Role of collagen VI in skeletal muscle regeneration and intestinal homeostasis

During my PhD I focused on the role of collagen VI in skeletal muscle regeneration and in intestinal homeostasis. Collagen VI is a glycoprotein of the extracellular matrix (ECM) containing three genetically distinct chains and forming an extended microfilamentous network that interacts with the cells and with other ECM components. Mutations of collagen VI genes in humans cause several muscle diseases, including Bethlem myopathy and Ullrich congenital muscle dystrophy. The generation of a collagen VI knockout mouse model was fundamental for clarifying the pathomolecular defects caused by the absence of this protein and provided a valuable tool for developing novel therapeutic opportunities in patients. During the past decade, studies on collagen VI null mice revealed an increasingly important role for this ECM component in a plethora of different cell and tissue processes, including apoptosis and oxidative damage, autophagy, cell differentiation, maintenance of stemness, regulation of tissue regeneration and biomechanical properties. In the first part of my PhD work, I was involved in a large project aimed at characterizing the role of collagen VI in muscle regeneration and in the regulation of the activities of satellite cells (SCs), the main adult stem cell population of skeletal muscles. Our studies revealed that collagen VI is a key component of the SC niche, and its lack affects muscle regeneration and impairs SC self-renewal in collagen VI null mice. Interestingly, we found that the absence of collagen VI affects the in vivo mechanical properties of skeletal muscles. Furthermore, in vitro studies revealed that SC stemness and regenerative capabilities are strongly compromised when SCs are cultured on biomimetic substrates with the abnormal stiffness displayed by collagen VI deficient muscles, compared to SCs cultured on biomimetic substrates with the normal muscle stiffness. Both the biomechanical properties value and the regenerative capability of collagen VI null muscle are improved after in vivo grafting with wild-type muscle fibroblasts, the main cell type producing collagen VI, thus pointing out that a key mechanism by which collagen VI regulates SC activity is via modulation of muscle mechanical properties. In a subsequent work, we found that pharmacological treatment of collagen VI null mice with cyclosporin A is able to stimulate myogenesis in physiological conditions by increasing the percentage of regenerating myofibers, and to improve muscle regeneration and SC homeostasis after cardiotoxin-induced injury. In the second part of my PhD, I investigated the role of collagen VI in intestine. Despite its broad distribution within the mucosa and the smooth muscle layers of the digestive tract, lack of collagen VI does not trigger any gross abnormality in the intestinal architecture of knockout mice. However, studies of the gastrointestinal functionality revealed that the lack of this ECM component lead to increased motility and decreased paracellular permeability. Induction of experimental acute colitis by administration of dextran sodium sulphate showed that collagen VI deficient mice have a decreased responsiveness and severity to acute colitis, with a lower body weight loss and decreased colonic inflammation when compared to wild-type subjected to the same treatment. Moreover, wild-type mice displayed an increased recruitment of inflammatory cells, in association with increased macrophage number and neutrophil activity, which decreased during 10 days of recovery subsequent to acute colitis, thus allowing proper tissue repair. Conversely, collagen VI deficient mice were unable to efficiently turn off inflammation during post-colitis recovery, displaying a high number of pro-inflammatory M1 colonic macrophages, an increased neutrophil activity and a higher body weight loss when compared to wild-type. Moreover, lack of collagen VI affected both macrophage polarization and activity in physiological conditions and during mild inflammation. Further studies allowed to reveal that lack of collagen VI affects the behavior of intestinal macrophages, both in physiological conditions and during mild inflammation, whose activity is essential to ensure intestinal mucosa homeostasis. These findings point at a role for collagen VI as a chemoattractant during acute inflammation and in tissue regeneration in the subsequent recovery phase. Immunofluorescence studies revealed an increased deposition of collagen VI in the colonic mucosa during acute colitis, and the protein was found in contact with macrophages. Interestingly, ileal biopsies of Crohn’s disease patients displayed increased expression and deposition of collagen VI, in association with a high number of macrophages, suggesting that the dysregulation of this ECM component may play an active role in the onset and/or maintenance of inflammatory bowel diseases. In conclusion, my PhD work provided novel information on the in vivo roles of collagen VI in cell and tissue homeostasis. In more general terms, these findings highlight the importance of a specific defined ECM microenvironment to ensure tissue homeostasis, and demonstrate that lack of one of the major ECM component may have a severe impact on cell behavior.Durante il mio percorso di dottorato mi sono occupata di studiare il ruolo del collagene VI nella rigenerazione del muscolo scheletrico e nell’omeostasi dell’intestino. Il collagene VI è una glicoproteina della matrice extracellulare (MEC) costituita da tre catene geneticamente distinte, le quali si organizzano in modo da formare un’estesa rete di microfilamenti in grado di connettere cellule e altri componenti della MEC. Mutazioni a carico dei geni codificanti le catene del collagene VI causano diverse patologie muscolari, principalmente la miopatia di Bethlem e la distrofia muscolare congenita di Ullrich. Gli studi condotti sul modello knockout murino privo di collagene VI hanno permesso di chiarire i difetti patomolecolari causati dall’assenza di questa proteina, dimostrandosi utile anche per l’identificazione di nuovi trattamenti farmacologici per le malattie umane. Nel coso degli anni, molteplici studi hanno messo in luce le diverse funzioni esercitate dal collagene VI nel regolare diversi eventi cellulari e tissutali, tra cui l’apoptosi e il danno ossidativo, l’autofagia, il differenziamento cellulare, il mantenimento della staminalità ai fini rigenerativi e le proprietà biomeccaniche. Nel corso del mio dottorato ho partecipato inizialmente alla caratterizzazione del ruolo del collagene VI durante la rigenerazione del muscolo scheletrico e la sua influenza sull’attività delle cellule satelliti, la popolazione principale di cellule staminali adulte nei muscoli scheletrici. Da tali studi è emerso che il collagene VI è un componente essenziale della nicchia delle cellule satelliti. La mancanza di tale proteina determina una ridotta rigenerazione tissutale e una diminuita capacità delle cellule satelliti di compiere self-renewal in seguito a danni muscolari multipli. I muscoli dei topi privi di collagene VI sono caratterizzati da una minore stiffness e approfondite analisi condotte in vitro hanno rivelato che le proprietà staminali e rigenerative delle cellule satelliti sono fortemente compromesse quando coltivate su biomateriali con un modulo elastico che mima la condizione patologica. Le capacità rigenerative e le proprietà meccaniche dei muscoli di topi privi di collagen VI vengono ripristinate in seguito alla deposizione di collagene VI, ristabilita tramite grafting di fibroblasti muscolari isolati da topi wild-type. Complessivamente, questi studi hanno dimostrato che modulando le proprietà meccaniche del muscolo, il collagene VI è in grado di regolare l’attività delle cellule satelliti. Abbiamo inoltre dimostrato che la somministrazione di ciclosporina A è in grado di stimolare la miogenesi in condizioni fisiologiche, inducendo la formazione di nuove fibre muscolari, e di migliorare la rigenerazione muscolare e l’omeostasi delle cellule satelliti in seguito a danni muscolari nei topi privi di collagene VI. Successivamente mi sono dedicata ad indagare il ruolo del collagene VI nell’omeostasi dell’intestino. Sebbene questa proteina sia ampiamente distribuita nella mucosa e nello strato muscolare, la sua assenza sembra non comportare alterazioni macroscopiche sull’architettura intestinale. L’analisi della funzionalità del sistema gastrointestinale ha evidenziato un’aumentata motilità e una ridotta permeabilità paracellulare in assenza di collagene VI. Esperimenti di induzione di colite acuta mediante sodio solfato destano hanno rivelato che i topi privi di collagene VI presentano una ridotta risposta e severità, associate ad una minore perdita di peso corporeo e minore infiammazione della mucosa del colon rispetto ai topi wild-type. Inoltre, durante la fase di colite acuta il reclutamento di cellule infiammatorie è risultato essere aumentato nei topi wild-type, comportando un aumento del numero di macrofagi e di attività dei neutrofili, mentre si riduce durante la fase di recupero seguente la colite acuta, favorendo la rigenerazione tissutale. Di contro, nei topi privi di collagene VI l’infiammazione è risultata essere ancora attiva durante la fase di recupero, con un elevato numero di macrofagi pro-infiammatori M1, un’alta attività dei neutrofili e un peggioramento della perdita di peso. Inoltre l’assenza di collagene VI ha dimostrato influenzare il comportamento dei macrofagi della mucosa del colon, sia in condizioni fisiologiche sia durante i primi giorni di infiammazione acuta, la cui attività è essenziale per assicurare l’omeostasi della mucosa intestinale. Nel complesso, da questi studi è emerso che il collagene VI esercita un ruolo da chemoattrattore per le cellule infiammatorie durante la fase di colite acuta, mentre nella successiva fase di risoluzione la sua presenza è necessaria nell’indurre una corretta rigenerazione tissutale. Studi di immunofluorescenza hanno inoltre rivelato nei topi wild-type un’elevata espressione di collagene VI in stretto contatto con i macrofagi della mucosa del colon durante la fase di colite acuta. L’evidenza di un’aumentata espressione di collagene VI su biopsia di ileo di paziente affetto da morbo di Crohn, associata ad un elevato numero di macrofagi rispetto al controllo sano, suggerisce un coinvolgimento di questo componente della MEC nel decorso delle malattie infiammatorie intestinali. In conclusione, le evidenze emerse in questo mio lavoro di tesi avvalorano l’importanza del ruolo della matrice extracellulare nell’omeostasi tissutale

Role of collagen VI in skeletal muscle regeneration and intestinal homeostasis

During my PhD I focused on the role of collagen VI in skeletal muscle regeneration and in intestinal homeostasis. Collagen VI is a glycoprotein of the extracellular matrix (ECM) containing three genetically distinct chains and forming an extended microfilamentous network that interacts with the cells and with other ECM components. Mutations of collagen VI genes in humans cause several muscle diseases, including Bethlem myopathy and Ullrich congenital muscle dystrophy. The generation of a collagen VI knockout mouse model was fundamental for clarifying the pathomolecular defects caused by the absence of this protein and provided a valuable tool for developing novel therapeutic opportunities in patients. During the past decade, studies on collagen VI null mice revealed an increasingly important role for this ECM component in a plethora of different cell and tissue processes, including apoptosis and oxidative damage, autophagy, cell differentiation, maintenance of stemness, regulation of tissue regeneration and biomechanical properties. In the first part of my PhD work, I was involved in a large project aimed at characterizing the role of collagen VI in muscle regeneration and in the regulation of the activities of satellite cells (SCs), the main adult stem cell population of skeletal muscles. Our studies revealed that collagen VI is a key component of the SC niche, and its lack affects muscle regeneration and impairs SC self-renewal in collagen VI null mice. Interestingly, we found that the absence of collagen VI affects the in vivo mechanical properties of skeletal muscles. Furthermore, in vitro studies revealed that SC stemness and regenerative capabilities are strongly compromised when SCs are cultured on biomimetic substrates with the abnormal stiffness displayed by collagen VI deficient muscles, compared to SCs cultured on biomimetic substrates with the normal muscle stiffness. Both the biomechanical properties value and the regenerative capability of collagen VI null muscle are improved after in vivo grafting with wild-type muscle fibroblasts, the main cell type producing collagen VI, thus pointing out that a key mechanism by which collagen VI regulates SC activity is via modulation of muscle mechanical properties. In a subsequent work, we found that pharmacological treatment of collagen VI null mice with cyclosporin A is able to stimulate myogenesis in physiological conditions by increasing the percentage of regenerating myofibers, and to improve muscle regeneration and SC homeostasis after cardiotoxin-induced injury. In the second part of my PhD, I investigated the role of collagen VI in intestine. Despite its broad distribution within the mucosa and the smooth muscle layers of the digestive tract, lack of collagen VI does not trigger any gross abnormality in the intestinal architecture of knockout mice. However, studies of the gastrointestinal functionality revealed that the lack of this ECM component lead to increased motility and decreased paracellular permeability. Induction of experimental acute colitis by administration of dextran sodium sulphate showed that collagen VI deficient mice have a decreased responsiveness and severity to acute colitis, with a lower body weight loss and decreased colonic inflammation when compared to wild-type subjected to the same treatment. Moreover, wild-type mice displayed an increased recruitment of inflammatory cells, in association with increased macrophage number and neutrophil activity, which decreased during 10 days of recovery subsequent to acute colitis, thus allowing proper tissue repair. Conversely, collagen VI deficient mice were unable to efficiently turn off inflammation during post-colitis recovery, displaying a high number of pro-inflammatory M1 colonic macrophages, an increased neutrophil activity and a higher body weight loss when compared to wild-type. Moreover, lack of collagen VI affected both macrophage polarization and activity in physiological conditions and during mild inflammation. Further studies allowed to reveal that lack of collagen VI affects the behavior of intestinal macrophages, both in physiological conditions and during mild inflammation, whose activity is essential to ensure intestinal mucosa homeostasis. These findings point at a role for collagen VI as a chemoattractant during acute inflammation and in tissue regeneration in the subsequent recovery phase. Immunofluorescence studies revealed an increased deposition of collagen VI in the colonic mucosa during acute colitis, and the protein was found in contact with macrophages. Interestingly, ileal biopsies of Crohn’s disease patients displayed increased expression and deposition of collagen VI, in association with a high number of macrophages, suggesting that the dysregulation of this ECM component may play an active role in the onset and/or maintenance of inflammatory bowel diseases. In conclusion, my PhD work provided novel information on the in vivo roles of collagen VI in cell and tissue homeostasis. In more general terms, these findings highlight the importance of a specific defined ECM microenvironment to ensure tissue homeostasis, and demonstrate that lack of one of the major ECM component may have a severe impact on cell behavior

Cyclosporin A Promotes in vivo Myogenic Response in Collagen VI-Deficient Myopathic Mice.

Mutations of genes encoding for collagen VI cause various muscle diseases in humans, including Bethlem myopathy and Ullrich congenital muscular dystrophy. Collagen VI null (Col6a1 (-/-)) mice are affected by a myopathic phenotype with mitochondrial dysfunction, spontaneous apoptosis of muscle fibers, and defective autophagy. Moreover, Col6a1 (-/-) mice display impaired muscle regeneration and defective self-renewal of satellite cells after injury. Treatment with cyclosporin A (CsA) is effective in normalizing the mitochondrial, apoptotic, and autophagic defects of myofibers in Col6a1 (-/-) mice. A pilot clinical trial with CsA in Ullrich patients suggested that CsA may increase the number of regenerating myofibers. Here, we report the effects of CsA administration at 5\u2009mg/kg body weight every 12\u2009h in Col6a1 (-/-) mice on muscle regeneration under physiological conditions and after cardiotoxin (CdTx)-induced muscle injury. Our findings indicate that CsA influences satellite cell activity and triggers the formation of regenerating fibers in Col6a1 (-/-) mice. Data obtained on injured muscles show that under appropriate administration, regimens CsA is able to stimulate myogenesis in Col6a1 (-/-) mice by significantly increasing the number of myogenin (MyoG)-positive cells and of regenerating myofibers at the early stages of muscle regeneration. CsA is also able to ameliorate muscle regeneration of Col6a1 (-/-) mice subjected to multiple CdTx injuries, with a concurrent maintenance of the satellite cell pool. Our data show that CsA is beneficial for muscle regeneration in Col6a1 (-/-) mic

Conjugates of oligonucleotides and bile acids and their derivatives for pharmaceutical active molecules delivery

The present invention refers to conjugates of bile acids or their derivatives with oligonucleotides, in particular for the treatment of Duchenne muscular dystrophy

Collagen VI null mice as a model for early onset muscle decline in aging

Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null (Col6a1−/−) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1−/− mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1−/− mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1−/− mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1−/− mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1−/− diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1−/− gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B light chain 3B (LC3B) lipidation are hallmarks of the aging process. Altogether these data indicate that the diaphragm of Col6a1−/− animal model can be considered as a model of early skeletal muscle aging

Collagen VI regulates satellite cell self-renewal and muscle regeneration.

Adult muscle stem cells, or satellite cells play essential roles in homeostasis and regeneration of skeletal muscles. Satellite cells are located within a niche that includes myofibers and extracellular matrix. The function of specific extracellular matrix molecules in regulating SCs is poorly understood. Here we show that the extracellular matrix protein collagen VI is a key component of the satellite cell niche. Lack of collagen VI in Col6a1(–/–) mice causes impaired muscle regeneration and reduced satellite cell self-renewal capability after injury. Collagen VI null muscles display significant decrease of stiffness, which is able to compromise the in vitro and in vivo activity of wild-type satellite cells. When collagen VI is reinstated in vivo by grafting wild-type fibroblasts, the biomechanical properties of Col6a1(–/–) muscles are ameliorated and satellite cell defects rescued. Our findings establish a critical role for an extracellular matrix molecule in satellite cell self-renewal and open new venues for therapies of collagen VI-related muscle diseases

Glycolytic-to-oxidative fiber-type switch and mTOR signaling activation are early-onset features of SBMA muscle modified by high-fat diet

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The mechanism by which expansion of polyglutamine in AR causes muscle atrophy is unknown. Here, we investigated pathological pathways underlying muscle atrophy in SBMA knock-in mice and patients. We show that glycolytic muscles were more severely affected than oxidative muscles in SBMA knock-in mice. Muscle atrophy was associated with early-onset, progressive glycolytic-to-oxidative fiber-type switch. Whole genome microarray and untargeted lipidomic analyses revealed enhanced lipid metabolism and impaired glycolysis selectively in muscle. These metabolic changes occurred before denervation and were associated with a concurrent enhancement of mechanistic target of rapamycin (mTOR) signaling, which induced peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1 alpha) expression. At later stages of disease, we detected mitochondrial membrane depolarization, enhanced transcription factor EB (TFEB) expression and autophagy, and mTOR-induced protein synthesis. Several of these abnormalities were detected in the muscle of SBMA patients. Feeding knock-in mice a high-fat diet (HFD) restored mTOR activation, decreased the expression of PGC1 alpha, TFEB, and genes involved in oxidative metabolism, reduced mitochondrial abnormalities, ameliorated muscle pathology, and extended survival. These findings show early-onset and intrinsic metabolic alterations in SBMA muscle and link lipid/glucose metabolism to pathogenesis. Moreover, our results highlight an HFD regime as a promising approach to support SBMA patients