A3 plays a crucial role in modulating various aspects of the progression of osteosarcoma, which would provide a potentially effective treatment for osteosarcoma.

RESUMO OBJETIVO Neste estudo, investigamos a função do COL6A3 na mobilidade celular e na via PI3K/AKT em osteossarcomas. METODOLOGIA A expressão relativa do COL6A3 foi obtida a partir de dados GEO em tecidos de osteossarcoma. O RNA de interferência (siRNA) foi utilizado para reduzir a expressão do COL6A3 nas células, e o teste de contagem de células kit-8 (CCK-8) e a análise de formação de colônias foram realizados para examinar o potencial de proliferação celular. Além disso, o Transwell comprovou os efeitos do si-COL6A3 na invasão celular e migração em células de osteossarcoma. Para medir os níveis de expressão das proteínas e mRNAs, utilizamos transcriptase reversa quantitativa (qRT-PCR) e western blot. RESULTADOS O COL6A3 foi regulado nos tecidos e células do osteossarcoma quando comparado com o controle normal. A redução de COL6A3 reduziu a proliferação e a capacidades de formação de colônias em relação ao COL6A3 sem interferência. Do Mesmo modo, ao contrário do observado no grupo si-con, a invasão e migração celular foram inibidas no grupo si-COL6A3. Além disso, o resultado do western blot sugere que a via PI3K/AKT foi manipulada, medindo a expressão proteica dos marcadores relacionados à PI3K/AKT, devido à inibição do COL6A3. CONCLUSÃO O COL6A3 desempenha um papel crucial na modulação de vários aspectos da progressão do osteossarcoma, o que pode representar um possível tratamento eficaz para a doença.

COL6A1 knockdown suppresses cell proliferation and migration in human aortic vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) migration is an important pathophysiological signature of neointimal hyperplasia. The aim of the present study was to investigate the effects of collagen type VI α1 chain (COL6A1) on VSMC migration. COL6A1 expression was silenced in platelet-derived growth factor (PDGF-BB)-stimulated VSMCs. Cell counting kit-8, wound healing and Transwell assays were used to measure cell viability, migration and invasion, respectively. Reverse transcription-quantitative PCR and western blot analysis were performed to analyze the expression of factors associated with metastasis. COL6A1 silencing attenuated PDGF-BB-induced increases in cell viability and invasive abilities of VSMCs, in addition to partially reversing the increased expression of fibronectin (FN), matrix metalloproteinase (MMP)-2 and MMP-9 induced by PDGF-BB stimulation. The silencing of COL6A also overturned PDGF-BB-induced reduction in tissue inhibitor of metalloproteinase 2 expression in VSMCs. PDGF-BB activated the AKT/mTOR pathway, which was also inhibited by COL6A1 knockdown. Taken together, these findings suggest that COL6A1 silencing inhibited VSMC viability and migration by inhibiting AKT/mTOR activation.

Bethlem myopathy: a series of 16 patients and description of seven new associated mutations.

BACKGROUND: Bethlem myopathy represents the milder phenotype of collagen type VI-related myopathies. However, clinical manifestations are highly variable among patients and no phenotype-genotype correlation has been described. We aim to analyse the clinical, pathological and genetic features of a series of patients with Bethlem myopathy, and we describe seven new mutations. METHODS: A series of 16 patients with the diagnosis of Bethlem myopathy were analyzed retrospectively from their medical records for clinical, creatine kinase (CK), muscle biopsy, and muscle magnetic resonance (MRI) data. Genetic testing was performed through next-generation sequencing of custom amplicon-based targeted genes panel of myopathies. Mutations were confirmed by Sanger sequencing. RESULTS: The most frequent phenotype consisted of proximal limb weakness associated with interphalangeal and wrists contractures. However, cases with isolated contractures or isolated myopathy were found. CK levels did not correlate with severity of the disease. The most frequent mutation was the COL6A3 variant c.7447A>G, p.Lys2486Glu, with either an homozygous or compound heterozygous presentation. Five new mutations were found in COL6A1 gene and other two in COL6A3 gene, all of them with a dominant heritability pattern. From these, a new COL6A1 mutation (c.1657G>A, p.Glu553Arg) was related to an oligosymptomatic phenotype with predominating contractures in the absence of weakness and a normal muscle MRI. Finally, the most common COL6A1 mutation reported to date that leads to an Ullrich phenotype (c. 868G>A, p.Gly290Arg), has been found here as Bethlem presentation. CONCLUSIONS: Manifestations of Bethlem myopathy are quite variable, so either contractures or weakness may be lacking, and no phenotype-genotype associations can be brought.

Alterations in non-type I collagen biomarkers in osteogenesis imperfecta.

Osteogenesis imperfecta [1] is a rare disorder of connective tissue caused by abnormalities in the synthesis or processing of type I collagen. Type I collagen is the most abundant type of collagen and is expressed in almost all connective tissues. Given that type I collagen interacts with other collagens based in the extracellular matrix (ECM), we hypothesized changes in type I collagen in OI would result in perturbations in the homeostasis of other collagen types. We measured serum biomarkers of several non-type I collagens in patients with mild (type I) and moderate-to-severe (type III/IV) OI. Compared to controls, those with moderate-to severe OI had a higher mean level of the synthesis markers of collagen III (ProC3) (P = 0.02), and levels of collagen V (ProC5) (P = 0.07) were slightly, but not significantly, higher. Degradation markers of collage type IV (C4M2) (P = 0.04) and type VI (C6M) (P = 0.003) were also higher. In each case, a test for trend suggested levels were higher in moderate-to-severe OI, intermediate in mild OI, and lowest in controls (P = 0.06-0.002). These changes supports the hypothesis that mutations in type I collagen induce a widespread alteration in the ECM, and that the diverse clinical manifestations of OI reflect an extensive disruption in ECM biology.

Stromal collagen type VI associates with features of malignancy and predicts poor prognosis in salivary gland cancer.

PURPOSE: Collagen Type VI (COLVI) is an extracellular matrix protein that is upregulated in various solid tumours during tumour progression and has been shown to stimulate proliferation, suppress apoptosis and promote invasion and metastasis. It has also been described as a mediator of chemotherapy resistance and as a therapeutic target in preclinical cancer models. Here, we aimed to analyse the prognostic role of COLVI in salivary gland cancer (SGC). METHODS: Stromal COLVI protein expression was assessed in primary SGC specimens of 91 patients using immunohistochemistry (IHC). The IHC expression patterns obtained were subsequently correlated with various survival and clinicopathological features, including Ki-67 and p53 expression. RESULTS: We found that COLVI was expressed in all SGC specimens. High expression was found to be associated with features of malignancy such as high histologic grades, advanced and invasive T stages and metastatic lymph node involvement (p < 0.05 for all variables). COLVI expression was also found to correlate with both Ki-67 and p53 expression (p < 0.01). We found that high COLVI expression predicted a significantly inferior 5-year overall survival (38.3%, 55.1% and 93.8%; p = 0.002) and remained a significant predictor of prognosis in a multivariate Cox regression analysis (hazard ratio, 2.62; 95% confidence interval, 1.22-5.61; p = 0.013). In all low-risk subgroups COLVI expression identified patients with an adverse outcome. Patients receiving adjuvant radiotherapy had a poor survival when expressing high levels of COLVI. CONCLUSIONS: Our data indicate that stromal COLVI expression associates with key features of malignancy, represents a novel independent prognostic factor and may affect response to radiotherapy in SGC. Although our results warrant validation in an independent cohort, assessing stromal COLVI expression may be suitable for future diagnostic and therapeutic decision making in patients with SGC.

Immunolocalization and distribution of proteoglycans in carious dentine.

BACKGROUND: Collagen type I, proteoglycans (PG) and non-collagenous proteins represent important building blocks of the dentine matrix. While different PGs have been identified in dentine, changes in the distribution of these macromolecules with the progression of caries have been poorly characterized. The aim of this study was to compare the immunolocalization of three small collagen-binding PGs (biglycan, fibromodulin and lumican) as well as collagen (types I and VI) in healthy versus carious dentine. METHODS: Longitudinal demineralized sections of extracted teeth were stained with antibodies recognizing specific PG core proteins and collagens, as well as glycosaminoglycans (GAGs) with toluidine blue. RESULTS: In healthy dentine, PGs appeared to be more abundant near the tubule walls and directly under the cusps. Conversely, in carious dentine, specific locations appeared to be more prone to PG degradation than others. These degradation patterns were well correlated with the progression of caries into the tissue, and also appeared to trigger interesting morphological changes in the tissue structure, such as the deformation of dentine tubules near highly infected areas and the lower concentration of PG in tertiary dentine. CONCLUSIONS: This study presents new insights into the involvement of PGs in the progression of caries.