Defining the Molecular Mechanisms of the Relaxant Action of Adiponectin on Murine Gastric Fundus Smooth Muscle: Potential Translational Perspectives on Eating Disorder Management.

Adiponectin (ADPN), a hormone produced by adipose tissue, facilitates gastric relaxation and can be a satiety signal in the network connecting peripheral organs and the central nervous system for feeding behavior control. Here, we performed preclinical research by morpho-functional analyses on murine gastric fundus smooth muscle to add insights into the molecular mechanisms underpinning ADPN action. Moreover, we conducted a clinical study to evaluate the potential use of ADPN as a biomarker for eating disorders (ED) based on the demonstrated gastric alterations and hormone level fluctuations that are often associated with ED. The clinical study recruited patients with ED and healthy controls who underwent blood draws for ADPN dosage and psychopathology evaluation tests. The findings of this basic research support the ADPN relaxant action, as indicated by the smooth muscle cell membrane pro-relaxant effects, with mild modifications of contractile apparatus and slight inhibitory effects on gap junctions. All of these actions engaged the ADPN/nitric oxide/guanylate cyclase pathway. The clinical data failed to unravel a correlation between ADPN levels and the considered ED, thus negating the potential use of ADPN as a valid biomarker for ED management for the moment. Nevertheless, this adipokine can modulate physiological eating behavior, and its effects deserve further investigation.

Platelet-rich plasma affects gap junctional features in myofibroblasts in vitro via vascular endothelial growth factor (VEGF)-A/VEGF receptor.

NEW FINDINGS: What is the central question of this study? It is a challenge to discover effective therapies for fibrosis. Increasing evidence supports the antifibrotic potential of platelet-rich plasma (PRP) as a source of bioactive molecules, such as vascular endothelial growth factor (VEGF)-A. However, the effects and mechanisms of action of PRP need to be clarified. What is the main finding and its importance? This report clarifies the mechanisms mediating the antifibrotic action of PRP, strengthening the role of VEGF-A/VEGF receptor, and identifies gap junction currents and connexin 43 as novel targets of this pathway in the fibroblast-to-myofibroblast transition induced by the transforming growth factor-ß1. ABSTRACT: Despite increasing experimental evidence, the antifibrotic potential of platelet-rich plasma (PRP) remains controversial, and its mechanisms of action are not fully clarified. This short report extends our previous research on the capability of PRP to prevent the in vitro differentiation of fibroblasts toward myofibroblasts, the key effectors of fibrosis, induced by the profibrotic agent transforming growth factor-ß1 (TGF-ß1). In particular, we focused on the involvement of signalling mediated by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) in the PRP-induced fibroblast response, highlighting gap junction features. Electrophysiological and morphological analyses revealed that PRP hindered morphofunctional differentiation of both murine NIH/3T3 and human primary adult skin fibroblasts toward myofibroblasts as judged by the analysis of membrane phenomena, α-smooth muscle actin and vinculin expression and cell morphology. Neutralization of VEGF-A by blocking antibodies or pharmacological inhibition of VEGFR by KRN633 in TGF-ß1-treated fibroblasts prevented the PRP-promoted effects, such as the reduction of voltage-dependent transjunctional currents in cell pairs and a decreased expression of connexin 43, the typical connexin isoform forming voltage-dependent connexons. The role of VEGF-A in inhibiting these events was confirmed by treating TGF-ß1-stimulated fibroblasts with soluble VEGF-A. The results obtained when cells were differentiated using KRN633 alone suggest an antagonistic cross-talk between TGF-ß1 and VEGFR. In conclusion, this study identifies, for the first time, gap junction currents as crucial targets in the VEGF-A/VEGFR-mediated antifibrotic pathway and provides new insights into mechanisms behind the action of PRP in preventing differentiation of fibroblasts to myofibroblasts.

Bone Marrow-Mesenchymal Stromal Cell Secretome as Conditioned Medium Relieves Experimental Skeletal Muscle Damage Induced by Ex Vivo Eccentric Contraction.

Bone marrow-mesenchymal stem/stromal cells (MSCs) may offer promise for skeletal muscle repair/regeneration. Growing evidence suggests that the mechanisms underpinning the beneficial effects of such cells in muscle tissue reside in their ability to secrete bioactive molecules (secretome) with multiple actions. Hence, we examined the effects of MSC secretome as conditioned medium (MSC-CM) on ex vivo murine extensor digitorum longus muscle injured by forced eccentric contraction (EC). By combining morphological (light and confocal laser scanning microscopies) and electrophysiological analyses we demonstrated the capability of MSC-CM to attenuate EC-induced tissue structural damages and sarcolemnic functional properties' modifications. MSC-CM was effective in protecting myofibers from apoptosis, as suggested by a reduced expression of pro-apoptotic markers, cytochrome c and activated caspase-3, along with an increase in the expression of pro-survival AKT factor. Notably, MSC-CM also reduced the EC-induced tissue redistribution and extension of telocytes/CD34+ stromal cells, distinctive cells proposed to play a "nursing" role for the muscle resident myogenic satellite cells (SCs), regarded as the main players of regeneration. Moreover, it affected SC functionality likely contributing to replenishment of the SC reservoir. This study provides the necessary groundwork for further investigation of the effects of MSC secretome in the setting of skeletal muscle injury and regenerative medicine.

Influence of Platelet-Rich and Platelet-Poor Plasma on Endogenous Mechanisms of Skeletal Muscle Repair/Regeneration.

The morpho-functional recovery of injured skeletal muscle still represents an unmet need. None of the therapeutic options so far adopted have proved to be resolutive. A current scientific challenge remains the identification of effective strategies improving the endogenous skeletal muscle regenerative program. Indeed, skeletal muscle tissue possesses an intrinsic remarkable regenerative capacity in response to injury, mainly thanks to the activity of a population of resident muscle progenitors called satellite cells, largely influenced by the dynamic interplay established with different molecular and cellular components of the surrounding niche/microenvironment. Other myogenic non-satellite cells, residing within muscle or recruited via circulation may contribute to post-natal muscle regeneration. Unfortunately, in the case of extended damage the tissue repair may become aberrant, giving rise to a maladaptive fibrotic scar or adipose tissue infiltration, mainly due to dysregulated activity of different muscle interstitial cells. In this context, plasma preparations, including Platelet-Rich Plasma (PRP) and more recently Platelet-Poor Plasma (PPP), have shown advantages and promising therapeutic perspectives. This review focuses on the contribution of these blood-derived products on repair/regeneration of damaged skeletal muscle, paying particular attention to the potential cellular targets and molecular mechanisms through which these products may exert their beneficial effects.

Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stromal Cells Prevent TGF-ß1-Induced Myofibroblast Generation but Are Not Synergistic when Combined: Morphological in vitro Analysis.

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-ß1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-ß1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated "from bench to bedside," could potentially offer advantages over the single treatments.

Red (635 nm), Near-Infrared (808 nm) and Violet-Blue (405 nm) Photobiomodulation Potentiality on Human Osteoblasts and Mesenchymal Stromal Cells: A Morphological and Molecular In Vitro Study.

Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, 808 ± 10 nm) diode lasers and violet-blue (405 ± 5 nm) light-emitting diode operating in a continuous wave with a 0.4 J/cm² energy density, on human osteoblast and mesenchymal stromal cell (hMSC) viability, proliferation, adhesion and osteogenic differentiation. PBM treatments did not alter viability (PI/Syto16 and MTS assays). Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67. Interestingly, osteoblast responses to red light were mediated by Akt signaling activation, which seems to positively modulate reactive oxygen species levels. Violet-blue light-irradiated cells behaved essentially as untreated ones and NIR irradiated ones displayed modifications of cytoskeleton assembly, Runx-2 expression and mineralization pattern. Although within the limitations of an in vitro experimentation, this study may suggest PBM with 635 nm laser as potential effective option for promoting/improving bone regeneration.

Mesenchymal stromal cell and osteoblast responses to oxidized titanium surfaces pre-treated with λ = 808 nm GaAlAs diode laser or chlorhexidine: in vitro study.

Preservation of implant biocompatibility following peri-implantitis treatments is a crucial issue in odontostomatological practice, being closely linked to implant re-osseointegration. Our aim was to assess the responses of osteoblast-like Saos2 cells and adult human bone marrow-mesenchymal stromal cells (MSCs) to oxidized titanium surfaces (TiUnite®, TiU) pre-treated with a 808 ± 10 nm GaAlAs diode laser operating in non-contact mode, in continuous (2 W, 400 J/cm2; CW) or pulsed (20 kHz, 7 µs, 0.44 W, 88 J/cm2; PW) wave, previously demonstrated to have a strong bactericidal effect and proposed as optional treatment for peri-implantitis. The biocompatibility of TiU surfaces pre-treated with chlorhexidine digluconate (CHX) was also evaluated. In particular, in order to mimic the in vivo approach, TiU surfaces were pre-treated with CHX (0.2%, 5 min); CHX and rinse; and CHX, rinse and air drying. In some experiments, the cells were cultured on untreated TiU before being exposed to CHX. Cell viability (MTS assay), proliferation (EdU incorporation assay; Ki67 confocal immunofluorescence analysis), adhesion (morphological analysis of actin cytoskeleton organization), and osteogenic differentiation (osteopontin confocal immunofluorescence analysis; mineralized bone-like nodule formation) analyses were performed. CHX resulted cytotoxic in all experimental conditions. Diode laser irradiation preserved TiU surface biocompatibility. Notably, laser treatment appeared even to improve the known osteoconductive properties of TiU surfaces. Within the limitations of an in vitro experimentation, this study contributes to provide additional experimental basis to support the potential use of 808 ± 10 nm GaAlAs diode laser at the indicated irradiation setting, in the treatment of peri-implantitis and to discourage the use of CHX.

Effects of photodynamic laser and violet-blue led irradiation on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide attached to moderately rough titanium surface: in vitro study.

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This study investigates the in vitro efficacy of photodynamic treatment (PDT) with methylene blue (MB) photoactivated with λ 635 nm diode laser and of λ 405 nm violet-blue LED phototreatment for the reduction of bacterial biofilm and lipopolysaccharide (LPS) adherent to titanium surface mimicking the bone-implant interface. Staphylococcus aureus biofilm grown on titanium discs with a moderately rough surface was subjected to either PDT (0.1% MB and λ 635 nm diode laser) or λ 405 nm LED phototreatment for 1 and 5 min. Bactericidal effect was evaluated by vital staining and residual colony-forming unit count. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, discs coated with Escherichia coli LPS were treated as above before seeding with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Both PDT and LED phototreatment induced a statistically significant (p < 0.05 or higher) reduction of viable bacteria, up to -99 and -98% (5 min), respectively. Moreover, besides bactericidal effect, PDT and LED phototreatment also inhibited LPS bioactivity, assayed as nitrite formation, up to -42%, thereby blunting host inflammatory response. Non-invasive phototherapy emerges as an attractive alternative in the treatment of peri-implantitis to reduce bacteria and LPS adherent to titanium implant surface without causing damage of surface microstructure. Its efficacy in the clinical setting remains to be investigated.

Low intensity 635 nm diode laser irradiation inhibits fibroblast-myofibroblast transition reducing TRPC1 channel expression/activity: New perspectives for tissue fibrosis treatment.

BACKGROUND AND OBJECTIVE: Low-level laser therapy (LLLT) or photobiomodulation therapy is emerging as a promising new therapeutic option for fibrosis in different damaged and/or diseased organs. However, the anti-fibrotic potential of this treatment needs to be elucidated and the cellular and molecular targets of the laser clarified. Here, we investigated the effects of a low intensity 635 ± 5 nm diode laser irradiation on fibroblast-myofibroblast transition, a key event in the onset of fibrosis, and elucidated some of the underlying molecular mechanisms. MATERIALS AND METHODS: NIH/3T3 fibroblasts were cultured in a low serum medium in the presence of transforming growth factor (TGF)-ß1 and irradiated with a 635 ± 5 nm diode laser (continuous wave, 89 mW, 0.3 J/cm(2) ). Fibroblast-myofibroblast differentiation was assayed by morphological, biochemical, and electrophysiological approaches. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 and of Tissue inhibitor of MMPs, namely TIMP-1 and TIMP-2, after laser exposure was also evaluated by confocal immunofluorescence analyses. Moreover, the effect of the diode laser on transient receptor potential canonical channel (TRPC) 1/stretch-activated channel (SAC) expression and activity and on TGF-ß1/Smad3 signaling was investigated. RESULTS: Diode laser treatment inhibited TGF-ß1-induced fibroblast-myofibroblast transition as judged by reduction of stress fibers formation, α-smooth muscle actin (sma) and type-1 collagen expression and by changes in electrophysiological properties such as resting membrane potential, cell capacitance and inwardly rectifying K(+) currents. In addition, the irradiation up-regulated the expression of MMP-2 and MMP-9 and downregulated that of TIMP-1 and TIMP-2 in TGF-ß1-treated cells. This laser effect was shown to involve TRPC1/SAC channel functionality. Finally, diode laser stimulation and TRPC1 functionality negatively affected fibroblast-myofibroblast transition by interfering with TGF-ß1 signaling, namely reducing the expression of Smad3, the TGF-ß1 downstream signaling molecule. CONCLUSION: Low intensity irradiation with 635 ± 5 nm diode laser inhibited TGF-ß1/Smad3-mediated fibroblast-myofibroblast transition and this effect involved the modulation of TRPC1 ion channels. These data contribute to support the potential anti-fibrotic effect of LLLT and may offer further informations for considering this therapy as a promising therapeutic tool for the treatment of tissue fibrosis.

The effects of diode laser on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide adherent to titanium oxide surface of dental implants. An in vitro study.

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This in vitro study aims at providing the experimental basis for possible use of diode laser (λ 808 nm) in the treatment of peri-implantitis. Staphylococcus aureus biofilm was grown for 48 h on titanium discs with porous surface corresponding to the bone-implant interface and then irradiated with a diode laser (λ 808 nm) in noncontact mode with airflow cooling for 1 min using a Ø 600-µm fiber. Setting parameters were 2 W (400 J/cm2) for continuous wave mode; 22 µJ, 20 kHz, 7 µs (88 J/cm2) for pulsed wave mode. Bactericidal effect was evaluated using fluorescence microscopy and counting the residual colony-forming units. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, the titanium discs were coated with Escherichia coli lipopolysaccharide (LPS), laser-irradiated and seeded with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Diode laser irradiation in both continuous and pulsed modes induced a statistically significant reduction of viable bacteria and nitrite levels. These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inflammatory response. The λ 808-nm diode laser emerges as a valuable tool for decontamination/detoxification of the titanium implant surface and may be used in the treatment of peri-implantitis.

Inhibitory effects of relaxin on cardiac fibroblast-to-myofibroblast transition: an electrophysiological study.

NEW FINDINGS: What is the central question of this study? Fibroblast-to-myofibroblast transition is a key mechanism in the reparative response to tissue damage, but myofibroblast persistence in the wound leads to fibrosis and organ failure. The role of relaxin as an antifibrotic agent capable of counteracting the acquisition of biophysical features of differentiated myofibroblasts deserves further investigation. What is the main finding and its importance? Electrophysiological analysis showed that relaxin, administered during profibrotic treatment, hyperpolarizes the membrane potential and attenuates delayed rectifier and inwardly rectifying K(+) currents, which usually increase in the transition to myofibroblasts. These findings provide further clues to the therapeutic potential of relaxin in fibrosis. The hormone relaxin (RLX) is produced by the heart and may be involved in endogenous mechanisms of cardiac protection against ischaemic injury and fibrosis. Recent findings in cultured cardiac stromal cells suggest that RLX can inhibit fibroblast-to-myofibroblast transition, thereby counteracting fibrosis. In order to explore its efficiency as an antifibrotic agent further, we designed the present study to investigate whether RLX may influence the electrophysiological events associated with differentiation of cardiac stromal cells to myofibroblasts. Primary cardiac proto-myofibroblasts and NIH/3T3 fibroblasts were induced to myofibroblasts by transforming growth factor-ß1, and the electrophysiological features of both cell populations were investigated by whole-cell patch clamp. We demonstrated that proto-myofibroblasts and myofibroblasts express different membrane passive properties and K(+) currents. Here, we have shown, for the first time, that RLX (100 ng ml(-1) ) significantly reduced both voltage- and Ca(2+) -dependent delayed-rectifier and inward-rectifying K(+) currents that are typically increased in myofibroblasts compared with proto-myofibroblasts, suggesting that this hormone can antagonize the biophysical effects of transforming growth factor-ß1 in inducing myofibroblast differentiation. These newly recognized effects of RLX on the electrical properties of cardiac stromal cell membrane correlate well with its well-known ability to suppress myofibroblast differentiation, further supporting the possibility that RLX may be used for the treatment of cardiac fibrosis.

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells.

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7(+) satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.

Photoactivation of bone marrow mesenchymal stromal cells with diode laser: effects and mechanisms of action.

Mesenchymal stromal cells (MSCs) are a promising cell candidate in tissue engineering and regenerative medicine. Their proliferative potential can be increased by low-level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. With the aim of expanding the therapeutic application of LLLI to MSC therapy, in the present study we investigated the effects of 635 nm diode laser on mouse MSC proliferation and investigated the underlying cellular and molecular mechanisms, focusing the attention on the effects of laser irradiation on Notch-1 signal activation and membrane ion channel modulation. It was found that MSC proliferation was significantly enhanced after laser irradiation, as judged by time lapse videomicroscopy and EdU incorporation. This phenomenon was associated with the up-regulation and activation of Notch-1 pathway, and with increased membrane conductance through voltage-gated K(+) , BK and Kir, channels and T- and L-type Ca(2+) channels. We also showed that MSC proliferation was mainly dependent on Kir channel activity, on the basis that the cell growth and Notch-1 up-regulation were severely decreased by the pre-treatment with the channel inhibitor Ba(2+) (0.5 mM). Interestingly, the channel inhibition was also able to attenuate the stimulatory effects of diode laser on MSCs, thus providing novel evidence to expand our knowledge on the mechanisms of biostimulation after LLLI. In conclusions, our findings suggest that diode laser may be a valid approach for the preconditioning of MSCs in vitro prior cell transplantation.

Development of accessible platforms to promote myofibroblast differentiation by playing on hydrogel scaffold composition.

Mechanomimetic materials are particularly attractive for modeling in vitro fibroblast to myofibroblast (Myof) transition, a key process in the physiological repair of damaged tissue, and recognized as the core cellular mechanism of pathological fibrosis in different organs. In vivo, mechanical stimuli from the extracellular matrix (ECM) are crucial, together with cell-cell contacts and the pro-fibrotic transforming growth factor (TGF)-ß1, in promoting fibroblast differentiation. Here, we explore the impact of hydrogels made by polyacrylamide with different composition on fibroblast behavior. By appropriate modulation of the hydrogel composition (e.g. adjusting the crosslinker content), we produce and fully characterize three kinds of scaffolds with different Young modulus (E). We observe that soft hydrogels (E < 1 kPa) induced fibroblast differentiation better than stiffer ones, also in the absence of TGF-ß1. This study provides a readily accessible biomaterial platform to promote Myof generation. The easy approach used and the commercial availability of the monomers make these hydrogels suitable to a wide range of biomedical applications combined with high reproducibility and simple preparation protocols.

HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro.

Hypoxia-inducible factor (HIF)-1α represents an oxygen-sensitive subunit of HIF transcriptional factor, which is usually degraded in normoxia and stabilized in hypoxia to regulate several target gene expressions. Nevertheless, in the skeletal muscle satellite stem cells (SCs), an oxygen level-independent regulation of HIF-1α has been observed. Although HIF-1α has been highlighted as a SC function regulator, its spatio-temporal expression and role during myogenic progression remain controversial. Herein, using biomolecular, biochemical, morphological and electrophysiological analyses, we analyzed HIF-1α expression, localization and role in differentiating murine C2C12 myoblasts and SCs under normoxia. In addition, we evaluated the role of matrix metalloproteinase (MMP)-9 as an HIF-1α effector, considering that MMP-9 is involved in myogenesis and is an HIF-1α target in different cell types. HIF-1α expression increased after 24/48 h of differentiating culture and tended to decline after 72 h/5 days. Committed and proliferating mononuclear myoblasts exhibited nuclear HIF-1α expression. Differently, the more differentiated elongated and parallel-aligned cells, which are likely ready to fuse with each other, show a mainly cytoplasmic localization of the factor. Multinucleated myotubes displayed both nuclear and cytoplasmic HIF-1α expression. The MMP-9 and MyoD (myogenic activation marker) expression synchronized with that of HIF-1α, increasing after 24 h of differentiation. By means of silencing HIF-1α and MMP-9 by short-interfering RNA and MMP-9 pharmacological inhibition, this study unraveled MMP-9's role as an HIF-1α downstream effector and the fact that the HIF-1α/MMP-9 axis is essential in morpho-functional cell myogenic commitment.

Adiponectin Modulates Smooth Muscle Cell Morpho-Functional Properties in Murine Gastric Fundus via Sphingosine Kinase 2 Activation.

Adipokines are peptide hormones produced by the adipose tissue involved in several biological functions. Among adipokines, adiponectin (ADPN) has antidiabetic and anti-inflammatory properties. It can also modulate food intake at central and peripheral levels, acting on hypothalamus and facilitating gastric relaxation. ADPN exerts its action interacting with two distinct membrane receptors and triggering some well-defined signaling cascades. The ceramidase activity of ADPN receptor has been reported in many tissues: it converts ceramide into sphingosine. In turn, sphingosine kinase (SK) phosphorylates it into sphingosine-1 phosphate (S1P), a crucial mediator of many cellular processes including contractility. Using a multidisciplinary approach that combined biochemical, electrophysiological and morphological investigations, we explored for the first time the possible role of S1P metabolism in mediating ADPN effects on the murine gastric fundus muscle layer. By using a specific pharmacological inhibitor of SK2, we showed that ADPN affects smooth muscle cell membrane properties and contractile machinery via SK2 activation in gastric fundus, adding a piece of knowledge to the action mechanisms of this hormone. These findings help to identify ADPN and its receptors as new therapeutic targets or as possible prognostic markers for diseases with altered energy balance and for pathologies with fat mass content alterations.

Human Recombinant Relaxin (Serelaxin) as Anti-fibrotic Agent: Pharmacology, Limitations and Actual Perspectives.

Relaxin (recombinant human relaxin-2 hormone; RLX-2; serelaxin) had raised expectations as a new medication for fibrotic diseases. A plethora of in vitro and in vivo studies have offered convincing demonstrations that relaxin promotes remodeling of connective tissue extracellular matrix mediated by inhibition of multiple fibrogenic pathways, especially the downstream signaling of transforming growth factor (TGF)-ß1, a major pro-fibrotic cytokine, and the recruitment and activation of myofibroblasts, the main fibrosis-generating cells. However, all clinical trials with relaxin in patients with fibrotic diseases gave inconclusive results. In this review, we have summarized the molecular mechanisms of fibrosis, highlighting those which can be effectively targeted by relaxin. Then, we have performed a critical reappraisal of the clinical trials performed to date with relaxin as an anti-fibrotic drug, in order to highlight their key points of strength and weakness and to identify some future opportunities for the therapeutic use of relaxin, or its analogues, in fibrotic diseases and pathologic scarring which, in our opinion, deserve to be investigated.

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis.

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca(2+)-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.

Low pulse energy Nd:YAG laser irradiation exerts a biostimulative effect on different cells of the oral microenvironment: "an in vitro study".

BACKGROUND AND OBJECTIVES: Dental lasers represent a promising therapeutic tool in the treatment of periodontal and peri-implant diseases. However, their clinical application remains still limited. Here, we investigated the potential biostimulatory effect of low pulse energy neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation on different cells representative of the oral microenvironment and elucidated the underlying molecular mechanisms. MATERIALS AND METHODS: Saos-2 osteoblasts, H-end endothelial cells, and NIH/3T3 fibroblasts pre-treated or not with photosensitizing dye methylene blue (MB), were irradiated with low pulse energy (20 mJ) and high repetition rate (50-70 Hz) Nd:YAG laser, and evaluated for cell viability and proliferation as well as for the expression of specific differentiation markers by confocal immunofluorescence and real-time RT-PCR. Changes in intracellular Ca(2+) levels after laser exposure were also evaluated in living osteoblasts. RESULTS: Nd:YAG laser irradiation did not affect cell viability in all the tested cell types, even when combined with pre-treatment with MB, and efficiently stimulated cell growth in the non-sensitized osteoblasts. Moreover, a significant induction in the expression of osteopontin, ALP, and Runx2 in osteoblasts, type I collagen in fibroblasts, and vinculin in endothelial cells could be observed in the irradiated cells. Pre-treatment with MB negatively affected cell differentiation in the unstimulated and laser-stimulated cells. Notably, laser irradiation also caused an increase in the intracellular Ca(2+) in osteoblasts through the activation of TRPC1 ion channels. Moreover, the pharmacologic or genetic inhibition of these channels strongly attenuated laser-induced osteopontin expression, suggesting a role for the laser-mediated Ca(2+) influx in regulating osteoblast differentiation. CONCLUSION: Low pulse energy and high repetition rate Nd:YAG laser irradiation may exert a biostimulative effect on different cells representative of the oral microenvironment, particularly osteoblasts. Pre-treatment with MB prior to irradiation hampers this effect and limits the potential clinical application of photosensitizing dyes in dental practice.