Absence of RAGE in an animal experimental model of Duchenne muscular dystrophy results in reduced muscle necrosis and inflammation

Duchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disorder characterized by progressive muscle degeneration due to lack of dystrophin, a protein essential for the integrity of sarcolemma during contraction. Chronic inflammation is a hallmark of muscles in DMD subjects, and contributes to progressive muscle wasting. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in physiological and pathological processes including inflammation and myogenesis [1]. While absent in healthy adult muscle tissue, RAGE is expressed in regenerating myofibers during muscle regeneration [2,3], in dystrophic muscles and activated immune cells. To have information about the role of RAGE in the pathophysiology of DMD we generated a double mutant mouse lacking dystrophin and RAGE (mdx/Ager–/– mouse) by cross-breeding dystrophic (mdx) mice with RAGE-null (Ager-/-) mice. Comparison of Quadriceps femoris of mdx and mdx/Ager–/– mice at different ages (i.e., 2, 3, 4 and 5 weeks, and 6 and 12 months of age) showed that the absence of RAGE in dystrophic mice did not affect the onset of the pathology. However, compared with age-matched mdx mice, muscles of 5 week- and 6 and 12 month-old mdx/Ager–/– mice showed i) significantly reduced numbers of necrotic myofibers, ii) a shift towards higher values of the cross-sectional areas (CSA) of myofibers, which was also evident in regenerating (centrally-nucleated) myofibers, and iii) reduced areas of immune cell infiltrate. The expression of MAC3, a marker of activated macrophages, was strongly reduced in muscles of mdx/Ager–/– mice compared with mdx mice. Moreover, muscles of mdx/Ager–/– mice exhibited significantly reduced PAX7+ve and myogenin+ve cell numbers, suggesting a reduced recruitment of muscle precursor cells and more efficient regeneration in dystrophic mice lacking RAGE. Our results suggest that RAGE may sustain inflammatory and degenerative processes in dystrophic muscles, and the inhibition of its expression/activity might represent a potential therapeutic approach in DMD patients.This work was supported by grants from MIUR 2012N8YJC3, AFM-Téléthon 16812 and Fondazione CRP 2015.0325.021

S100B protein regulates myoblast and macrophage functions in skeletal muscle regeneration

Regeneration of acutely injured skeletal muscles relies on a tightly controlled chain of cellular and molecular events, but a complete picture of factors concurring to the regeneration process is still missing. Extracellular S100B protein inhibits myoblast differentiation and stimulates myoblast proliferation by activating its canonical receptor, RAGE (receptor for advanced glycation endproducts), or bFGF/FGFR1 depending on myoblast density (1-4). S100B is released by damaged muscle tissue early after injury in advance of bFGF release, with declining release thereafter (4). We show that S100B is required for correct timing of skeletal muscle regeneration after acute injury. S100B expands the myoblast population, attracts macrophages to damage sites, promotes macrophage polarization into M2 (pro-regenerative) phenotype and reduces fibroblast proliferation. Also, S100B is transiently induced in and released by infiltrating macrophages under the action of proinflammatory and antiinflammatory cytokines, and effects of macrophage-derived S100B sum up with those of myofiber-released S100B. S100B’s effects are mediated by RAGE during the first 3 days after injury, however during the myoblast proliferation phase/macrophage M2 phase (i.e. at days 4-6 post-injury) S100B also activates bFGF-FGFR1 to stimulate myoblast proliferation and macrophage M1/M2 transition. Thus, S100B is a major molecular determinant of timed muscle regeneration after acute injury by virtue of its regulatory effects on myoblasts and macrophages.This work was supported by grants from MIUR PRIN-2010R8JK2X_004, AFM-Téléthon 16260 and Fondazione CRP 2012.0241.021

Transplantation of microencapsulated Sertoli cells in a mouse model of Duchenne muscular dystrophy (DMD) reduces inflammation and rescues muscle performance

Duchenne muscular dystrophy (DMD), a progressive muscle degenerative disease associated with chronic inflammation, necrosis and fibrosis, is currently treated with antiinflammatory steroids, despite their limited efficacy and undesired side effects. Testicular Sertoli cells (SCs) have been successfully implanted to treat many experimental diseases due to their ability to secrete trophic, antiinflammatory and immunomodulatory molecules (Mital et al., 2010). We transplanted microencapsulated SCs, within highly biocompatible microcapsules (Luca et al., 2007) into the peritoneal cavity of mdx mice, an animal model of DMD. Three weeks after transplantation, skeletal muscles from SC-treated mice, compared with muscles from mock-treated mice, showed: i) dramatically reduced number of infiltrated cells, including (MAC3+) macrophages; ii) a marked decrease in necrotic myofibers, and an increased number of regenerated (normally sized and centrally nucleated) myofibers; and, iii) a significant decrease in fibrous tissue infiltration. Moreover, SC-treated, but not mock-treated mdx mice showed recovery of muscle performance in treadmill endurance tests and a comparable resistance to exercise-induced muscle damage to that of untreated wildtype mice. These preliminary results suggest that our transplant product creates a suitable microenvironment for muscle regeneration and growth potentially applicable to DMD patients

Hyperactivated RAGE in Comorbidities as a Risk Factor for Severe COVID-19—The Role of RAGE-RAS Crosstalk

The receptor for advanced glycation-end products (RAGE) is a multiligand receptor with a role in inflammatory and pulmonary pathologies. Hyperactivation of RAGE by its ligands has been reported to sustain inflammation and oxidative stress in common comorbidities of severe COVID-19. RAGE is essential to the deleterious effects of the renin–angiotensin system (RAS), which participates in infection and multiorgan injury in COVID-19 patients. Thus, RAGE might be a major player in severe COVID-19, and appears to be a useful therapeutic molecular target in infections by SARS-CoV-2. The role of RAGE gene polymorphisms in predisposing patients to severe COVID-19 is discussed.&nbsp

RAGE in the pathophysiology of skeletal muscle

Abstract Emerging evidence suggests that the signalling of the Receptor for Advanced Glycation End products (RAGE) is critical for skeletal muscle physiology controlling both the activity of muscle precursors during skeletal muscle development and the correct time of muscle regeneration after acute injury. On the other hand, the aberrant re‐expression/activity of RAGE in adult skeletal muscle is a hallmark of muscle wasting that occurs in response to ageing, genetic disorders, inflammatory conditions, cancer, and metabolic alterations. In this review, we discuss the mechanisms of action and the ligands of RAGE involved in myoblast differentiation, muscle regeneration, and muscle pathological conditions. We highlight potential therapeutic strategies for targeting RAGE to improve skeletal muscle function

Employment of Microencapsulated Sertoli Cells as a New Tool to Treat Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked pathology due to lack of dystrophin and characterized by progressive muscle degeneration, impaired locomotion and premature death. The chronic presence of inflammatory cells, fibrosis and fat deposition are hallmarks of DMD muscle tissue. Many different therapeutic approaches to DMD have been tested, including cell-based and gene-based approaches, exon skipping, induction of expression of the dystrophin paralogue, utrophin, and, most recently the application of the CASPR/Cas9 genome editing system. However, corticosteroid treatment remains the gold standard therapy, even if corticosteroids have shown multiple undesirable side effects. Sertoli cells (SeC) have long been known for their ability to produce immunomodulatory and trophic factors, and have been used in a plethora of experimental models of disease. Recently, microencapsulated porcine SeC (MC-SeC) injected intraperitoneally in dystrophic mice produced morphological and functional benefits in muscles thanks to their release into the circulation of anti-inflammatory factors and heregulin β1, a known inducer of utrophin expression, thus opening a new avenue in the treatment of DMD. In order to stress the potentiality of the use of MC-SeC in the treatment of DMD, here, we examine the principal therapeutic approaches to DMD, and the properties of SeC (either nude or encapsulated into alginate-based microcapsules) and their preclinical and clinical use. Finally, we discuss the potential and future development of this latter approach

Equisetum arvense standardized dried extract hinders age-related osteosarcopenia

: Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ's anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia

KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK

The imbalance in osteoblast (OB)-dependent bone formation in favor of osteoclast (OC)-dependent bone resorption is the main cause of loss of tissue mineral mass during bone remodeling leading to osteoporosis conditions. Thus, the suppression of OC activity together with the improvement in the OB activity has been proposed as an effective therapy for maintaining bone mass during aging. We tested the new dietary product, KYMASIN UP containing standardized Withania somnifera, Silybum marianum and Trigonella foenum-graecum herbal extracts or the single extracts in in vitro models mimicking osteoclastogenesis (i.e., RAW 264.7 cells treated with RANKL, receptor activator of nuclear factor kappa-Beta ligand) and OB differentiation (i.e., C2C12 myoblasts treated with BMP2, bone morphogenetic protein 2). We found that the dietary product reduces RANKL-dependent TRAP (tartrate-resistant acid phosphatase)-positive cells (i.e., OCs) formation and TRAP activity, and down-regulates osteoclastogenic markers by reducing Src (non-receptor tyrosine kinase) and p38 MAPK (mitogen-activated protein kinase) activation. Withania somnifera appears as the main extract responsible for the anti-osteoclastogenic effect of the product. Moreover, KYMASIN UP maintains a physiological release of the soluble decoy receptor for RANKL, OPG (osteoprotegerin), in osteoporotic conditions and increases calcium mineralization in C2C12-derived OBs. Interestingly, KYMASIN UP induces differentiation in human primary OB-like cells derived from osteoporotic subjects. Based on our results, KYMASIN UP or Withania somnifera-based dietary supplements might be suggested to reverse the age-related functional decline of bone tissue by re-balancing the activity of OBs and OCs, thus improving the quality of life in the elderly and reducing social and health-care costs