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Couples at risk for an inherited disorder often have several pregnancy interruptions because of affected fetuses and difficulty in achieving their desired family. We evaluated the efficiency and acceptability of selective fetal reduction after chorionic villus sampling (CVS) of multiple pregnancy induced by ovarian stimulation and gamete intra-Fallopian transfer (GIFT). This approach has been offered to nine patients at risk of Mendelian diseases and one patient carrier of reciprocal translocation. The acceptance has been very high (90%). One patient at risk of an autosomic recessive disease opted for artificial donor insemination, one conceived spontaneously, and one was a poor responder to ovarian stimulation. Seven patients actually underwent a single GIFT procedure with six achieving pregnancy (86%), all but two being a multiple pregnancy (67%). All pregnancies concluded uneventfully at term and newborns were alive and healthy. Prenatal diagnosis, including fetal karyotyping in all cases, was performed at 8.5–11.5 weeks of gestation and confirmed either on amniotic fluid aspirated at reduction or at birth. The number of fetuses was reduced to one or two because the genetic disease was present and/or to reduce the risk of premature delivery and improve the likelihood of successful pregnancy. The new approach seems to be highly effective and might be considered a practical and useful alternative to preimplantation genetic diagnosis. [ABSTRACT FROM PUBLISHER]

A single attempt of gamete intra-Fallopian transfer was performed in 106 patients, who had previously failed to become pregnant after a certain number of artificial donor inseminations. A maximum of six unselected oocytes were transferred into the tubes. Fifty-five clinical pregnancies (51.9%) were obtained. There were 11 multiple pregnancies (20%) and 10 miscarriages (18.1%). Only one ectopic pregnancy occurred (1.8%). The age of the patients and the previous number of failed inseminations did not have any significant influence on the outcome of the procedure, whereas the number of transferred oocytes had a proportional, significant correlation with the pregnancy rate. [ABSTRACT FROM PUBLISHER]

Growing interest in preimplantation genetic diagnosis has indicated uterine flushing as one method for obtaining human preimplantation embryos. To date, our institution has performed non-surgical uterine flushing to donate the recovered embryos to infertile recipients. We performed 127 flushings in 127 cycles using a modified urinary bladder catheter. Using the donors' natural cycles, a single ovum was recuperated in 37 out of 88 flushings. In 17 flushings, clomiphene citrate was given to the donors and 14 ova were found in nine positive recoveries. Human menopausal gonado-trophins were administered to the donors in 22 flushings and 22 ova were located in 14 positive recoveries. In total, 22 blastocysts, 11 morulae and 13 pre-embryos at the 2- to 16-cell stages were found. When tranferred, these embryos gave rise to 18 clinical pregnancies in the recipients (40.9% of the transfers; 14.1% of the flushings). In comparison with natural cycles, superovulation of donors did not significantly increase the recipients' pregnancy rate. At present, non-surgical recovery of uterine pre-embryos does not seem to carry much potential as a tool for infertility treatment, or for genetic diagnosis. This is because currently available alternative methods are more successful. [ABSTRACT FROM PUBLISHER]

Thirteen procedures of oocyte donation by the gamete intra-Fallopian transfer (GIFT) technique are described. The patients included six women with premature ovarian failure, four normally cycling women with unexplained infertility who responded poorly to super-ovulation induction in preparation for GIFT, and lastly one woman carrier of a 16/21 balanced translocation. Two patients had oocytes donated on two occasions. Oocyte donors were recruited either among the patients' relatives ( = 4), or among GIFT or IVF patients ( = 8), who altruistically donated their extra oocytes. Donors were superovulated and oocytes collected laparoscopically or vaginally under ultrasound guidance. Donors did not suffer any complications. Recipients were given exogenous oestrogens, and exogenous progesterone was added from the day of donation. Seven clinical pregnancies were obtained (53.8% per attempt); one set of triplets aborted at 14 weeks. Donation took place on replacement day 12–18 and pregnancies were obtained in patients receiving oocytes throughout this temporal window. The increasing availability of embryo-freezing facilities will probably reduce the number of ova available for donation. Therefore, the patients' families may become a precious source of donated eggs, especially for those patients having large families, with strong family ties. [ABSTRACT FROM PUBLISHER]

This study describes eleven procedures of non-surgical recovery of preimplantation conceptuses from the uteri of eight fertile donor women and the transfer of the recovered pre-embryos into the uteri of infertile recipients with primary ovarian fallure, or with hypothalamic-pituitary failure. Conjugated oestrogens and progesterone were administered to recipients as replacement steroids during the first 20 weeks of pregnancy, in order to produce a secretory endometrium allowing nidation and development of the transferred conceptuses. Five pre-embryo transfers resulted in two viable pregnancies and one early abortion. Non-surgical pre-embryo transfer is a simple and practical method for providing donated ova to women lacking efficient ovarian function. [ABSTRACT FROM PUBLISHER]

In consideration of the close relationship between radicular cysts and alveolar bone, it is important to evaluate the potential involvement of the neighbouring bone tissue in such lesions. In the present study, using cytochemical, immunocytochemical and morphological analyses, presumptive osteoclast precursors were revealed in the connective tissue of radicular cyst capsules. The osteoclastic nature of these cells was postulated by their positive staining for the enzyme tartrate-resistant acid phosphatase (TRAP) and by the expression of vitronectin receptor (VnR) on their cell surface. However, these cells did not express the vacuolar-type proton pump, suggesting that they may represent early osteoclast precursors infiltrating the cyst capsule. Cysts also contained activated small blood vessels whose endothelial cells expressed the VnR. This integrin receptor is important in the adhesion of preosteoclasts to the endothelial lining, a necessary step for their emigration out of the vasculature. Therefore, the intracystic vessels could represent a substrate for preosteoclast recruitment. These precursor cells may then reach the perialveolar bone surface and contribute to bone demolition together with those recruited by the resorbing surfaces. The bone-destroying potential of radicular cysts was confirmed by the presence of numerous osteoclasts with large resorption areas on the perialveolar bone surfaces exposed to the cyst capsules. The resorbed surfaces were usually located around the vascular canals of the Haversian systems. [ABSTRACT FROM AUTHOR]

In an attempt to verify the nature of bone resorption processes on the bone surrounding radicular cysts, fragments of cysts with the adjacent bone tissue were studied by morphological, cytochemical and ultrastructural methods. Cyst fluid was analysed for its content of cytokines with osteolytic activity. The cyst wall exhibited several connective tissue extensions which penetrated the adjacent bone. Numerous multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells were seen at the tip of the intraosseous extensions of the cyst capsule and in direct contact with the bone tissue. Typical resorption lacunae were identified on the bone surface by scanning electron microscopy. Moreover, mononuclear TRAP-positive cells were seen within the cyst capsule. High levels of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) were detected in the cyst fluids. In conclusion, active bone resorption may contribute significantly to the growth of these lesions within the jaws. [ABSTRACT FROM AUTHOR]

A procedure has been devised in order to establish a pregnancy in a patient with severe, untreatable tubal infertility who had unsuccessfully undergone tubal reconstructive surgery and repeated in-vitro fertilization procedures. This technique, which involves a volunteer, fertile woman, results from the combination of two infertility treatments: namely gamete intra-Fallopian transfer and uterine flushing. The gametes of the infertile couple were introduced into the distal tube of a synchronized volunteer woman; after 5 days, the embryos, recovered by lavage of the volunteer's uterus, were transferred into the patient's uterus with a resulting pregnancy and delivery. [ABSTRACT FROM PUBLISHER]

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]

Objective: We previously showed that photoablative laser therapy followed by multiple photodynamic cycles (PAPD) after scaling/root planing (SRP) improved healing of periodontitis patients as compared with conventional SRP after 1-year follow-up. This study reports the favorable results of PAPD plus SRP in patients with chronic periodontitis at a 4-year follow-up., Materials and Methods: Twenty-four patients were studied. Maxillary left or right quadrants were randomly assigned to PAPD laser treatment or sham-treatment and SRP. PAPD consisted of: (1) photoablative intra/extrapocket de-epithelization with diode laser (λ 810 nm, 1 W), and (2) photodynamic treatments (4-10 weekly) with diode laser (λ 635 nm, 100 mW) and 0.3% methylene blue as photoactive antiseptic, performed after SRP. Sham treatment was performed with switched- off laser. Probing depth (PD), clinical attachment level (CAL), and bleeding-on-probing (BOP) were evaluated. Additional disease markers, namely polymorphonuclear leukocytes (PMN), erythrocytes (RBC), damaged epithelial cells (DEC), and bacteria were assayed by cytofluorescence on gingival exfoliative samples., Results: At 4-year follow-up, PAPD plus SRP significantly improved PD, CAL, and BOP, as well as bacterial contamination and PMN-RBC shedding in the exfoliative samples, compared with sham treatment plus SRP. This effect was greater than that observed at 1-year follow-up., Conclusions: PAPD plus SRP provided significant, durable improvement of chronic periodontitis over sham treatment plus SRP alone.

Big efforts have been dedicated up to now to identify novel targets for cancer treatment. The peculiar biophysical profile and the atypical ionic channels activity shown by diverse types of human cancers suggest that ion channels may be possible targets in cancer therapy. Earlier studies have shown that melatonin exerts an oncostatic action on different tumors. In particular, it was shown that melatonin was able to inhibit growth/viability and proliferation, to reduce the invasiveness and metastatic properties of human estrogen-sensitive breast adenocarcinoma MCF-7 cell line cultured in growth medium, with substantial impairments of epidermal growth factor (EGF) and Notch-1-mediated signaling. The purpose of this work was to evaluate on MCF-7 cells the possible effects of melatonin on the biophysical features known to have a role in proliferation and differentiation, by using the patch-clamp technique. Our results show that in cells cultured in growth as well as in differentiation medium melatonin caused a hyperpolarization of resting membrane potential paralleled by significant changes of the inward Ca(2+) currents (T- and L-type), outward delayed rectifier K(+) currents and cell capacitance. All these effects are involved in MCF-7 growth and differentiation. These findings strongly suggest that melatonin, acting as a modulator of different voltage-dependent ion channels, might be considered a new promising tool for specifically disrupting cell viability and differentiation pathways in tumour cells with possible beneficial effects on cancer therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.)

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.)

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., (© 2014 The Authors. Wound Repair and Regeneration published by . Wiley Periodicals, Inc. on behalf of The Wound Healing Society.)

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., (© FASEB.)

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.

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.

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., (Copyright © 2014. Published by Elsevier Inc.)

Background: The use of lasers in periodontology is a matter of debate, mainly because of the lack of consensual therapeutic protocols. In this randomized, split-mouth trial, the clinical efficacy of two different photoablative dental lasers, erbium:yttrium-aluminum-garnet (Er:YAG) and diode, for the treatment of gingival hyperpigmentation is compared., Methods: Twenty-one patients requiring treatment for mild-to-severe gingival hyperpigmentation were enrolled. Maxillary or mandibular left or right quadrants were randomly subjected to photoablative deepithelialization with either Er:YAG or diode laser. Masked clinical assessments of each laser quadrant were made at admission and days 7, 30, and 180 postoperatively by an independent observer. Histologic examination was performed before and soon after treatment and 6 months after irradiation. Patients also compiled a subjective evaluation questionnaire., Results: Both diode and Er:YAG lasers gave excellent results in gingival hyperpigmentation. However, Er:YAG laser induced deeper gingival tissue injury than diode laser, as judged by bleeding at surgery, delayed healing, and histopathologic analysis. The use of diode laser showed additional advantages compared to Er:YAG in terms of less postoperative discomfort and pain., Conclusions: This study highlights the efficacy of diode laser for photoablative deepithelialization of hyperpigmented gingiva. It is suggested that this laser can represent an effective and safe therapeutic option for gingival photoablation.

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin 43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects is independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.