Relaxin prevents cardiac fibroblast-myofibroblast transition via Notch-1-mediated Inhibition of TGF-β/Smad3 signaling

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects

Bone marrow mesenchymal stromal cells stimulate skeletal myoblast proliferation through the paracrine release of VEGF.

Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies

Relaxin antagonizes the effects of TGF-β/Smad3 axis on cardiac fibroblast-myofibroblast transition through the upregulation of Notch-1 signalling

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-b1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate a-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-b1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed,Notch-1 expression was significantly decreased in TGF-b1-stimulated fibroblasts and this reduction was prevented by the addition of RLX to TGF-b1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by DAPT, a g-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-b1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-b1 signaling, since the addition of RLX to TGF-b1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-b1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-b1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects

Mesenchymal stromal cells stimulate C2C12 myoblast cell proliferation: potential relevance in skeletal muscle regeneration

Skeletal muscle tissue harbors of a population of resident myoblastic stem cells which are capable of regenerating the damaged tissue. However, in case of extended injury, their myogenic potential is compromised by excessive inflammatory response and collagen deposition which hamper the environmental conducivity to muscle regeneration. Therefore, the identification of new strategies aimed at potentiating the proliferative attitude of myoblasts may have potential therapeutic application for muscle repair. In this line, we have previously demonstrated in a co-culture system, that adult mouse bone marrow-derived mesenchymal stromal/stem cells (MSCs) stimulate mouse neonatal cardiomyocyte proliferation. On these premises, in the present study, we searched whether MSCs were also able to influence C2C12 myoblast cells proliferation. We found that myoblast proliferation was significantly enhanced in co-culture as compared with the single culture, and that this response involved the activation of Notch-1 signalling in the myoblastic cells. These data were confirmed by the finding that Hes1 transcriptional regulator, the major downstream effector of Notch-1, was also upregulated in C2C12 cells cultured in the presence of MSCs or their derived conditioned medium. In particular, MSCs were able to release growth factors, including FGF and VEGF, thus underscoring potential paracrine mechanisms involved in the regulation of myoblast proliferation and Notch-1 expression by MSCs. In conclusion, the results of the present study provide strong evidence for a role of MSCs in stimulating myoblast cell proliferation and suggest that the functional interaction betweeen the two cell types may be exploited to the development of new and more efficient cell-based skeletal muscle repair strategies

Effect of Red Mud Addition on Electrical and Magnetic Properties of Hemp-Derived-Biochar-Containing Epoxy Composites

Waste stream valorization is a difficult task where the economic and environmental issues must be balanced. The use of complex metal-rich waste such as red mud is challenging due to the wide variety of metal oxides present such as iron, aluminum, and titanium. The simple separation of each metal is not economically feasible, so alternative routes must be implemented. In this study, we investigated the use of red mud mixed with hemp waste to produce biochar with high conductivity and good magnetic properties induced by the reduction of the metal oxides present in the red mud through carbothermal processes occurring during the co-pyrolysis. The resulting biochar enriched with thermally-reduced red mud is used for the preparation of epoxy-based composites that are tested for electric and magnetic properties. The electric properties are investigated under DC (direct current) regime with or without pressure applied and under AC (alternating current) in a frequency range from 0.5 up to 16 GHz. The magnetic measurements show the effective tailoring of hemp-derived biochar with magnetic structures during the co-pyrolytic process

Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodeling

In the post-infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin-producing C2C12 myoblasts displayed greater efficacy to engraft the post-ischaemic scar and to induce extracellular matrix re-modelling and angiogenesis as compared with the control cells. By echocardio-graphy, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post-infarcted patients

Effect of Red Mud Addition on Electrical and Magnetic Properties of Hemp-Derived-Biochar-Containing Epoxy Composites

Waste stream valorization is a difficult task where the economic and environmental issues must be balanced. The use of complex metal-rich waste such as red mud is challenging due to the wide variety of metal oxides present such as iron, aluminum, and titanium. The simple separation of each metal is not economically feasible, so alternative routes must be implemented. In this study, we investigated the use of red mud mixed with hemp waste to produce biochar with high conductivity and good magnetic properties induced by the reduction of the metal oxides present in the red mud through carbothermal processes occurring during the co-pyrolysis. The resulting biochar enriched with thermally-reduced red mud is used for the preparation of epoxy-based composites that are tested for electric and magnetic properties. The electric properties are investigated under DC (direct current) regime with or without pressure applied and under AC (alternating current) in a frequency range from 0.5 up to 16 GHz. The magnetic measurements show the effective tailoring of hemp-derived biochar with magnetic structures during the co-pyrolytic process

A Comprehensive Review of Electromagnetic Interference Shielding Composite Materials

The interaction between matter and microwaves assumes critical significance due to the ubiquity of wireless communication technology. The selective shielding of microwaves represents the only way to achieve the control on crucial technological sectors. The implementation of microwave shielding ensures the proper functioning of electronic devices. By preventing electromagnetic pollution, shielding safeguards the integrity and optimal performances of devices, contributing to the reliability and efficiency of technological systems in various sectors and allowing the further step forwards in a safe and secure society. Nevertheless, the microwave shielding research is vast and can be quite hard to approach due to the large number and variety of studies regarding both theory and experiments. In this review, we focused our attention on the comprehensive discussion of the current state of the art of materials used for the production of electromagnetic interference shielding composites, with the aim of providing a solid reference point to explore this research field

Characterization of the Holliday Junction Resolving Enzyme Encoded by the <em>Bacillus subtilis</em> Bacteriophage SPP1

<div><p>Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (<em>ori</em>L), and when replication progresses, the fork might stall by the binding of the origin binding protein G<em>38</em>P to the late replication origin (<em>ori</em>R<em>).</em> Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from <em>P</em>_E2 and <em>P</em>_E3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the <em>P</em>_E2 operon (genes <em>44</em> and <em>45</em>) showed that they are in fact a single gene, re-annotated here as gene <em>44</em>, that encodes a single polypeptide, named gene <em>44</em> product (G<em>44</em>P, 27.5 kDa). G<em>44</em>P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G<em>44</em>P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G<em>44</em>P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in <em>B. subtilis</em> cells. These <em>in vitro</em> and <em>in vivo</em> data suggest a role for G<em>44</em>P in replication restart during the transition to concatemeric viral replication.</p> </div