Giovanni Nicolao Berta

Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 μM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by N(G)-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 μM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 μM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area (P < 0.001) and limited the postischemic myocardial contracture (P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.

Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch by topical application of resveratrol complexed with 2-hydroxypropyl-β-cyclodextrin.

Oral squamous cell carcinoma (OSCC) develops slowly and it is usually preceded by identifiable oral preneoplastic lesions (OPLs): chemoprevention could be a promising approach. Resveratrol (RV) is a plant-based agent characterized by a strong in vitro antineoplastic action, but this effect has not been clinically confirmed owing to its metabolic inactivation. In order to circumvent this limitation and to improve RV efficacy, it was locally applied and complexed with a protective and solubilising vehicle (2-hydroxypropyl-beta-cyclodextrin, HPbetaCD). The experimentation was performed in vitro on 7,12-dimethylbenz[a]anthracene-induced hamster OSCC cell line (HCPC I) and in vivo in the related animal model, by comparison of two RV-HPbetaCD formulations (cream and mouthwash) and RV alone. Vehicles and RV-formulations were free from toxicity. Antiproliferative action of RV on HCPC I was concentration- and time-dependent, and was improved in HPbetaCD-formulations. In vivo, RV prevented OPL and OSCC appearance and growth. Here, too, HPbetaCD-formulations (mainly mouthwash) demonstrated the best chemopreventive effects in terms of lesions prevalence, multiplicity, dimension, and histological signs of malignancy. HPLC detection of RV corroborated that its action is concentration-correlated and is improved by its inclusion in HPbetaCDs. In summary, our study demonstrates that RV is effective in the chemoprevention of DMBA-induced oral carcinogenesis and when it is complexed with HPbetaCDs its efficacy is significantly improved.

Benefits and drawbacks of open partial horizontal laryngectomies, part B: Intermediate and selected advanced stage laryngeal carcinoma

Cancer of the larynx in the intermediate/advanced stage still presents a major challenge in terms of controlling the disease and preserving the organ. Among therapeutic options, open partial horizontal laryngectomy is proposed as a function‐sparing surgical technique.

ErbB2 Receptor Over-Expression Improves Post-Traumatic Peripheral Nerve Regeneration in Adult Mice

In a transgenic mice (BALB-neuT) over-expressing ErbB2 receptor, we investigated the adult mouse median nerve in physiological and pathological conditions. Results showed that, in physiological conditions, the grip function controlled by the median nerve in BALB-neuT mice was similar to wild-type (BALB/c). Stereological assessment of ErbB2-overexpressing intact nerves revealed no difference in number and size of myelinated fibers compared to wild-type mice. By contrast, after a nerve crush injury, the motor recovery was significantly faster in BALB-neuT compared to BALB/c mice. Moreover, stereological assessment revealed a significant higher number of regenerated myelinated fibers with a thinner axon and fiber diameter and myelin thickness in BALB-neuT mice. At day-2 post-injury, the level of the mRNAs coding for all the ErbB receptors and for the transmembrane (type III) Neuregulin 1 (NRG1) isoforms significantly decreased in both BALB/c and BALB-neuT mice, as shown by quantitative real time PCR. On the other hand, the level of the mRNAs coding for soluble NRG1 isoforms (type I/II, alpha and beta) increased at the same post-traumatic time point though, intriguingly, this response was significantly higher in BALB-neuT mice with respect to BALB/c mice. Altogether, these results suggest that constitutive ErbB2 receptor over-expression does not influence the physiological development of peripheral nerves, while it improves nerve regeneration following traumatic injury, possibly through the up-regulation of soluble NRG1 isoforms.

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents.

Among the oxidative breakdown products of omega-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor beta1 (TGFbeta1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFbeta1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFbeta1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Isolation and Characterization of a Spontaneously Immortalized Multipotent Mesenchymal Cell Line Derived from Mouse Subcutaneous Adipose Tissue

The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ (null) mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ (null) monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.

Injured cardiomyocytes promote dental pulp mesenchymal stem cell homing

BACKGROUND The heart is unable to regenerate its tissues after severe injuries. Stem cell therapy appears to be one of the most promising approaches, though preclinical results are hitherto contradictory and clinical trials scanty and/or limited to phase-I. The limited knowledge about stem cell early homing in infarcted cardiac tissues can concur to this scenario. METHODS The stem cell migration was assessed in in-vitro and ex-vivo models of heart ischemia, employing a rat dental pulp stem cell line (MUR-1) that shares the same ontogenic progenitors with portions of the heart, expresses markers typical of cardiac/vascular-like progenitors and is able to differentiate into cardiomyocytes in-vitro. RESULTS Here, we demonstrated that the MUR-1 can reach the injured cells/tissue and make contacts with the damaged cardiomyocytes, likely through Connexin 43, N-cadherin and von Willebrand Factor mediated cell-cell interactions, both in in-vitro and ex-vivo models. Furthermore, we found that SDF-1, FGF-2 and HGF, but not VEGF are involved as chemotactic factors in MUR-1 migration, notifying a similarity with neural crest cell behavior during the organogenesis of both the splanchnocranium and the heart. CONCLUSIONS Herein we found a similarity between what happens during the heart organogenesis and the early migration and homing of MUR-1 cells in ischemic models. GENERAL SIGNIFICANCE The comprehension of molecular aspects underlying the early phases of stem cell migration and interaction with damaged organ contributes to the future achievement of the coveted stem cell-mediated organ regeneration and function preservation in-vivo.

Oncologic outcomes of supratracheal laryngectomy: Critical analysis

Laryngeal cancer management should pursue function‐sparing therapeutic options. Even though demolitive surgery provides better control of disease at intermediate to advanced stages when compared to chemoradiotherapy, it does not preserve laryngeal function. Supratracheal partial laryngectomy has been described as a function‐sparing surgical technique for laryngeal cancer with subglottic extension.

Potentiation of amyloid-β peptide neurotoxicity in human dental-pulp neuron-like cells by the membrane lipid peroxidation product 4-hydroxynonenal

Lipid peroxidation is generally considered as primarily implicated in the pathogenesis of Alzheimers disease (AD); one of its more reactive end products, 4-hydroxynonenal (HNE), has been shown to cause neuron dysfunction and degeneration. HNE production in the brain is stimulated by the amyloid-β peptide (Aβ), whose excessive accumulation in specific brain areas is a hallmark of AD. Conversely, Aβ production is up-regulated by this multifunctional aldehyde. Findings reported here point to the ability of HNE and Aβ to interact, with consequent potentiation of Aβs cytotoxicity as determined in vitro using neuron-like cells derived from human dental-pulp progenitor cells. Preincubation of cells with the aldehyde markedly up-regulated Aβ uptake and intracellular accumulation, by overexpressing two of the three components of the plasma membrane multireceptor complex CD36/CD47/β1-integrin: experimental and clinical data indicate that intraneuronal accumulation of Aβ is an early event possibly playing a primary role in AD pathogenesis. That HNE-mediated overexpression of CD36 and β1-integrin, which plays a key role in HNEs potentiating Aβ neurotoxicity, in terms of necrosis, was confirmed when this effect was prevented by specific antibodies against the two receptors.

Self-Renewal and Multipotency Coexist in a Long-Term Cultured Adult Rat Dental Pulp Stem Cell Line: An Exception to the Rule?

The stemness state is characterized by self-renewal and differentiation properties. However, stem cells are not able to preserve these characteristics in long-term culture because of the intrinsic fragility of their phenotype easily undergoing senescence or neoplastic transformation. Furthermore, although isolated from the same original tissue using similar protocols, adult stem cells can display dissimilar phenotypes and important cell clone/species contamination. Finally, the lack of a clear standardization contributes to complicate the comprehension about the stemness condition. In this context, cell lines displaying a particularly stable phenotype must be identified to define one or multiple benchmarks against which other stem cell lines could be reliably assessed. The present paper demonstrates that it is possible to isolate from the rat dental pulp a stem cell line (MUR-1) that does not display neoplastic transformation in long-term culture. MUR-1 cells stably express a broad range of stemness markers and are able to differentiate into adipogenic, osteogenic, chondrogenic, neurogenic, and cardiomyogenic lineages independently of the culture passages. Moreover, serial in vitro passages have not changed their immunophenotype, proliferation capacity, or differentiation potential. The uniqueness of these characteristics candidates MUR-1 as a model to reliably improve the understanding of the mechanisms governing the stem cell fate in the same as well as in other stem cell populations.