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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. [ABSTRACT FROM AUTHOR]

Our In-Vitro Fertilisation Centre is situated in a large developing country, Madagascar, with very bad roads and low income patients. Therefore we try to find ways to reduce as much as possible the number of attempts to obtain a pregnancy. Poor or no response to ovarian stimulation in In Vitro Fertilization (IVF) cycles is a great challenge. Here we describe a method whereby we shift from IVF to Oocyte Donation (OD) during the same cycle for patients whose ovaries do not respond properly to ovulation stimulation. Patients were superovulated with a long protocol agonist treatment and ultrasonically monitored for IVF/ICSI. When, at half way of the stimulation, it was clear that there was a no or poor response, gonadotropin administration was stopped and immediately replaced by estrogens; when the endometrium was considered to be sufficiently receptive, some donated oocytes from our concomitant oocyte donation (OD) program were fertilized with the patient's husband sperm and progesterone was added to the patients' treatment. After 48 hours the resulting embryos were transferred. Five poor responders patients underwent the described procedure. Three conceived, one of which aborted at 9 weeks, while the other two are ongoing. These patients signed the consent form accepting the possibility to shift from IVF to OD during the same cycle and three clinical pregnancies were obtained. OD through this technique seems more acceptable by poor responders than planned OD. This is a preliminary report and to our knowledge it is the first report of such a method.

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-β1, 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-β1 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., (© 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.)

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., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.

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.

The notion that the adult heart of mammals including humans contain a small population of resident cardiac progenitor/stem cells (CSCs) have questioned the traditional paradigm of the myocardium as a post-mitotic terminally differentiated tissue. These cells, however, are relatively scarce and unable to be recruited in large number at the site of tissue damage. This has sparkled novel interest in the identification of molecules capable of stimulating the regenerative potentials of CSCs in their microenvironment. In this context, the peptide hormone relaxin (RLX), recently validated as a cardiovascular hormone, deserves a key place. This article summarizes the most recent findings of our group on the ability of RLX to modulate growth and differentiation of mouse neonatal cardiomyocytes, suggesting that this hormone, for which the heart is both a source and target organ, may participate in the endogenous mechanisms of myocardial repair/regeneration. Moreover, we have recently observed that RLX, by a Notch-1-mediated mechanism, inhibits cardiac myofibroblast differentiation and function, suggesting that the known anti-fibrotic effects of RLX may be part of a complex network of actions whereby this hormone facilitates cardiac stem cell growth and improves myocardial regeneration.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) represent the leading candidate cell in tissue engineering and regenerative medicine. These cells can be easily isolated, expanded in vitro and are capable of providing significant functional benefits after implantation in the damaged muscle tissues. Despite their plasticity, the participation of BM-MSCs to new muscle fiber formation is controversial; in fact, emerging evidence indicates that their therapeutic effects occur without signs of long-term tissue engraftment and involve the paracrine secretion of cytokines and growth factors with multiple effects on the injured tissue, including modulation of inflammation and immune reaction, positive extracellular matrix (ECM) remodeling, angiogenesis and protection from apoptosis. Recently, a new role for BM-MSCs in the stimulation of muscle progenitor cells proliferation has been demonstrated, suggesting the potential ability of these cells to influence the fate of local stem cells and augment the endogenous mechanisms of repair/regeneration in the damaged tissues.

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., (© 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

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.

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., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

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.

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., (© 2010 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

The multidrug resistance (MDR) phenotype is characterized by the overexpression of a few transport proteins at the plasma membrane level, one of which is the breast cancer resistance protein (BCRP). These proteins are expressed in excretory organs, in the placenta and blood-brain barrier, and are involved in the transport of drugs and endogenous compounds. Because some of these proteins are expressed in the mitochondria, this study was designed to determine whether BCRP is expressed at a mitochondrial level and to investigate its function in various MDR and parental drug-sensitive cell lines. By using Western blot analysis, immunofluorescence confocal and electron microscopy, flow cytometry analysis, and the BCRP (ABCG-2) small interfering RNA, these experiments showed that BCRP is expressed in the mitochondrial cristae, in which it is functionally active. Mitoxantrone accumulation was significantly reduced in mitochondria and in cells that overexpress BCRP, in comparison to parental drug-sensitive cells. The specific inhibitor of BCRP, fumitremorgin c, increased the accumulation of mitoxantrone significantly in comparison with basal conditions in both whole cells and in mitochondria of BCRP-overexpressing cell lines. In conclusion, this study shows that BCRP is overexpressed and functionally active in the mitochondria of MDR-positive cancer cell lines. However, its presence in the mitochondria of parental drug-sensitive cells suggests that BCRP can be involved in the physiology of cancer cells.

Transient receptor potential canonical (TRPC) channels provide cation and Ca(2+) entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channel's activity and stretch-induced Ca(2+) influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, C(m)) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, C(m), used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce C(m) and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.

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 echocardiography, 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.

Although androgens and the androgen receptor (AR) are involved in tumorigenesis of prostate cancer (PC) in initial phases, less clear is the role played in advanced androgen-independent (AI) stages of the disease. Several recent reports indicated that re-expression of AR in PC-derived cell lines determines a less aggressive phenotype of the cells. We have previously demonstrated that re-expression of AR decreases the invasion ability of PC3 cells in vitro by affecting signalling and internalization processes of epidermal growth factor receptor (EGFR). Here, we show that reduced EGFR internalization is also a characteristic of AR positive PC cell lines LNCaP and 22Rv1. Reduced internalization in PC3-AR cells is associated to a defective interaction between the EGFR and two adaptor proteins which mediate the endocytotic process, Grb2 and c-Cbl. As a consequence of such reduced interaction, ubiquitination of the receptor, which is mainly mediated by c-Cbl, is also altered. In addition, we show that internalized EGFR co-localizes with early endosome antigen-1, a marker of clathrin-mediated endocytosis, in PC3-Neo cells but not in AR positive cell lines. Conversely, EGFR maintains co-localization with caveolin-1 after EGF stimulation in PC3-AR cells. These data suggest that expression of AR affects clathrin-mediated endocytosis pathway of EGFR, which, according to recent findings, plays an essential role in the completeness of signalling of the receptor. Taken together, these data emphasize the role of AR in the regulation of EGFR endocytotic trafficking and active signalling in PC cells. In view of the role of EGFR signalling in invasion of carcinoma cells, our data may explain the lower invasive phenotype observed in AR-positive cell lines.

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion.

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.

Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF-N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF-N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl-2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro-apoptotic stimuli, which are counteracted by upregulation of Bcl-2.

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is abundantly present in the serum and mediates multiple biological responses. With the aim of extending our knowledge on the role played by S1P in the regulation of cytoskeletal reorganization, native as well as C2C12 myoblasts stably transfected with green fluorescent protein (GFP)-tagged alpha- and beta-actin constructs were stimulated with S1P (1 microM) and observed under confocal and multiphoton microscopes. The addition of S1P induced the appearance of actin stress fibres and focal adhesions through Rho- and phospholipase D (PLD)-mediated pathways. The cytoskeletal response was dependent on the extracellular action of S1P through its specific surface receptors, since the intracellular delivery of the sphingolipid by microinjection was unable to modify the actin cytoskeletal assembly. Interestingly, it was revealed by whole-cell patch-clamp that S1P-induced stress fibre formation was associated with increased ion currents and conductance through stretch-activated channels (SACs), thereby suggesting a possible regulatory role for organized actin in channel sensitivity. Experiments aimed at stretching the plasma membrane of C2C12 cells, using the cantilever of an atomic force microscope, indicated that there was a Ca2+ influx through putative SACs. In conclusion, the present data suggest novel mechanisms of S1P signalling involving actin cytoskeletal reorganization and Ca2+ elevation through SACs that might influence myoblastic functions.

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

The permeability transition pore (PTP) is a mitochondrial channel whose opening causes the mitochondrial membrane potential (deltapsi) collapse that leads to apoptosis. Some ubiquinone analogues have been demonstrated previously to modulate the PTP open-closed transition in isolated mitochondria and thought to act through a common PTP-binding site rather than through oxidation-reduction reactions. We have demonstrated recently both in vitro and in vivo that the ubiquitous free radical scavenger and respiratory chain coenzyme Q10 (CoQ10) prevents keratocyte apoptosis induced by excimer laser irradiation more efficiently than other antioxidants. On this basis, we hypothesized that the antiapoptotic property of CoQ10 could be independent of its free radical scavenging ability and related to direct inhibition of PTP opening. In this study, we have verified this hypothesis by evaluating the antiapoptotic effects of CoQ10 in response to apoptotic stimuli, serum starvation, antimycin A, and ceramide, which do not generate free radicals, in comparison to control, free radical-generating UVC irradiation. As hypothesized, CoQ10 dramatically reduced apoptotic cell death, attenuated ATP decrease, and hindered DNA fragmentation elicited by all apoptotic stimuli. This was accompanied by inhibition of mitochondrial depolarization, cytochrome c release, and caspase 9 activation. Because these events are consequent to mitochondrial PTP opening, we suggest that the antiapoptotic activity of CoQ10 could be related to its ability to prevent this phenomenon.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C(2)C(12) myoblasts SPP elicited significant Ca(2+) mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca(2+) response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca(2+) transients performed with a spectrophotofluorimeter showed that the rise in Ca(2+) was strictly dependent on availability of extracellular Ca(2+). Cell treatment with pertussis toxin partially prevented the Ca(2+) response induced by SPP, indicating that G(i)-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca(2+) mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca(2+), although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca(2+) increase, supporting the notion that Ca(2+) mobilization in C(2)C(12) cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Background: Neutrophils are the predominant phagocytes in the early stages of myocardial ischemia-reperfusion response and are also implicated in the development of tissue damage. This study examined the role of recruited macrophages in the evolution of this tissue injury., Methods: Farm pigs were subjected to 30 minutes of myocardial ischemia followed by 30 minutes of reperfusion. Biopsy samples were taken from the control, ischemic, and ischemic-reperfused left ventricle wall and processed for both morphologic and biochemical analyses. In situ production of tumor necrosis factor-alpha was evaluated by Western blot and immunofluorescence. A full hemodynamic evaluation was also performed., Results: Myocardial ischemia and early reperfusion caused marked neutrophil and macrophage tissue accumulation and tumor necrosis factor-alpha production by the injured tissue. Immunofluorescence studies allowed us to localize tumor necrosis factor-alpha predominantly in tissue-infiltrating macrophages. No depression in the global myocardial contractile function was observed, either during ischemia or after reperfusion., Conclusions: These data suggest that the newly recruited macrophages within the ischemic and early post-ischemic myocardium may play a role in promoting neutrophil tissue infiltration and subsequent neutrophil-induced tissue dysfunction by producing tumor necrosis factor-alpha.

Neutrophil accumulation and the consequent production of oxygen-derived free radicals are involved in the pathogenesis of Ischemia-Reperfusion syndrome. In this study we investigated whether a treatment with Vitamin E, which has antioxidant properties, could attenuate the tissue damage by interfering with the influx of neutrophils within the ischemic and reperfused human skeletal muscle. To this purpose, patients undergoing aortic cross-clamping during the surgical repair of aortic abdominal aneurysm were studied as a model of ischemia-reperfusion of the lower limb muscles. Muscle biopsies from the right femoral quadriceps of patients not receiving and receiving Vitamin E pretreatment before surgery were taken: a) after the induction of anaesthesia, as control samples, and b) after a period of ischemia followed by 30 min of reperfusion. The tissue samples were either routinely processed for morphological study and immunohistochemical analysis to detect an altered expression of specific endothelial adhesion proteins, such as E-selectin and ICAM-1. The results obtained showed that Vitamin E administration was able to prevent the accumulation of neutrophils within the ischemic and reperfused muscle. This beneficial effect of Vitamin E was due to its ability to hinder the expression of E-selectin and ICAM-1, molecules known to increase the adhesiveness of endothelium to circulating neutrophils. After treatment with Vitamin E a marked attenuation of the reperfusion injury was also evident. In conclusion, Vitamin E treatment may be considered a valuable tool for protection against the ischemia-reperfusion damage of human skeletal muscle.

Nonsurgical recovery of a preimplantation conceptus from the uterus of a fertile donor woman with transfer to an infertile recipient woman and subsequent pregnancy has been described previously. In this report, we describe the performance of 56 nonsurgical uterine lavages, recovery of 23 ova, and transfer of 17 ova with production of 8 viable pregnancies. Four pregnancies resulted in healthy infants, two aborted, and two are progressing normally. We describe a case of a woman donating an ovum to her sister, with birth of a healthy child. We also describe a viable pregnancy resulting from nonsurgical ovum transfer to a woman with no ovaries. Donation of ova by uterine lavage is a new method of overcoming human infertility; it is nonsurgical, has a low complication rate, and may be repeated several times until it produces pregnancy.