Myocardial hypothermia increases autophagic flux, mitochondrial mass and myocardial function after ischemia-reperfusion injury.

Animal studies have demonstrated beneficial effects of therapeutic hypothermia on myocardial function, yet exact mechanisms remain unclear. Impaired autophagy leads to heart failure and mitophagy is important for mitigating ischemia/reperfusion injury. This study aims to investigate whether the beneficial effects of therapeutic hypothermia are due to preserved autophagy and mitophagy. Under general anesthesia, the left anterior descending coronary artery of 19 female farm pigs was occluded for 90 minutes with consecutive reperfusion. 30 minutes after reperfusion, we performed pericardial irrigation with warm or cold saline for 60 minutes. Myocardial tissue analysis was performed one and four weeks after infarction. Therapeutic hypothermia induced a significant increase in autophagic flux, mitophagy, mitochondrial mass and function in the myocardium after infarction. Cell stress, apoptosis, inflammation as well as fibrosis were reduced, with significant preservation of systolic and diastolic function four weeks post infarction. We found similar biochemical changes in human samples undergoing open chest surgery under hypothermic conditions when compared to the warm. These results suggest that autophagic flux and mitophagy are important mechanisms implicated in cardiomyocyte recovery after myocardial infarction under hypothermic conditions. New therapeutic strategies targeting these pathways directly could lead to improvements in prevention of heart failure

PKC Theta Ablation Improves Healing in a Mouse Model of Muscular Dystrophy

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ−/−, where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ−/− mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells

Characterization of the PKCθ-dependent Immune Response in Muscular Dystrophy

Our previous observations showed that lack of PKCθ in mdx, greatly improved muscle maintenance, regeneration and strength. The observed phenotype was primarily due to lack of PKCθ in BM derived cells. PKCθ is currently proposed as a target to selectively manipulate T cell functions. Scid/mdx mice, which lack functional T and B-cells in the mdx background, present a milder dystrophic phenotype than the mdx, supporting the pathogenic role of T cells in mdx muscle. The aims we addressed were: (1) characterize the PKCθ-dependent immune response in the pathogenesis of muscular dystrophy in mdx, using mdx/θ-/- mice; (2) investigate the T-cell dependent contribution to the pathogenesis of muscular dystrophy, using Scid/mdx mice as recipient of PKCθ expressing or not expressing T cells; (3) investigate the T/macrophage cells interactions by using an in vitro system. We found that PKCθ depletion is sufficient to counteract dystrophic features progression, already from the early stages, decreasing muscle inflammation and preventing fibrotic tissue deposition. PKCθ depletion in mdx avoided an excessive accumulation of M1 or M2 macrophages in muscle, modifying macrophage balance. Wild type T cells reintroduced into Scid/mdx mice specifically invaded dystrophic muscle, co-localizing with infiltrate and surrounding necrotic fibers, thus worsening dystrophic phenotype. Exogenous wild type T cells increased M1 macrophages in muscle, cell infiltrate and fibrosis. PKCθ inhibition decreases T cells activation in vitro, increasing Treg differentiation. By contrast, PKCθ-/- T cells reintroduction into Scid/mdx mice did not worsen dystrophic phenotype, neither increasing cell infiltrate nor affecting M1/M2 balance. By co-culturing T cells and Raw 264.7 macrophage cell line we found that lack of PKCθ in T cells affects macrophage responsiveness to LPS or IL-4. By PKCθ inhibition in macrophages we found that PKCθ is necessary for MMP-9 activity, but not for iNOS expression, in response to LPS, while its inhibition increased CD206 and CD163 expression in response to IL-4. According to our studies T cells and macrophages are key players inmuscular dystrophy. Taken together these results suggest that lack of PKCθ in mdx may improve Treg activity in the inflammatory infiltrate, which, in turn, reduces M1 pro-inflammatory activity while enhancing M2 pro-healing activity, but avoiding accumulation of M2 pro-fibrotic macrophages in late stages, resulting in attenuation of dystrophic features. For its role in T cells, particularly in Treg, in macrophages and in T/macrophage cross-talk, PKCθ could be a good pharmacological target in immune disorders

Three-dimensional imaging technologies: a priority for the advancement of tissue engineering and a challenge for the imaging community

Tissue engineering/regenerative medicine (TERM) is an interdisciplinary field that applies the principle of engineering and life sciences to restore/replace damaged tissues/organs with in vitro artificially-created ones. Research on TERM quickly moves forward. Today newest technologies and discoveries, such as 3D-/bio-printing, allow in vitro fabrication of ex-novo made tissues/organs, opening the door to wide and probably never-ending application possibilities, from organ transplant to drug discovery, high content screening and replacement of laboratory animals. Imaging techniques are fundamental tools for the characterization of tissue engineering (TE) products at any stage, from biomaterial/scaffold to construct/organ analysis. Indeed, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular features, allowing three-dimensional (3D) and time-lapse in vivo analysis, in a non-destructive, quantitative, multidimensional analysis of TE constructs, to analyze their pre-implantation quality assessment and their fate after implantation. This review focuses on the newest developments in imaging technologies and applications in the context of requirements of the different steps of the TERM field, describing strengths and weaknesses of the current imaging approaches

Exploring the roles of MSCs in infections: focus on bacterial diseases

Despite human healthcare advances, some microorganisms continuously react evolving new survival strategies, choosing between a commensal fitness and a pathogenic attitude. Many opportunistic microbes are becoming an increasing cause of clinically evident infections while several renowned infectious diseases sustain a considerable number of deaths. Besides the primary and extensively investigated role of immune cells, other cell types are involved in the microbe-host interaction during infection. Interestingly, mesenchymal stem cells (MSCs), the current leading players in cell therapy approaches, have been suggested to contribute to tackling pathogens and modulating the host immune response. In this context, this review critically explores MSCs’ role in E. coli, S. aureus, and polymicrobial infections. Summarizing from various studies, in vitro and in vivo results support the mechanistic involvement of MSCs and their derivatives in fighting infection and in contributing to microbial spreading. Our work outlines the double face of MSCs during infection, disease, and sepsis, highlighting potential pitfalls in MSC-based therapy due to the MSCs’ susceptibility to pathogens’ weapons. We also identify potential targets to improve infection treatments, and propose the potential applications of MSCs for vaccine research

Targeting PKCθ in skeletal muscle and muscle diseases: good or bad?

Protein kinase C-theta (PKCθ) is a member of the novel calcium-indipendent protein kinase C (PKC) family, with a relatively selective tissue distribution. Most studies have focussed on its unique role in T lymphocyte activation and suggest that inhibition of PKC could represent a novel therapeutic approach in the treatment of chronic inflammation, autoimmunity and allograft rejection. However, considering that PKC is also expressed in other cell types, including skeletal muscle cells, it is important to understand its function in different tissues before proposing it as a molecular target for the treatment of immune mediated diseases. A number of studies have highlighted the role of PKC in mediating several intracellular pathways regulating muscle cell development, homeostasis and remodelling, although a comprehensive picture is still lacking. Moreover, we recently showed that lack of PKC in a mouse model of Duchenne Muscular Dystrophy ameliorates the progression of the disease. Here, we review new developments in our understanding of the involvement of PKC in intracellular mechanisms regulating skeletal muscle development, growth and maintenance under physiological conditions, and recent advances showing a hitherto unrecognized role of PKC in promoting muscular dystrophy

Lack of PKCθ in <i>mdx</i> mice reduces cell infiltrate in muscle.

<p>(<b>A</b>) Hematoxylin/Eosin staining of TA crysosections derived from 2 mo old <i>mdx</i> (<b>a, c</b>) and <i>mdx/θ^−/−</i> (<b>b, d</b>). The insets in <b>a</b> and <b>b</b> indicate the areas shown in <b>c</b> and <b>d</b>, respectively, at higher magnification; bar = 100 µm. (<b>B</b>) Myofiber variability coefficient in TA muscles derived from 2 mo old WT, <i>mdx</i> and <i>mdx/θ^−/−</i>, determined as described in the material and methods sections. (n = 3/genotype). (<b>C</b>) Percentage of centrally nucleated myofibers in TA muscles derived from 2 mo old <i>mdx</i> and <i>mdx/θ^−/−</i>, expressed as percentage over the total number of myofibers (n = 3/genotype). (<b>D</b>) Esterase histochemical staining of TA cryosections derived from 2 mo old <i>mdx</i> and <i>mdx/θ^−/−</i> mice, as indicated. Arrows indicate cell infiltrates, arrows indicate neuromuscular junctions. Bar = 200 µm (<b>E</b>) FACS analysis of CD45^+ve/Mac3^+ve mononucleated cells isolated from TA muscle derived from 2 mo old <i>mdx</i> and <i>mdx/θ^−/−</i> mice, as indicated, expressed as percentage of the total number of cells examined. The percentage of reduction in <i>mdx/θ^−/−</i>muscle, in respect to <i>mdx</i>, is also shown (n = 3/genotype). (<b>F</b>) Gel zymography of MMP9 activity in TA muscle derived from 2 mo old <i>mdx</i> and <i>mdx/θ^−/−</i> mice, as indicated; media collected from differentiating muscle cell cultures was used as positive control (+).</p

Lack of PKCθ in <i>mdx</i> mice reduces muscle degeneration.

<p>(<b>A</b>) Representative western blot analysis of total protein fraction of TA muscles derived from 2 mo old WT, PKCθ^−/−, <i>mdx</i> and <i>mdx/θ^−/−</i> mice, as indicated. The blot was incubated with the indicated primary antibodies. GAPDH expression level is shown in the bottom for equal loading. PKCθ activation in muscle derived from <i>mdx</i> (black bar) mice, expressed as fold induction in respect to WT (white bar, assumed as 1), is shown as the ratio of pPKCθ/PKCθ (right panel), as determined by densitometric analysis from three independent experiments (*<i>p<0.05</i>). (<b>B</b>) EBD uptake in diaphragm derived from 2 mo old <i>mdx</i> or <i>mdx/θ^−/−</i> mice, as indicated, shown under light (<b>a, c</b>) and epifluorescence (<b>b, d</b>) microscopy. (<b>C</b>) EBD uptake in TA muscle derived from 2 mo old <i>mdx</i> (<b>a</b>) or <i>mdx/θ^−/−</i> (<b>b</b>) mice, as indicated; immunofluorescence analysis of IgG accumulation in <i>mdx</i> (<b>c</b>) or <i>mdx/θ^−/−</i> (<b>d</b>) mice; bar = 200 µm. (<b>D</b>) Mononuclear cells accumulation, revealed as Hoechst staining, around single degenerating fiber, detected as EBD uptake (<b>a–b</b>) and IgG immunofluorescence (<b>c–d</b>), in TA muscles from <i>mdx</i> (<b>a</b> and <b>c</b>) or <i>mdx/θ^−/−</i> (<b>b</b> and <b>d</b>). (<b>E</b>) Representative western Blot analysis of IgG accumulation in TA muscles from <i>mdx</i> or <i>mdx/θ^−/−</i> (two mice/genotype), as indicated. Densitometric analysis is shown in the bottom (<i>mdx</i>, black bar; <i>mdx/θ^−/−</i>, grey bar, *<i>p<0.05</i>).</p

Lack of PKCθ in <i>mdx</i> mice improves muscle regeneration.

<p>(<b>A</b>) eMyHC immunofluorescence (green) in TA cryosections derived from <i>mdx</i> (<b>a</b> and <b>d</b>) and <i>mdx/θ^−/−</i> (<b>b</b> and <b>e</b>) mice, as indicated. Merge with EBD uptake (red) is shown in <b>d</b> (<i>mdx</i>) and <b>e</b> (.<i>mdx/θ^−/−</i>). Bar = 200 µm. Extension of regenerating, eMyHC^+ve, area (<b>c</b>) and of necrotic, EBD^+ve, area (<b>f</b>) in <i>mdx/θ^−/−</i>, expressed as the percentage in respect to the respective areas in <i>mdx</i> (assumed as 1); *<i>p<0.01</i>, n = 3/genotype. Bar = 200 µm. (<b>B</b>) Representative Western Blot analysis of total protein fraction of TA muscles derived from 2 mo old WT, <i>mdx</i> and <i>mdx/θ^−/−</i> mice, as indicated, incubated with the α-myogenin antibody; Red Ponceau staining of the membrane is shown for equal loading. Up-regulation of myogenin expression in <i>mdx/θ^−/−</i>, in respect to <i>mdx</i> (assumed as 1), muscles, as determined by densitometric analysis of three independent experiments is shown in the bottom (n = 3/genotype) (<b>C</b>) Representative Wright staining of <i>mdx</i>- and <i>mdx/θ^−/−</i>- muscle derived cells, as indicated, cultured in DM for 48 hrs. The mean number of nuclei contained within each myotube is shown, as well as the percentage of reduction in <i>mdx/θ^−/−</i> in respect to <i>mdx</i>, as determined from three independent experiments.</p

Rescue of PKCθ expression in hematopoietic cells in <i>mdx/θ^−/−</i> mice restores <i>mdx</i> mice phenotype.

<p>(<b>A</b>) H/E (<b>a</b> and <b>e</b>) and esterase (<b>b</b> and <b>f</b>, arrows indicate cell infiltrates, asterisks indicate neuromuscular junctions) as well as EBD uptake (<b>c</b> and <b>g</b>) and eMyHC immunofluorescence (<b>d</b> and <b>h</b>, Hoechst was used to counterstain the nuclei) of TA cryosections derived from <i>mdx/θ^{−/−BMmdx/θ−/−}</i> (<b>a–d</b>) and from <i>mdx/θ^−/−BMmdx</i> (<b>e–h</b>) mice. Bar = 200 µm. (<b>B</b>) Extension of regenerating, eMyHC^+ve, area and of necrotic, EBD^+ve, area in <i>mdx/θ^{−/−BMmdx/θ−/−}</i> and in <i>mdx/θ^−/−BMmdx</i> expressed as the percentage in respect to the respective areas in <i>mdx</i> (assumed as 1), *<i>p<0.05</i>, n = 3/genotype; (<b>C</b>) Western Blot analysis of iNOS expression, and of NFkB and JNK expression and activation (phosphorylation) in protein extracts from TA muscle derived from <i>mdx/θ^{−/−BMmdx/θ−/−}</i> and from <i>mdx/θ^−/−BMmdx</i>; red Ponceau staining of the membrane is shown (loading) for equal loading. Representative blots are shown. The level of expression or of the activation of the above molecules was determined by densitometric analysis and expressed as fold induction in <i>mdx/θ^−/−BMmdx</i> in respect to <i>mdx/θ^{−/−BMmdx/θ−/−}</i>, assumed as 1, evaluated from 3 separate experiments (bottom). *<i>p<0.05</i> (<b>D</b>) Treadmill exercise test performed on <i>mdx/θ^{−/−BMmdx/θ−/−}</i> (squares, dotted grey line) and <i>mdx/θ^−/−BMmdx</i> (circles, grey.line) mice for a 30 min running, twice a week, for 3 weeks, as above, starting 6 weeks after transplantation (n = 3/genotype). Performance of <i>mdx</i> (triangles, black line) and <i>mdx/θ^−/−</i> (rhombi, dotted black line) mice is also reported for comparison.</p