Martina Margheri

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

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

Carbamylated erythropoietin is neuroprotective in an experimental model of traumatic brain injury.

Objective:The well-documented neuroprotective effects of recombinant human erythropoietin (rhEPO) are commonly associated with untoward erythrocyte-stimulating effects (polycythemia), with subsequent risk of thromboembolic complications. A carbamylated-rhEPO (CEPO) derivative, which is neuroprotective but lacks hematopoietic activity, has been recently developed. In this study, we evaluated the neuroprotective capability of CEPO in an in vitro model of cerebral trauma in which rhEPO was previously shown to reduce posttraumatic cell death. Design:Prospective, controlled experiment. Setting:Animal, basic science laboratory. Subjects:Wistar rats, 8 days old. Interventions:Organotypic hippocampal slices, obtained from rat brains, were subjected to a well-characterized model of mechanical injury followed by addition of 10 IU/mL rhEPO, 10–100 IU/mL CEPO, or vehicle (injured control) to the incubation medium at different times to assess the temporal window of therapeutic neuroprotection. Measurements and Main Results:Posttraumatic cell death was quantified at 12, 24, or 48 hrs after injury by measuring propidium iodide fluorescence in the selectively vulnerable CA1 hippocampal area. Posttraumatic injury, observed in injured, vehicle-treated hippocampal slices, was significantly attenuated by addition of either 10 IU/mL rhEPO or 10 IU/mL CEPO. The neuroprotective efficacy of 10 IU/mL rhEPO or CEPO remained intact even when administration was delayed 1 hr after trauma. Qualitative microscopy in semithin sections showed that both rhEPO and CEPO exerted a marked pyramidal neuron-sparing effect. Conclusion:Our study shows that 10 IU/mL CEPO exerts neuroprotective effects comparable with those of rhEPO in an in vitro model of mechanical cerebral trauma. Because CEPO lacks hematopoietic effects and seems to possess a prolonged therapeutic time window, this erythropoietin derivative may represent an exciting new pharmacologic tool in treating patients with mechanical injury to the brain.

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 hearts 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.

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-&agr; 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-&agr; 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.

Integra® dermal matrix bioengineered with platelet rich plasma (PRP) and mesenchymal stromal cells to serve as niche for skin regeneration

Regenerative medicine strategies represent one of the main challenges to improve tissue healing and repair after damage or chronic pathologies. In this perspective, the setting of bioengineered scaffolds, namely synthetic matrices enriched with growth factors and stem cells, is considered a hot issue by numerous research groups. In a previous “in vitro” study we have demonstrated that rat bone marrow-derived mesenchymal stem cells (MSCs) seeded on an artificial dermal matrix Integra®, enriched with platelet-rich plasma (PRP) displayed enhanced proliferative attitude as compared with those cultured in the presence of PRP or on the scaffold alone. To this purpose, in this study we wanted to extend the experimentation by evaluating the efficacy of the bioengineered Integra® in an in vivo model of skin damage in rats. In particular, we used MSC derived from genetically modified rats overexpressing green fluorescent protein (GFP). Rats were divided into different groups: those receiving Integra® or PRP alone, Integra® plus PRP, Integra® plus PRP and MSC, and injured and untreated rats. Skin biopsies, obtained at different times from the injury and the implant, were examined to evaluate the regeneration process and neovascularization pattern of the substrate at light an confocal immunofluorescence microscopy. In parallel experiments we evaluated the ability of MSC to release growth factors, namely VEGF and FGF, and immunomodulatory cytokines, to underscore the paracrine effects of these cells on the surrounding host tissue.

Sialic acid expression in rat kidney urinary tubule after experimental polymicrobic sepsis

Investigations have demonstrated that sialic acid play an important role in maintaining the structure and functionality of the kidney tubular wall. In several studies, performed on some renal pathologies, morpho-functional changes of the tubules showed a correlation with altered expression of sialic acid. In sepsis, morpho-functional changes in the tubules and proteoglycans alteration were also observed; no data are available on the expression of sialic acid in these kidney components. Therefore, the aim of this study was to evaluate the expression of sialic acid in the different tubular regions of the kidney in the early stages of an experimental animal model of polymicrobial sepsis. For this purpose experiments were performed on adult male rats which were randomly assigned to two groups: 1) sham-operated (n=15); 2) Caecal Ligation and Puncture (CLP) (clinically relevant model of polymicrobial infection that mimics human sepsis) (n=19). The groups were divided into 3 subgroups related to 3 time points after CLP or sham-operated: t1=0 h, t2=3 h and t3=7 h. For evidence of sepsis TNF-a plasma level was measured and microbiology of peritoneal fluid was examined with bacteriologic techniques. Urinary protein levels were measured to test the functional damage of the kidney. Sialic acid in glomerular barrier were investigated using MAA, SNA and PNA lectins, in addition to the use of enzymatic and chemical treatments to characterize different glycosidic linkages of the sialoderivatives and to obtain information on their structure. The results showed plasma TNF-a level significantly increased after the CLP induction when compared to sham-operated animals. Bacteriologic techniques revealed a polymicrobial infection after the CLP. The proteinuria was significantly increased in CLP group. Lectin histochemistry showed an increase of sialic acids with linkage α-2,3 and a decrease with linkage α-2,6 in the tubules of septic rats with respect to the control ones. Sialic acids with acetylic groups were in major amount in sepsis group. These findings suggest a correlation between sialic acids altered expression and the characteristics morpho-functional changes in the kidney tubules during sepsis. Moreover, the increase of acetylated sialic acids could be a compensatory response in attempt to impede desialylation by bacterial sialidase and/or probably the action of circulating proinflammatory molecules, such as TNF-a.

Synergistic effects of melatonin and other biological molecules on breast cancer MCF-7 cell functions: possible implications in anticancer therapy

In the recent years, the antineoplastic action of melatonin has been extensively described, including oncostatic effects on different tumors and improvement of chemotherapeutic regimens. However, the mechanisms underlying these phenomena remain unclear and a matter of debate. The aim of the present study was to further investigate the effects of melatonin, administered alone or in combination with other molecules, such as somatostatin and retinoic acid, on MCF-7 human breast cancer cells, in terms of cell viability, proliferation and death. Melatonin 1 μM, 10 μM, 100 μM was administered for 24, 48, 72 hours. It was found that melatonin induced a dose and time-dependent inhibition of cell viability, as judged by MTS assay. This effect was particularly evident at pharmacologic concentration and was greatly potentiated when melatonin was given in association with somatostatin and retinoic acid, suggesting a synergistic effect of these molecules. We also showed that the reduced cell viability in the cells treated with the highest concentration of melatonin, either alone or in combination with somatostatin and retinoic acid, was associated with alteration in the cell cycle, with a reduced number of cells expressing cyclin A. Consistent with these latter data, the treated cells showed alteration in Notch/ Jagged signalling and a significant induction of necrotic cell death. The electrical properties of the treated cells were also analyzed by whole patch clamp in voltageclamp condition. Melatonin induced a decrease of the fast delayed rectifier K+ current and an increase of the slower activating Ca2+-dependent K+ current. Such effects were potentiated when melatonin was given in association with somatostatin and retinoic acid. Taken together, these findings demonstrate, for the first time, that the oncostatic effect of melatonin on MCF-7 cells may be greatly increased by its association with somatostatin and retinoic acid and suggest that these effects are related, at least in part, to changes in the K+ channels type expression and functionality, in cyclin A expression and Notch pathway. These novel findings rise the intriguing hypothesis that melatonin in association with somatostatin and retinoic acid might prove helpful as adjuvant in breast cancer therapy.