Effect of mechanical strain on the collagen VI pericellular matrix in anterior cruciate ligament fibroblasts.

Cell-extracellular matrix interaction plays a major role in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. Collagen VI is a widely expressed non-fibrillar collagen, which regulates tissues homeostasis. The objective of the present investigation was to extend our understanding of the role of collagen VI in human ACL. This study shows that collagen VI is associated both in vivo and in vitro to the cell membrane of knee ACL fibroblasts, contributing to the constitution of a microfibrillar pericellular matrix. In cultured cells the localization of collagen VI at the cell surface correlated with the expression of NG2 proteoglycan, a major collagen VI receptor. The treatment of ACL fibroblasts with anti-NG2 antibody abolished the localization of collagen VI indicating that collagen VI pericellular matrix organization in ACL fibroblasts is mainly mediated by NG2 proteoglycan. In vitro mechanical strain injury dramatically reduced the NG2 proteoglycan protein level, impaired the association of collagen VI to the cell surface, and promoted cell cycle withdrawal. Our data suggest that the injury-induced alteration of specific cell-ECM interactions may lead to a defective fibroblast self-renewal and contribute to the poor regenerative ability of ACL fibroblasts.

Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy.

Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies.

Collagen VI-NG2 axis in human tendon fibroblasts under conditions mimicking injury response.

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI-NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI-NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor ß1 (TGFß1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFß1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair.

Aggresome-Autophagy Involvement in a Sarcopenic Patient with Rigid Spine Syndrome and a p.C150R Mutation in FHL1 Gene.

The four-and-half LIM domain protein 1 (FHL1) is highly expressed in skeletal and cardiac muscle. Mutations of the FHL1 gene have been associated with diverse chronic myopathies including reducing body myopathy, rigid spine syndrome (RSS), and Emery-Dreifuss muscular dystrophy. We investigated a family with a mutation (p.C150R) in the second LIM domain of FHL1. In this family, a brother and a sister were affected by RSS, and their mother had mild lower limbs weakness. The 34-year-old female had an early and progressive rigidity of the cervical spine and severe respiratory insufficiency. Muscle mass evaluated by DXA was markedly reduced, while fat mass was increased to 40%. CT scan showed an almost complete substitution of muscle by fibro-adipose tissue. Muscle biopsy showed accumulation of FHL1 throughout the cytoplasm and around myonuclei into multiprotein aggregates with aggresome/autophagy features as indicated by ubiquitin, p62, and LC3 labeling. DNA deposits, not associated with nuclear lamina components and histones, were also detected in the aggregates, suggesting nuclear degradation. Ultrastructural analysis showed the presence of dysmorphic nuclei, accumulation of tubulofilamentous and granular material, and perinuclear accumulation of autophagic vacuoles. These data point to involvement of the aggresome-autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation.

Melanocytes from Patients Affected by Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy have Dysfunctional Mitochondria That Can be Rescued with Cyclophilin Inhibitors.

Ullrich congenital muscular dystrophy and Bethlem myopathy are caused by mutations in collagen VI (ColVI) genes, which encode an extracellular matrix protein; yet, mitochondria play a major role in disease pathogenesis through a short circuit caused by inappropriate opening of the permeability transition pore, a high-conductance channel, which causes a shortage in ATP production. We find that melanocytes do not produce ColVI yet they bind it at the cell surface, suggesting that this protein may play a trophic role and that its absence may cause lesions similar to those seen in skeletal muscle. We show that mitochondria in melanocytes of Ullrich congenital muscular dystrophy and Bethlem myopathy patients display increased size, reduced matrix density, and disrupted cristae, findings that suggest a functional impairment. In keeping with this hypothesis, mitochondria (i) underwent anomalous depolarization after inhibition of the F-ATP synthase with oligomycin, and (ii) displayed decreased respiratory reserve capacity. The non-immunosuppressive cyclophilin inhibitor NIM811 prevented mitochondrial depolarization in response to oligomycin in melanocytes from both Ullrich congenital muscular dystrophy and Bethlem myopathy patients, and partially restored the respiratory reserve of melanocytes from one Bethlem myopathy patient. These results match our recent findings on melanocytes from patients affected by Duchenne muscular dystrophy (Pellegrini et al., 2013), and suggest that skin biopsies may represent a minimally invasive tool to investigate mitochondrial dysfunction and to evaluate drug efficacy in ColVI-related myopathies and possibly in other muscle wasting conditions like aging sarcopenia.

Ultrastructural changes in muscle cells of patients with collagen VI-related myopathies.

Collagen VI is an extracellular matrix protein expressed in several tissues including skeletal muscle. Mutations in COL6A genes cause Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD) and Myosclerosis Myopathy (MM). Collagen VI deficiency causes increased opening of the mitochondrial permeability transition pore (mPTP), leading to ultrastructural and functional alterations of mitochondria, amplified by impairment of autophagy. Here we report for the first time ultrastructural studies on muscle biopsies from BM and UCMD patients, showing swollen mitochondria with hypodense matrix, disorganized cristae and paracrystalline inclusions, associated with dilated sarcoplasmic reticulum and apoptotic changes. These data were supported by scanning electron microscopy analysis on BM and UCMD cultured cells, showing alterations of the mitochondrial network. Morphometric analysis also revealed a reduced short axis and depicted swelling in about 3% of mitochondria. These data demonstrate that mitochondrial defects underlie the pathogenetic mechanism in muscle tissue of patients affected by collagen VI myopathies.

Osteoblasts display different responsiveness to TRAIL-induced apoptosis during their differentiation process.

Apoptosis can occur throughout the life span of osteoblasts (OBs), beginning from the early stages of differentiation and continuing throughout all stages of their working life. Here, we investigated the effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal human OBs showing for the first time that the expression of TRAIL receptors is modulated during OB differentiation. In particular, the TRAIL receptor ratio was in favor of the deaths because of the low expression of DcR2 in undifferentiated OBs, differently it was shifted toward the decoys in differentiated ones. Undifferentiated OBs treated with TRAIL showed reduced cell viability, whereas differentiated OBs displayed TRAIL resistance. The OB sensitiveness to TRAIL was due to the up-regulation of DR5 and the down-regulation of DcR2. The main death receptor involved in TRAIL-reduced OB viability was DR5 as demonstrated by the rescue of cell viability observed in the presence of anti-DR5 neutralizing antibody. Besides the ratio of TRAIL receptors, the sensitivity of undifferentiated OBs to TRAIL-cytotoxic effect was also associated with low mRNA levels of intracellular anti-apoptotic proteins, such as cFLIP, the activation of caspase-8 and -3, as well as the DNA fragmentation. This study suggests that apoptotic effect exerted by TRAIL/TRAIL-receptor system on normal human OB is strictly dependent upon cell differentiation status.

TRAIL effect on osteoclast formation in physiological and pathological conditions.

Although osteoclasts (OCs) differentiate under the control of RANK/RANKL/OPG system, a number of inflammatory cytokines can contribute to increase osteoclastogenesis in diseases associated with bone loss. Recently, different studies indicate that TRAIL is implicated in modulating osteoclastogenesis. Here, we investigated the effect of TRAIL on OC formation in physiological and pathological conditions with bone involvement utilizing osteoclastogenesis in vitro models represented by peripheral blood mononuclear cells (PBMCs) from healthy donors and patients affected by multiple myeloma or periodontal disease. We demonstrated that in PBMCs from healthy donors TRAIL can directly induce OC formation in the absence of RANKL, while exert an inhibitory effect when added concomitantly to RANKL. In PBMCs from the patients, in which media the levels of TRAIL, RANKL and OPG are elevated, the neutralization of TRAIL partially inhibits the OC formation, and this effect was reversed by RANKL addition. Finally, we detect high TRAIL levels in the sera from the patients. In conclusion, our results indicate that TRAIL could exert a different role in modulating OC differentiation in physiological and pathological conditions.