Relaxin protects cardiac muscle cells from hypoxia/reoxygenation injury: involvement of the Notch-1 pathway.

In animal models, the cardiotropic hormone relaxin has been shown to protect the heart against ischemia and reperfusion-induced damage, acting by multiple mechanisms that primarily involve the coronary vessels. This in vitro study evaluates whether relaxin also has a direct protective action on cardiac muscle cells. H9c2 rat cardiomyoblasts and primary mouse cardiomyocytes were subjected to hypoxia and reoxygenation. In some experiments, relaxin was added preventatively before hypoxia; in others, at reoxygenation. To elucidate its mechanisms of action, we focused on Notch-1, which is involved in heart pre- and postconditioning to ischemia. Inactivated RLX was used as negative control. Relaxin (17 nmol/L, EC50 4.7 nmol/L), added 24 h before hypoxia or at reoxygenation, protected against cardiomyocyte injury. In fact, relaxin significantly increased cell viability (assayed by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), decreased apoptosis (assayed by TUNEL and bax/bcl-2 ratio), and reduced nitroxidative damage (assayed by nitrotyrosine expression and 8-hydroxy-deoxyguanosine levels). These effects were partly attributable to the ability of relaxin to upregulate Notch-1 signaling; indeed, blockade of Notch-1 activation with the specific inhibitor DAPT reduced relaxin-induced cardioprotection during hypoxia and reoxygenation. This study adds new mechanistic insights on the cardioprotective role of relaxin on ischemic and oxidative damage.

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

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.

MSCs seeded on bioengineered scaffolds improve skin wound healing in rats.

Growing evidence has shown the promise of mesenchymal stromal cells (MSCs) for the treatment of cutaneous wound healing. We have previously demonstrated that MSCs seeded on an artificial dermal matrix, Integra (Integra Lifesciences Corp., Plainsboro, NJ) enriched with platelet-rich plasma (Ematrix) have enhanced proliferative potential in vitro as compared with those cultured on the scaffold alone. In this study, we extended the experimentation by evaluating the efficacy of the MSCs seeded scaffolds in the healing of skin wounds in an animal model in vivo. It was found that the presence of MSCs within the scaffolds greatly ameliorated the quality of regenerated skin, reduced collagen deposition, enhanced reepithelization, increased neo-angiogenesis, and promoted a greater return of hair follicles and sebaceous glands. The mechanisms involved in these beneficial effects were likely related to the ability of MSCs to release paracrine factors modulating the wound healing response. MSC-seeded scaffolds, in fact, up-regulated matrix metalloproteinase 9 expression in the extracellular matrix and enhanced the recruitment of endogenous progenitors during tissue repair. In conclusion, the results of this study provide evidence that the treatment with MSC-seeded scaffolds of cutaneous wounds contributes to the recreation of a suitable microenvironment for promoting tissue repair/regeneration at the implantation sites.

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.

Minocycline but not tigecycline is neuroprotective and reduces the neuroinflammatory response induced by the superimposition of sepsis upon traumatic brain injury.

OBJECTIVE: The development of sepsis in patients with traumatic brain injury increases mortality, exacerbates morphological and functional cerebral damage, and causes persistent neuroinflammation, including microglial activation. The administration of antibiotics possessing both antimicrobial and immunomodulatory activity might attenuate both sepsis and posttraumatic cerebral inflammation. We compared the potential therapeutic efficacy of two tetracyclines, minocycline and the newer generation tigecycline, on functional neurobehavioral impairment and regional histopathological damage in an experimental model of combined traumatic brain injury and sepsis. DESIGN: Prospective, experimental animal study. SETTING: University Research Laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Controlled cortical impact was used to induce traumatic brain injury and cecal ligation and puncture for sepsis. Immediately following injury, animals were treated with minocycline (45 mg/kg intraperitoneal), tigecycline (7.5 mg/kg intraperitoneal), or saline every 12 hours for 3 days. MEASUREMENTS AND MAIN RESULTS: The development of sepsis and cerebral inflammatory response were evaluated, respectively, by 1) growth of peritoneal microorganisms and clinical variables and 2) tumor necrosis factor-α expression in the perilesional cortex. To assess posttraumatic outcome, vestibulomotor and cognitive function were evaluated at different time points for 14 days post injury whereupon animals were killed and cerebral tissue analyzed for lesion volume, regional hippocampal (CA1/CA3) cell death, and microglial activation in the perilesional cortex, lesion core zone, and choroid plexus. Treatment with both antibiotics reduced microorganism growth, body weight loss, and mortality but had no effect on vestibulomotor or cognitive function. Minocycline alone attenuated postinjury cortical lesion volume, hippocampal CA3 neuronal cell loss, tumor necrosis factor-α expression, and the extent of microglial activation and infiltration. CONCLUSIONS: The significantly heightened mortality caused by the superimposition of sepsis upon traumatic brain injury can be reduced by administration of both antibiotics but only minocycline can decrease the extent of cell death in selectively cortical and hippocampal brain regions, via, in part, a reduction in cerebral inflammation.

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.

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1.

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.

Relaxin promotes growth and maturation of mouse neonatal cardiomyocytes in vitro: clues for cardiac regeneration.

The demonstration that the adult heart contains myocardial progenitor cells which can be recruited in an attempt to replace the injured myocardium has sparkled interest towards novel molecules capable of improving the differentiation of these cells. In this context, the peptide hormone relaxin (RLX), recently validated as a cardiovascular hormone, is a promising candidate. This study was designed to test the hypothesis that RLX may promote the growth and maturation of mouse neonatal immature cardiomyocytes in primary culture. The cultures were studied at 2, 12, 24 and 48 hrs after the addition of human recombinant H2 RLX (100 ng/ml), the main circulating form of the hormone, or plain medium by combining molecular biology, morphology and electrophysiology. RLX modulated cell proliferation, promoting it at 2 and 12 hrs and inhibiting it at 24 hrs; RLX also induced the expression of both cardiac-specific transcription factors (GATA-4 and Nkx2-5) and cardiac-specific structural genes (connexin 43, troponin T and HCN4 ion channel) at both the mRNA and protein level. Consistently, RLX induced the appearance of ultrastructural and electrophysiological signs of functionally competent, mature cardiomyocytes. In conclusion, this study provides novel circumstantial evidence that RLX specifically acts on immature cardiomyocytes by promoting their proliferation and maturation. This notion suggests that RLX, for which the heart is both a source and target organ, may be an endogenous regulator of cardiac morphogenesis during pre-natal life and could participate in heart regeneration and repair, both as endogenous myocardium-derived factor and exogenous cardiotropic drug, during adult life.

Combined photoablative and photodynamic diode laser therapy as an adjunct to non-surgical periodontal treatment: a randomized split-mouth clinical trial.

AIM: Comparing the efficacy of photoablative and photodynamic diode laser in adjunct to scaling -root planing (SRP) and SRP alone for the treatment of chronic periodontitis. MATERIALS AND METHODS: Twenty-six patients were studied. Maxillary left or right quadrants were randomly assigned to sham-laser treatment + SRP or laser + SRP. This consisted of photoablative intra/extra-pocket de-epithelization with diode laser (λ = 810 nm), followed by single SRP and multiple photodynamic treatments (once weekly, 4-10 applications, mean ± SD: 3.7 ± 2.4) using diode laser (λ = 635 nm) and 0.3% methylene blue as photosensitizer. The patients were monitored at days 0 and 365 by clinical assessment (probing depth, PD; clinical attachment level, CAL; bleeding on probing, BOP) and at days 0, 15, 30, 45, 60, 75, 90, 365 by cytofluorescence analysis of gingival exfoliative samples taken in proximity of the teeth to be treated (polymorphonuclear leukocytes, PMN; red blood cells, RBC; damaged epithelial cells, DEC; bacteria). RESULTS: At day 365, compared with the control quadrants, the laser + SRP therapy yielded a significant (p < 0.001) reduction in PD (-1.9 mm), CAL (-1.7 mm) and BOP (-33.2% bleeding sites), as well as in bacterial contamination - especially spirochetes - and PMN and RBC shedding in the gingival samples (p < 0.001). CONCLUSIONS: Diode laser treatment (photoablation followed by multiple photodynamic cycles) adjunctive to conventional SRP improves healing in chronic periodontitis patients.

Mesenchymal stromal cells affect cardiomyocyte growth through juxtacrine Notch-1/Jagged-1 signaling and paracrine mechanisms: clues for cardiac regeneration.

The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies.

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction.

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.

Sepsis induces albuminuria and alterations in the glomerular filtration barrier: a morphofunctional study in the rat.

INTRODUCTION: Increased vascular permeability represents one of the hallmarks of sepsis. In the kidney, vascular permeability is strictly regulated by the 'glomerular filtration barrier' (GFB), which is comprised of glomerular endothelium, podocytes, their interposed basement membranes and the associated glycocalyx. Although it is likely that the GFB and its glycocalyx are altered during sepsis, no study has specifically addressed this issue. The aim of this study was to evaluate whether albuminuria--the hallmark of GFB perm-selectivity--occurs in the initial stage of sepsis and whether it is associated with morphological and biochemical changes of the GFB. METHODS: Cecal ligation and puncture (CLP) was used to induce sepsis in the rat. Tumor necrosis factor (TNF)-alpha levels in plasma and growth of microorganisms in the peritoneal fluid were evaluated at 0, 3 and 7 hours after CLP or sham-operation. At the same times, kidney specimens were collected and structural and ultrastructural alterations in the GFB were assessed. In addition, several components of GFB-associated glycocalyx, syndecan-1, hyluronan (HA) and sialic acids were evaluated by immunofluorescence, immunohistochemistry and lectin histochemistry techniques. Serum creatinine and creatinine clearance were measured to assess kidney function and albuminuria for changes in GFB permeability. Analysis of variance followed by Tukey's multiple comparison test was used. RESULTS: Septic rats showed increased TNF-alpha levels and growth of microorganisms in the peritoneal fluid. Only a few renal corpuscles had major ultrastructural and structural alterations and no change in serum creatinine or creatinine clearance was observed. Contrarily, urinary albumin significantly increased after CLP and was associated with diffuse alteration in the glycocalyx of the GFB, which consisted in a decrease in syndecan-1 expression and in HA and sialic acids contents. Sialic acids were also changed in their structure, exhibiting a higher degree of acetylation. CONCLUSIONS: In its initial phase, sepsis is associated with a significant alteration in the composition of the GFB-associated glycocalyx, with loss of GFB perm-selectivity as documented by albumin leakage into urine.

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.

Telocytes as supporting cells for myocardial tissue organization in developing and adult heart.

Recent evidence indicates that the adult heart contains sub-epicardial cardiogenic niches where cardiac stem cells and stromal supporting cells reside together. Such stromal cells include a special population, previously identified as interstitial Cajal-like cells and recently termed telocytes because of their long, slender processes (telopodes) embracing the myocardial precursors. Specific stromal cells, presumptively originated from the epicardium, have been postulated to populate the developing heart where they are thought to play a role in its morphogenesis. This study is designed to investigate the occurrence of telocytes in the developing heart and provide clues to better understand their role as supporting cells involved in the architectural organization of the myocardium during heart development. Our results showed that stromal cells with the immunophenotypical (vimentin, CD34) and ultrastructural features of telocytes were present in the mouse heart since early embryonic to adult life, as well as in primary cultures of neonatal mouse cardiac cells. These cells formed an extended network of telopodes which closely embraced the growing cardiomyocytes and appeared to contribute to the aggregation of cardiomyocyte clusters in vitro. In conclusion, the present findings strongly suggest that, during heart development, stromal cells identifiable as telocytes could play a nursing and guiding role for myocardial precursors to form the correct three-dimensional tissue pattern and contribute to compaction of the embryonic myocardial trabeculae. It is tempting to speculate that telocytes could be a novel, possible target for therapeutic strategies aimed at potentiating cardiac repair and regeneration after ischemic injury.

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.

Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures.

The possibility that resident myocardial progenitor cells may be re-activated by transplantation of exogenous stem cells into the post-infarcted heart has been suggested as a possible mechanism to explain the heart's functional improvement after stem cell therapy. Here we studied whether differentiation of mouse neonatal immature cardiomyocytes in vitro was influenced by mouse skeletal myoblasts C2C12, wild type or engineered to secrete the cardiotropic hormone relaxin. The cultured cardiomyocytes formed spontaneously beating clusters and temporally exhibited cardiac immunophenotypical (cKit, atrial natriuretic peptide, troponin T, connexin-43, HCN4) and electrical features (inward voltage-dependent Na(+), T- and L-type Ca(2+) currents, outward and inward K(+) currents, I(f) pacemaker current). These clusters were functionally connected through nanotubular structures and undifferentiated cardiac cells in the form of flattened stripes, bridging the clusters through connexin-43-containing gap junctions. These findings suggested the existence of long distance cell-to-cell communications among the cardiomyocyte aggregates involved in the intercellular transfer of Ca(2+) signals and organelles, likely required for coordination of myocardial differentiation. Co-presence of the myoblasts greatly increased cardiomyocyte differentiation and the amount of intercellular connections. In fact, these cells formed a structural support guiding elongation of nanotubules and stripe-like cells. The secretion of relaxin by the engineered myoblasts accelerated and enhanced the cardiomyogenic potential of the co-culture. These findings underscore the possibility that grafted myoblasts and cardiotropic factors, such as relaxin, may influence regeneration of resident immature cardiac cells, thus adding a tile to the mosaic of mechanisms involved in the functional benefits of cell transplantation for cardiac repair.

In vitro evaluation of the effects of low-intensity Nd:YAG laser irradiation on the inflammatory reaction elicited by bacterial lipopolysaccharide adherent to titanium dental implants.

BACKGROUND: The bacterial endotoxin lipopolysaccharide (LPS) represents a prime pathogenic factor of peri-implantitis because of its ability to adhere tenaciously to dental titanium implants. Despite this, the current therapeutic approach to this disease remains based mainly on bacterial decontamination, paying little attention to the neutralization of bioactive bacterial products. The purpose of the present study was to evaluate whether irradiation with low-energy neodymium-doped:yttrium, aluminum, and garnet (Nd:YAG) laser, in addition to the effects on bacterial implant decontamination, was capable of attenuating the LPS-induced inflammatory response. METHODS: RAW 264.7 macrophages or human umbilical vein endothelial cells were cultured on titanium disks coated with Porphyromonas gingivalis LPS, subjected or not to irradiation with the Nd:YAG laser, and examined for the production of inflammatory cytokines and the expression of morphologic and molecular markers of cell activation. RESULTS: Laser irradiation of LPS-coated titanium disks significantly reduced LPS-induced nitric oxide production and cell activation by the macrophages and strongly attenuated intercellular adhesion molecule-1 and vascular cell adhesion molecule expression, as well as interleukin-8 production by the endothelial cells. CONCLUSION: By blunting the LPS-induced inflammatory response, Nd:YAG laser irradiation may be viewed as a promising tool for the therapeutic management of peri-implantitis.

Differentiation increases the resistance of neuronal cells to amyloid toxicity.

A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Abeta1-40, Abeta1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca(2+) and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca(2+)-ATPase equipment and a higher total antioxidant capacity. Finally, increasing the content of membrane cholesterol resulted in a remarkable reduction of vulnerability and ability to bind the aggregates in either undifferentiated and differentiated cells.

Prostate cancer: a model of integration of genomic and non-genomic effects of the androgen receptor in cell lines model.

Androgens and the androgen receptor (AR) are involved both in early tumorigenesis of prostate cancer (PCa) and in androgen-refractory disease. The role of AR signalling has also been highlighted by the fusion gene TMPRSS2:ERG recently identified in the majority of PCa. Several data indicate that re-expression of AR in PCa cell lines confers a less aggressive phenotype. We observed that re-expression of AR in the AR-negative cells PC3 decreases anchorage-independent growth and Matrigel invasiveness of PC3-AR cells where plasma membrane interaction between AR and EGFR led to an interference with downstream signalling and internalization of activated EGFR. Our data evidenced a shift of EGFR internalization pathway from the clathrin-coated pit one mediating signalling and recycling of EGFR to the lipid raft-mediated one mainly involved in lysosomal degradation of EGFR. These effects involved an altered recruitment to EGFR of the adaptor proteins Grb2 and c-Cbl followed by a reduced ubiquitination of EGFR. Our preliminary results suggest that in PC3-AR cells a pool of classical AR is located within cholesterol-rich membrane microdomains (namely as lipid rafts) and a population of EGFR is within cholesterol-rich membrane microdomains too. However, AR and EGFR membrane interaction that is increased by rapid androgen signalling is not within cholesterol-rich membrane microdomains. Our data enlighten that the crosstalk between genotropic and non-genotropic AR signalling interferes with signalling of EGFR in response to ligand leading to a lower invasive phenotype of AR-positive PCa cells.