Diabetic Cardiomyopathy: Role of Cell Death, Exosomes, Fibrosis and Epicardial Adipose Tissue.

Diabetic cardiomyopathy (DCM) represents one of the typical complications associated with diabetes. It has been described as anomalies in heart function and structure, with consequent high morbidity and mortality. DCM development can be described by two stages; the first is characterized by left ventricular hypertrophy and diastolic dysfunction, and the second by heart failure (HF) with systolic dysfunction. The proposed mechanisms involve cardiac inflammation, advanced glycation end products (AGEs) and angiotensin II. Furthermore, different studies have focused their attention on cardiomyocyte death through the different mechanisms of programmed cell death, such as apoptosis, autophagy, necrosis, pyroptosis and ferroptosis. Exosome release, adipose epicardial tissue and aquaporins affect DCM development. This review will focus on the description of the mechanisms involved in DCM progression and development.

Alternative therapeutic strategies in diabetes management.

Diabetes is a heterogeneous metabolic disease characterized by elevated blood glucose levels resulting from the destruction or malfunction of pancreatic ß cells, insulin resistance in peripheral tissues, or both, and results in a non-sufficient production of insulin. To adjust blood glucose levels, diabetic patients need exogenous insulin administration together with medical nutrition therapy and physical activity. With the aim of improving insulin availability in diabetic patients as well as ameliorating diabetes comorbidities, different strategies have been investigated. The first approaches included enhancing endogenous ß cell activity or transplanting new islets. The protocol for this kind of intervention has recently been optimized, leading to standardized procedures. It is indicated for diabetic patients with severe hypoglycemia, complicated by impaired hypoglycemia awareness or exacerbated glycemic lability. Transplantation has been associated with improvement in all comorbidities associated with diabetes, quality of life, and survival. However, different trials are ongoing to further improve the beneficial effects of transplantation. Furthermore, to overcome some limitations associated with the availability of islets/pancreas, alternative therapeutic strategies are under evaluation, such as the use of mesenchymal stem cells (MSCs) or induced pluripotent stem cells for transplantation. The cotransplantation of MSCs with islets has been successful, thus providing protection against proinflammatory cytokines and hypoxia through different mechanisms, including exosome release. The use of induced pluripotent stem cells is recent and requires further investigation. The advantages of MSC implantation have also included the improvement of diabetes-related comorbidities, such as wound healing. Despite the number of advantages of the direct injection of MSCs, new strategies involving biomaterials and scaffolds have been developed to improve the efficacy of mesenchymal cell delivery with promising results. In conclusion, this paper offered an overview of new alternative strategies for diabetes management while highlighting some limitations that will need to be overcome by future approaches.

Irisin Modulates Inflammatory, Angiogenic, and Osteogenic Factors during Fracture Healing.

Bone fractures are a widespread clinical event due to accidental falls and trauma or bone fragility; they also occur in association with various diseases and are common with aging. In the search for new therapeutic strategies, a crucial link between irisin and bone fractures has recently emerged. To explore this issue, we subjected 8-week-old C57BL/6 male mice to tibial fracture, and then we treated them with intra-peritoneal injection of r-Irisin (100 µg/kg/weekly) or vehicle as control. At day 10 post fracture, histological analysis showed a significant reduced expression of inflammatory cytokines as tumor necrosis factor-alpha (TNFα) (p = 0.004) and macrophage inflammatory protein-alpha (MIP-1α) (p = 0.015) in the cartilaginous callus of irisin-treated mice compared to controls, supporting irisin's anti-inflammatory role. We also found increased expressions of the pro-angiogenic molecule vascular endothelial growth factor (VEGF) (p = 0.002) and the metalloproteinase MMP-13 (p = 0.0006) in the irisin-treated mice compared to the vehicle ones, suggesting a myokine involvement in angiogenesis and cartilage matrix degradation processes. Moreover, the bone morphogenetic protein (BMP2) expression was also upregulated (p = 0.002). Taken together, our findings suggest that irisin can contribute to fracture repair by reducing inflammation and promoting vessel invasion, matrix degradation, and bone formation, supporting its possible role as a novel molecule for fracture treatment.

Cellular and Molecular Mechanisms Activated by a Left Ventricular Assist Device.

Left ventricular assist devices (LVADs) represent the final treatment for patients with end-stage heart failure (HF) not eligible for transplantation. Although LVAD design has been further improved in the last decade, their use is associated with different complications. Specifically, inflammation, fibrosis, bleeding events, right ventricular failure, and aortic valve regurgitation may occur. In addition, reverse remodeling is associated with substantial cellular and molecular changes of the failing myocardium during LVAD support with positive effects on patients' health. All these processes also lead to the identification of biomarkers identifying LVAD patients as having an augmented risk of developing associated adverse events, thus highlighting the possibility of identifying new therapeutic targets. Additionally, it has been reported that LVAD complications could cause or exacerbate a state of malnutrition, suggesting that, with an adjustment in nutrition, the general health of these patients could be improved.

Antidepressant Effect of Intermittent Long-Term Systemic Administration of Irisin in Mice.

Depression is a psychiatric disorder increasingly diffused worldwide. Evidence suggests that irisin, a myokine secreted by contracting muscle, mediates beneficial effects on several targets, including the brain. Here, the potential antidepressant properties of long-term intermittent systemic irisin administration (100 µg/kg/weekly for 1 month) were evaluated in mice by the Tail Suspension Test (TST), Forced Swim Test (FST), and Open Field Test (OFT). Furthermore, to deepen the molecular pathways underlying irisin treatment, the expression of irisin precursor, neurotrophic/growth factors, and cytokines was analyzed. Irisin treatment significantly decreased the immobility time in the TST and FST, suggesting an antidepressant effect. Additionally, irisin seemed to display an anxiolytic-like effect increasing the time spent in the OFT arena center. These findings were probably due to the modulation of endogenous brain factors as the gene expression of some neurotrophins, such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1), was upregulated only in irisin-treated mouse brain. Moreover, irisin modulated the expression of some cytokines (IL-1ß, IL-4, IL-6, and IL-10). To the best of our knowledge, this is the first study demonstrating that the irisin antidepressant effect may be observed even with a systemic administration in mice. This could pave the way toward intriguing preclinical research in humans.

Systemic Administration of Recombinant Irisin Accelerates Fracture Healing in Mice.

To date, pharmacological strategies designed to accelerate bone fracture healing are lacking. We subjected 8-week-old C57BL/6 male mice to closed, transverse, mid-diaphyseal tibial fractures and treated them with intraperitoneal injection of a vehicle or r-irisin (100 µg/kg/weekly) immediately following fracture for 10 days or 28 days. Histological analysis of the cartilaginous callus at 10 days showed a threefold increase in Collagen Type X (p = 0.0012) and a reduced content of proteoglycans (40%; p = 0.0018). Osteoclast count within the callus showed a 2.4-fold increase compared with untreated mice (p = 0.026), indicating a more advanced stage of endochondral ossification of the callus during the early stage of fracture repair. Further evidence that irisin induced the transition of cartilage callus into bony callus was provided by a twofold reduction in the expression of SOX9 (p = 0.0058) and a 2.2-fold increase in RUNX2 (p = 0.0137). Twenty-eight days post-fracture, microCT analyses showed that total callus volume and bone volume were increased by 68% (p = 0.0003) and 67% (p = 0.0093), respectively, and bone mineral content was 74% higher (p = 0.0012) in irisin-treated mice than in controls. Our findings suggest that irisin promotes bone formation in the bony callus and accelerates the fracture repair process, suggesting a possible use as a novel pharmacologic modulator of fracture healing.

The Novel Role of PGC1α in Bone Metabolism.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is a protein that promotes transcription of numerous genes, particularly those responsible for the regulation of mitochondrial biogenesis. Evidence for a key role of PGC1α in bone metabolism is very recent. In vivo studies showed that PGC1α deletion negatively affects cortical thickness, trabecular organization and resistance to flexion, resulting in increased risk of fracture. Furthermore, in a mouse model of bone disease, PGC1α activation stimulates osteoblastic gene expression and inhibits atrogene transcription. PGC1α overexpression positively affects the activity of Sirtuin 3, a mitochondrial nicotinammide adenina dinucleotide (NAD)-dependent deacetylase, on osteoblastic differentiation. In vitro, PGC1α overexpression prevents the reduction of mitochondrial density, membrane potential and alkaline phosphatase activity caused by Sirtuin 3 knockdown in osteoblasts. Moreover, PGC1α influences the commitment of skeletal stem cells towards an osteogenic lineage, while negatively affects marrow adipose tissue accumulation. In this review, we will focus on recent findings about PGC1α action on bone metabolism, in vivo and in vitro, and in pathologies that cause bone loss, such as osteoporosis and type 2 diabetes.

FNDC5/Irisin System in Neuroinflammation and Neurodegenerative Diseases: Update and Novel Perspective.

Irisin, the circulating peptide originating from fibronectin type III domain-containing protein 5 (FNDC5), is mainly expressed by muscle fibers under peroxisome proliferator-activated receptor gamma coactivator 1-alpha PGC1α control during exercise. In addition to several beneficial effects on health, physical activity positively affects nervous system functioning, particularly the hippocampus, resulting in amelioration of cognition impairments. Recently, FNDC5/irisin detection in hippocampal neurons and the presence of irisin in the cerebrospinal fluid opened a new intriguing chapter in irisin history. Interestingly, in the hippocampus of mice, exercise increases FNDC5 levels and upregulates brain-derived neurotrophic factor (BDNF) expression. BDNF, displaying neuroprotection and anti-inflammatory effects, is mainly produced by microglia and astrocytes. In this review, we discuss how these glial cells can morphologically and functionally switch during neuroinflammation by modulating the expression of a plethora of neuroprotective or neurotoxic factors. We also focus on studies investigating the irisin role in neurodegenerative diseases (ND). The emerging involvement of irisin as a mediator of the multiple positive effects of exercise on the brain needs further studies to better deepen this issue and the potential use in therapeutic approaches for neuroinflammation and ND.

The effect of Irisin on bone cells in vivo and in vitro.

The myokine Irisin, produced during physical exercise, has an anabolic effect on bone, both in vitro and in vivo. Very recently, using a controlled in vitro 3D cell model to mimic the bone microenvironment aboard the International Space Station, it has been shown that Irisin treatment in microgravity prevents the down-regulation of the transcription factors Atf4, Runx2 and Osterix, as well as Collagen I and Osteoprotegerin proteins, crucial for osteoblast differentiation in physiologic conditions. Irisin action has also been investigated in human subjects, in which it correlates with bone health status, supporting its physiological importance also in human bone, both in healthy subjects and in patients suffering from diseases related to bone metabolism, such as hyperparathyroidism and type 1 diabetes. Low levels of circulating Irisin have been found in post-menopausal women affected by hyperparathyroidism. Furthermore, Irisin is positively correlated with bone strength in athletes and bone mineral density in football players. Moreover, in healthy children, Irisin is positively associated with bone mineral status and in children with type 1 diabetes, Irisin is positively correlated with improved glycemic control and skeletal health. In this review, we will focus on recent findings about Irisin action on microgravity induced bone loss and on osteocyte activity and survival through its αV/ß5 integrin receptor.

AQP4ex is crucial for the anchoring of AQP4 at the astrocyte end-feet and for neuromyelitis optica antibody binding.

Brain water homeostasis is essential for the appropriate control of neuronal activity. Furthermore, the encasement of the central nervous system (CNS) by a hard structure, greatly limits its tolerance for the volume changes occurring with acute brain edema, which quickly leads to severe damage or death.The recent discovery of the extended isoform of AQP4 (AQP4ex), generated by translational readthrough, revealed a potential new mechanism of water transport regulation and polarization at the blood-brain-barrier level.In the present study we used CRISPR/Cas9 technology to generate an AQP4ex-/- mouse model and evaluate the effect on the overall AQP4 expression, polarization, supramolecular organization in orthogonal arrays of particles (OAPs) and neuromyelitis optica (NMO-IgG) autoantibodies binding.AQP4ex removal did not cause a decrease in total AQP4 protein expression but completely suppressed the specific location of AQP4 at the astrocyte endfeet. Without AQP4ex, AQP4 was mislocalized and α-syntrophin expression, the selective partner for AQP4 localization, was partially altered. The supramolecular organization of AQP4 in OAPs was subtly altered. Indeed, the absence of AQP4ex reduced the size of AQP4-OAPs but the number of AQP4-OAP pools remained largely the same. More importantly, AQP4ex resulted critical for the binding of pathogenic human NMO-IgG autoantibodies to the brain. Indeed, the absence of AQP4ex completely abolished the binding of NMO-IgG at the perivascular astrocyte endfeet.This study provides the first direct evidence in vivo on the specific role of AQP4ex in AQP4 perivascular OAPs assembly and confinement and reveals AQP4ex as new and important player in neuromyelitis optica.

Supramolecular aggregation of aquaporin-4 is different in muscle and brain: correlation with tissue susceptibility in neuromyelitis optica.

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by autoantibodies (NMO-IgG) against the water channel aquaporin-4 (AQP4). Though AQP4 is also expressed outside the CNS, for example in skeletal muscle, patients with NMO generally do not show clinical/diagnostic evidence of skeletal muscle damage. Here, we have evaluated whether AQP4 supramolecular organization is at the basis of the different tissue susceptibility. Using immunofluorescence we found that while the sera of our cohort of patients with NMO gave typical perivascular staining in the CNS, they were largely negative in the skeletal muscle. This conclusion was obtained using human, rat and mouse skeletal muscle including the AQP4-KO mouse. A biochemical analysis using a new size exclusion chromatography approach for AQP4 suprastructure fractionation revealed substantial differences in supramolecular AQP4 assemblies and isoform abundance between brain and skeletal muscle matching a lower binding affinity of NMO-IgG to muscle compared to the brain. Super-resolution microscopy analysis with g-STED revealed different AQP4 organization in native tissues, while in the brain perivascular astrocyte endfoot membrane AQP4 was mainly organized in large interconnected and raft-like clusters, in the sarcolemma of fast-twitch fibres AQP4 aggregates often appeared as small, relatively isolated linear entities. In conclusion, our results provide evidence that AQP4 supramolecular structure is different in brain and skeletal muscle, which is likely to result in different tissues susceptibility to the NMO disease.

Translational readthrough generates new astrocyte AQP4 isoforms that modulate supramolecular clustering, glial endfeet localization, and water transport.

Regulation of water homeostasis is a central feature of central nervous system pathophysiology. In this context, several lines of evidence suggest a crucial role for the water channel aquaporin-4 (AQP4) and its plasma membrane supramolecular organization as the key element. Here, we demonstrate the expression in tissues of additional isoforms of AQP4 characterized by a C-terminal extension generated by programmed translational readthrough. These extended isoforms (AQP4ex) display a perivascular polarization and expression in dystrophin-dependent pools. AQP4ex reduces supramolecular clustering tendency and allows AQP4 interactions with syntrophin. Furthermore, site-directed mutagenesis of two serines in the extended C-terminus of AQP4ex showed potential regulation of water permeability by phosphorylation. Finally, AQP4ex expression can be positively modulated by gentamicin treatment, demonstrating the possibility of regulating the AQP4 translational readthrough frequency. This novel regulatory mechanism could have important pathophysiological implications for conditions in which alternations have been reported in AQP4 structure.

The effects of bone pâté on human osteoblasts cell cultures.

The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone paté has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone paté taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone patè, with bone paté mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone paté (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone paté alone and bone pate mixed with fibrin glue. Treatment with bone patè upregulated transcription factors and components of the extracellular matrix. The present data show that bone paté has a high osteoinductive potential on human osteoblasts, enhancing their activity.

The myokine irisin increases cortical bone mass.

It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg(-1). We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg(-1) per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, ß-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle-bone connectivity.