Collagen VI sustains cell stemness and chemotherapy resistance in glioblastoma.

Microenvironmental factors are known fundamental regulators of the phenotype and aggressiveness of glioblastoma (GBM), the most lethal brain tumor, characterized by fast progression and marked resistance to treatments. In this context, the extracellular matrix (ECM) is known to heavily influence the behavior of cancer cells from several origins, contributing to stem cell niches, influencing tumor invasiveness and response to chemotherapy, mediating survival signaling cascades, and modulating inflammatory cell recruitment. Here, we show that collagen VI (COL6), an ECM protein widely expressed in both normal and pathological tissues, has a distinctive distribution within the GBM mass, strongly correlated with the most aggressive and phenotypically immature cells. Our data demonstrate that COL6 sustains the stem-like properties of GBM cells and supports the maintenance of an aggressive transcriptional program promoting cancer cell proliferation and survival. In particular, we identified a specific subset of COL6-transcriptionally co-regulated genes, required for the response of cells to replicative stress and DNA damage, supporting the concept that COL6 is an essential stimulus for the activation of GBM cell response and resistance to chemotherapy, through the ATM/ATR axis. Altogether, these findings indicate that COL6 plays a pivotal role in GBM tumor biology, exerting a pleiotropic action across different GBM hallmarks, including phenotypic identity and gene transcription, as well as response to treatments, thus providing valuable information for the understanding of the complex microenvironmental cues underlying GBM malignancy.

Collagen VI promotes recovery from colitis by inducing lymphangiogenesis and drainage of inflammatory cells.

Despite a number of studies providing evidence that the extracellular matrix (ECM) is an active player in the pathogenesis of intestinal inflammation, knowledge on the actual contribution of specific ECM molecules in the progression of inflammatory bowel disease (IBD) remains scant. Here, we investigated the role of a major ECM protein, collagen VI (ColVI), in gut homeostasis and elucidated the impact of its deregulation on the pathophysiology of IBD. To this end, we combined in vivo and ex vivo studies on wild type and ColVI-deficient (Col6a1-/- ) mice both under physiological conditions and during experimentally induced acute colitis and its subsequent recovery, by means of gut histology and immunostaining, gene expression, bone marrow transplantation, flow cytometry of immune cell subpopulations, and lymph flow assessment. We found that ColVI displayed dynamic expression and ECM deposition during the acute inflammatory and recovery phases of experimentally induced colitis, whereas the genetic ablation of ColVI in Col6a1 null mice impaired the functionality of lymphatic vessels, which in turn affected the resolution of inflammation during colitis. Based on these findings, we investigated ColVI expression and deposition in ileal specimens from two cohorts of patients affected by Crohn's disease (CD) and correlated ColVI abundance to clinical outcome. Our results show that high ColVI immunoreactivity in ileal biopsies of CD patients at diagnosis correlates with increased risk of surgery and that ColVI expression in biopsies taken at the resection margin during surgery, and showing inactive disease, predict disease recurrence. Our data unveil a key role for ColVI in the intestinal microenvironment, where it is involved in lymphangiogenesis and intestinal inflammation. Altogether, these findings point at the dysregulation of ColVI expression as a novel factor contributing to the onset and maintenance of inflammation in CD via mechanisms impinging on the modulation of inflammatory cell recruitment and function. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Lack of COL6/collagen VI causes megakaryocyte dysfunction by impairing autophagy and inducing apoptosis.

Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-/- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-/- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-/- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-/- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-/-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.

Collagen VI deficiency causes behavioral abnormalities and cortical dopaminergic dysfunction.

Mutations of genes coding for collagen VI (COL6) cause muscle diseases, including Ullrich congenital muscular dystrophy and Bethlem myopathy. Although COL6 genetic variants were recently linked to brain pathologies, the impact of COL6 deficiency in brain function is still largely unknown. Here, a thorough behavioral characterization of COL6-null (Col6a1-/-) mice unexpectedly revealed that COL6 deficiency leads to a significant impairment in sensorimotor gating and memory/attention functions. In keeping with these behavioral abnormalities, Col6a1-/- mice displayed alterations in dopaminergic signaling, primarily in the prefrontal cortex. In vitro co-culture of SH-SY5Y neural cells with primary meningeal fibroblasts from wild-type and Col6a1-/- mice confirmed a direct link between COL6 ablation and defective dopaminergic activity, through a mechanism involving the inability of meningeal cells to sustain dopaminergic differentiation. Finally, patients affected by COL6-related myopathies were evaluated with an ad hoc neuropsychological protocol, revealing distinctive defects in attentional control abilities. Altogether, these findings point towards a previously undescribed role for COL6 in the proper maintenance of dopamine circuitry function and its related neurobehavioral features in both mice and humans. This article has an associated First Person interview with the first author of the paper.

Ambra1 deficiency impairs mitophagy in skeletal muscle.

BACKGROUND: Maintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well-known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full-body Ambra1 deficiency. METHODS: To establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria-targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle-specific Ambra1 knockout (Ambra1fl/fl :Mlc1f-Cre) mice. Mitochondria-enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux. RESULTS: Our data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild-type muscles is sufficient to enhance mitochondria clearance through the autophagy-lysosome system. Consistently with this, Ambra1-deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6-7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6-7 mice; P < 0.01) and Parkin (n = 6-7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1-deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild-type muscles (n = 4; P < 0.05). Histologically, Ambra1-deficient muscles of both 3- and 6-month-old animals display a significant decrease of myofibre cross-sectional area and a 52% reduction in oxidative fibres (n = 6-7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle. CONCLUSIONS: Our study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

Emilin-2 is a component of bone marrow extracellular matrix regulating mesenchymal stem cell differentiation and hematopoietic progenitors.

BACKGROUND: Dissection of mechanisms involved in the regulation of bone marrow microenvironment through cell-cell and cell-matrix contacts is essential for the detailed understanding of processes underlying bone marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma. METHODS: Immunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry. RESULTS: Emilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging. CONCLUSION: Our data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.

The Polyphenol Pterostilbene Ameliorates the Myopathic Phenotype of Collagen VI Deficient Mice via Autophagy Induction.

The induction of autophagy, the catabolic pathway by which damaged or unnecessary cellular components are subjected to lysosome-mediated degradation and recycling, is impaired in Collagen VI (COL6) null mice and COL6-related myopathies. This autophagic impairment causes an accumulation of dysfunctional mitochondria, which in turn leads to myofiber degeneration. Our previous work showed that reactivation of autophagy in COL6-related myopathies is beneficial for muscle structure and function both in the animal model and in patients. Here we show that pterostilbene (Pt)-a non-toxic polyphenol, chemically similar to resveratrol but with a higher bioavailability and metabolic stability-strongly promotes in vivo autophagic flux in the skeletal muscle of both wild-type and COL6 null mice. Reactivation of autophagy in COL6-deficient muscles was also paralleled by several beneficial effects, including significantly decreased incidence of spontaneous apoptosis, recovery of ultrastructural defects and muscle remodeling. These findings point at Pt as an effective autophagy-inducing nutraceutical for skeletal muscle with great potential in counteracting the major pathogenic hallmarks of COL6-related myopathies, a valuable feature that may be also beneficial in other muscle pathologies characterized by defective regulation of the autophagic machinery.

Corrigendum: The Polyphenol Pterostilbene Ameliorates the Myopathic Phenotype of Collagen VI Deficient Mice via Autophagy Induction.

[This corrects the article DOI: 10.3389/fcell.2020.580933.].

Multimerin-2 maintains vascular stability and permeability.

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2-/- (Mmrn2-/-) mice. Although Mmrn2-/- mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2-/- mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2-/- vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2-/- mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

Fra-2-expressing macrophages promote lung fibrosis in mice.

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with Type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections and Fra-2 transgenic mice (Fra-2tg) exhibit spontaneous lung fibrosis. Here we show that Bleomycin-induced lung fibrosis is attenuated upon myeloid-inactivation of Fra-2 and aggravated in Fra-2tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is up-regulated in three lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2-dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine pro-fibrotic activity of macrophages. Importantly, ColVI knock-out mice (KO) and ColVI-KO bone marrow chimeras are protected from Bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2tg mice and in the Bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and co-localize in lung macrophages. Therefore, the Fra-2/ColVI pro-fibrotic axis is a promising biomarker and therapeutic target for lung fibrosis, and possibly other fibrotic diseases.

A novel murine model for arrhythmogenic cardiomyopathy points to a pathogenic role of Wnt signalling and miRNA dysregulation.

AIMS: Arrhythmogenic cardiomyopathy (AC) is one of the most common inherited cardiomyopathies, characterized by progressive fibro-fatty replacement in the myocardium. Clinically, AC manifests itself with ventricular arrhythmias, syncope, and sudden death and shows wide inter- and intra-familial variability. Among the causative genes identified so far, those encoding for the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are the most commonly mutated. So far, little is known about the molecular mechanism(s) behind such a varied spectrum of phenotypes, although it has been shown that the causative mutations not only lead to structural abnormalities but also affect the miRNA profiling of cardiac tissue. Here, we aimed at studying the pathogenic effects of a nonsense mutation of the desmoglein-2 gene, both at the structural level and in terms of miRNA expression pattern. METHODS AND RESULTS: We generated transgenic mice with cardiomyocyte-specific overexpression of a FLAG-tagged human desmoglein-2 harbouring the Q558* nonsense mutation found in an AC patient. The hearts of these mice showed signs of fibrosis, decrease in desmosomal size and number, and reduction of the Wnt/ß-catenin signalling. Genome-wide RNA-Seq performed in Tg-hQ hearts and non-transgenic hearts revealed that 24 miRNAs were dysregulated in transgenic animals. Further bioinformatic analyses for selected miRNAs suggested that miR-217-5p, miR-499-5p, and miR-708-5p might be involved in the pathogenesis of the disease. CONCLUSION: Down-regulation of the canonical Wnt/ß-catenin signalling might be considered a common key event in the AC pathogenesis. We identified the miRNA signature in AC hearts, with miR-708-5p and miR-217-5p being the most up-regulated and miR-499-5p the most down-regulated miRNAs. All of them were predicted to be involved in the regulation of the Wnt/ß-catenin pathway and might reveal the potential pathophysiology mechanisms of AC, as well as be useful as therapeutic targets for the disease.

AMBRA1 Controls Regulatory T-Cell Differentiation and Homeostasis Upstream of the FOXO3-FOXP3 Axis.

Regulatory T cells (Treg) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of Treg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human Treg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating Treg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis.

Loss of EMILIN-1 Enhances Arteriolar Myogenic Tone Through TGF-ß (Transforming Growth Factor-ß)-Dependent Transactivation of EGFR (Epidermal Growth Factor Receptor) and Is Relevant for Hypertension in Mice and Humans.

Objective- EMILIN-1 (elastin microfibrils interface located protein-1) protein inhibits pro-TGF-ß (transforming growth factor-ß) proteolysis and limits TGF-ß bioavailability in vascular extracellular matrix. Emilin1-/- null mice display increased vascular TGF-ß signaling and are hypertensive. Because EMILIN-1 is expressed in vessels from embryonic life to adulthood, we aimed at unravelling whether the hypertensive phenotype of Emilin1-/- null mice results from a developmental defect or lack of homeostatic role in the adult. Approach and Results- By using a conditional gene targeting inactivating EMILIN-1 in smooth muscle cells of adult mice, we show that increased blood pressure in mice with selective smooth muscle cell ablation of EMILIN-1 depends on enhanced myogenic tone. Mechanistically, we unveil that higher TGF-ß signaling in smooth muscle cells stimulates HB-EGF (heparin-binding epidermal growth factor) expression and subsequent transactivation of EGFR (epidermal growth factor receptor). With increasing intraluminal pressure in resistance arteries, the cross talk established by TGF-ß and EGFR signals recruits TRPC6 (TRP [transient receptor potential] classical type 6) and TRPM4 (TRP melastatin type 4) channels, lastly stimulating voltage-dependent calcium channels and potentiating myogenic tone. We found reduced EMILIN-1 and enhanced myogenic tone, dependent on increased TGF-ß-EGFR signaling, in resistance arteries from hypertensive patients. Conclusions- Taken together, our findings implicate an unexpected role of the TGF-ß-EGFR pathway in hypertension with current translational perspectives.

Collagen VI in healthy and diseased nervous system.

Collagen VI is a major extracellular matrix protein exerting a number of functions in different tissues, spanning from biomechanical to regulatory signals in the cell survival processes, and playing key roles in maintaining the stemness or determining the differentiation of several types of cells. In the last couple of years, emerging findings on collagen VI have led to increased interest in its role in the nervous system. The role of this protein in the peripheral nervous system was intensely studied and characterized in detail. Collagen VI acts as a regulator of Schwann cell differentiation and is required for preserving peripheral nerve myelination, function and structure, as well as for orchestrating nerve regeneration after injury. Although the role and distribution of collagen VI in the peripheral nervous system is now well established, the role of this distinctive extracellular matrix component in the central nervous system, along with its links to human neurological and neurodegenerative disorders, remains an open field of investigation. In this Review, we summarize and discuss a number of recent findings related to collagen VI in the central and peripheral nervous systems. We further link these findings to different aspects of the protein that are relevant to human diseases in these compartments in order to provide a comprehensive overview of the roles of this key matrix component in the nervous system.

The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth.

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

Targeting Interleukin-1ß Protects from Aortic Aneurysms Induced by Disrupted Transforming Growth Factor ß Signaling.

Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor ß (TGF-ß) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-ß. The results revealed that Smad4 inhibition activated interleukin-1ß (IL-1ß) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1ß antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-ß signaling, such as those driven by SMAD4 mutations.

Muscle proteomics reveals novel insights into the pathophysiological mechanisms of collagen VI myopathies.

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

EMILIN-3, peculiar member of elastin microfibril interface-located protein (EMILIN) family, has distinct expression pattern, forms oligomeric assemblies, and serves as transforming growth factor ß (TGF-ß) antagonist.

EMILIN-3 is a glycoprotein of the extracellular matrix belonging to a family that contains a characteristic N-terminal cysteine-rich EMI domain. Currently, EMILIN-3 is the least characterized member of the elastin microfibril interface-located protein (EMILIN)/Multimerin family. Using RNA, immunohistochemical, and protein chemistry approaches, we carried out a detailed characterization of the expression and biochemical properties of EMILIN-3 in mouse. During embryonic and postnatal development, EMILIN-3 showed a peculiar and dynamic pattern of gene expression and protein distribution. EMILIN-3 mRNA was first detected at E8.5-E9.5 in the tail bud and in the primitive gut, and at later stages it became abundant in the developing gonads and osteogenic mesenchyme. Interestingly and in contrast to other EMILIN/Multimerin genes, EMILIN-3 was not found in the cardiovascular system. Despite the absence of the globular C1q domain, immunoprecipitation and Western blot analyses demonstrated that EMILIN-3 forms disulfide-bonded homotrimers and higher order oligomers. Circular dichroism spectroscopy indicated that the most C-terminal part of EMILIN-3 has a substantial α-helical content and forms coiled coil structures involved in EMILIN-3 homo-oligomerization. Transfection experiments with recombinant constructs showed that the EMI domain contributes to the higher order self-assembly but was dispensable for homotrimer formation. EMILIN-3 was found to bind heparin with high affinity, a property mediated by the EMI domain, thus revealing a new function for this domain that may contribute to the interaction of EMILIN-3 with other extracellular matrix and/or cell surface molecules. Finally, in vitro experiments showed that EMILIN-3 is able to function as an extracellular regulator of the activity of TGF-ß ligands.

Cyclosporin A corrects mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies.

Ullrich congenital muscular dystrophy and Bethlem myopathy are skeletal muscle diseases that are due to mutations in the genes encoding collagen VI, an extracellular matrix protein forming a microfibrillar network that is particularly prominent in the endomysium of skeletal muscle. Myoblasts from patients affected by Ullrich congenital muscular dystrophy display functional and ultrastructural mitochondrial alterations and increased apoptosis due to inappropriate opening of the permeability transition pore, a mitochondrial inner membrane channel. These alterations could be normalized by treatment with cyclosporin A, a widely used immunosuppressant that desensitizes the permeability transition pore independently of calcineurin inhibition. Here, we report the results of an open pilot trial with cyclosporin A in five patients with collagen VI myopathies. Before treatment, all patients displayed mitochondrial dysfunction and increased frequency of apoptosis, as determined in muscle biopsies. Both of these pathologic signs were largely normalized after 1 month of oral cyclosporin A administration, which also increased muscle regeneration. These findings demonstrate that collagen VI myopathies can be effectively treated with drugs acting on the pathogenic mechanism downstream of the genetic lesion, and they represent an important proof of principle for the potential therapy of genetic diseases.