on Histology and histopathology
by Forte A, Della Corte A, Grossi M, Finicelli M, Bancone C, Provenzano R, Pepino P, Nappi GA, De Feo M, Galderisi U, Cotrufo M, Cipollaro M
Knowledge of the characteristics of the normal human aorta has been constrained by lack of data on fresh aortic tissue, especially from healthy individuals. In this study, the gene expression and morphological characteristics of the thoracic ascending aorta (AA) of healthy organ donors have been evaluated, with the aim of providing reference data for the analysis of pathological AAs. We analysed by RT-PCR the differential expression of mRNAs coding for myocardin, smoothelin, alpha-smooth muscle actin (alpha-SMA) and the ED-A isoform of fibronectin (ED-A FN) in AA specimens from donors, integrating the results with immunohistochemical analysis of the same targets. Morphological and morphometric characteristics of the AAs were also evaluated. In order to account for possible regional variations in wall structure, the convexity of the aortic profile was compared to the concavity. No differences in gene expression occurred for any of the target genes between the concavity and the convexity of AAs. Immunohistochemistry revealed a different distribution of total FN and of its ED-A isoform in the media and in the intima. Smoothelin is expressed by the majority of cells in the media, with some positive cells also in the intima. Alpha-SMA is expressed in all the tunicae. Immunohistochemistry also revealed in the convexity of 50% of AAs the presence of discrete areas in the subadventital media with altered structure and cell morphology and with altered gene expression, resulting positive for ED-A FN and alpha-SMA, but not for smoothelin, indicating the occurrence of early lesions also in macroscopically healthy AAs.
on Journal of cellular physiology
by Di Bernardo G, Alessio N, Dell'Aversana C, Casale F, Teti D, Cipollaro M, Altucci L, Galderisi U
Histone deacetylase inhibitors (HDACis) have received considerable attention for their anti-tumoral properties. We report here the effects of two HDACis, SAHA and MS-275, on the biology of mesenchymal stem cells (MSCs). It is well known that HDACis trigger both DNA damage responses and actual DNA damage in cancer cells. On this premise, we evaluated HDACis influence on DNA damage pathways in MSCs. We analyzed a panel of genes involved in the regulation of base and nucleotide excision repair, mismatch repair, and double strand break repair. That a majority of the analyzed genes displayed significant expression changes upon incubation with SAHA or MS-275 suggested that regulation of their expression is greatly affected by HDACis. The complex expression pattern, with some genes up-regulated and other under-expressed, did not allow to foresee whether these changes allow cells cope with stressful DNA damaging stimuli. Furthermore, we evaluated the biological outcome following treatment of MSCs with DNA damaging agents (H(2)O(2) and UV) in presence of HDACis. In these settings, MSCs treated with H(2)O(2) or UV radiation underwent apoptosis and/or senescence, and pre-incubation with HDACi exacerbated cell death phenomena. Accordingly, the number of cells harboring 8-oxo-7,8-dihydroguanine (8oxodG), a hallmark of DNA oxidative damage, was significantly higher in samples incubated with HDACis compared to controls. In summary, our findings suggest that SAHA and MS-275, even at low effective doses, can alter the biology of MSCs, diminishing their ability to survive the effects of DNA-damaging agents.
on Journal of stem cells & regenerative medicine
by Siniscalco D, Giordano C, Galderisi U, Luongo L, Alessio N, Di Bernardo G, de Novellis V, Rossi F, Maione S
by Alessio N, Squillaro T, Cipollaro M, Bagella L, Giordano A, Galderisi U
We focused our attention on brahma-related gene 1 (BRG1), the ATPase subunit of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, and analyzed its role in mesenchymal stem cell (MSC) biology. We hypothesized that deviation from the correct concentration of these proteins, which act at the highest level of gene regulation, may be deleterious for cells. We wanted to know what would happen if a cell had to cope with altered regulation of gene expression, either by upregulation or downregulation of BRG1. We assumed that cells would try to restore homeostasis or, alternatively, that the event could trigger senescence/apoptosis phenomena. To this end, in MSCs, we silenced BRG1gene. Knockdown of BRG1 expression induced a significant increase in senescent cells and decrease in apoptotic cells. It is interesting that BRG1 downregulation also induced an increase in heterochromatin. At the molecular level, these phenomena were associated with activation of retinoblastoma-like protein 2 (RB2)/P130- and P53-related pathways. Senescence was accompanied by reduced expression of some stemness-related genes. This is consistent with our previous research, which showed that BRG1 upregulation by ectopic expression also induced senescence processes. Together, these data suggest that BRG1 belongs to a class of genes whose expression is tightly regulated; hence, subtle alterations in BRG1 activity seem to negatively affect mechanisms regulating chromatin status and, in turn, impair cellular physiology.
on The neuroradiology journal
by Conforti R, Porto A, Cirillo M, Sgambato A, Galderisi S, Cirillo S
A 16-year-old female who manifested psychotic symptoms underwent CT and MRI for the evaluation of an incidentally discovered asymptomatic palpable mass of the right occipital region of the skull. The correlation between clinical and radiological data and biopsy data led to the diagnosis of eosinophilic granuloma. The radiological finding is discussed and reviewed in relation to clinical aspects and literature data.
on Journal of cellular biochemistry
by Calarco A, Petillo O, Bosetti M, Torpedine A, Cannas M, Perrone L, Galderisi U, Melone MA, Peluso G
Growth factors and other regulatory molecules are required to direct differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) along specific lineages. However, the therapeutic use of growth factors is limited by their susceptibility to degradation, and the need to maintain prolonged local release of growth factor at levels sufficient to stimulate hMSC. The aim of this study was to investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus enhance hMSC stimulation. To this aim, we synthesized cationic polyelectrolyte polymers covalently and non-covalently anchored to HS and evaluated their effect on hMSC proliferation. Polymers non-covalently bound to HS resulted in the release of an HS/FGF-2 complex rather than FGF-2 alone. The release of this complex significantly restored hMSC proliferation, which was abolished in serum-free medium and only partially restored by the release of FGF-2 alone as occurred with polymer covalently bound to HS. We also demonstrate that exposure to HS/FGF-2 during early growth but not during post-confluence is essential for hMSC differentiation down the fibroblast lineage, which suggests that both factors are required to establish the correct stem cell commitment that is necessary to support subsequent differentiation. In conclusion, the delivery platform described here is a step towards the development of a new class of biomaterial that enables the prolonged, non-covalent binding and controlled delivery of growth factors and cofactors without altering their potency.
on FASEB journal : official publication of the Federation of American Societies for Experimental Biology
by Squillaro T, Alessio N, Cipollaro M, Renieri A, Giordano A, Galderisi U
DNA methylation is an epigenetic modification that occurs almost exclusively on CpG dinucleotides. MECP2 is a member of a family of proteins that preferentially bind to methylated CpGs. We analyzed the contribution of MECP2 to the physiology of mesenchymal stem cells (MSCs). Partial silencing of MECP2 in human MSCs induced a significant reduction of S-phase cells, along with an increase in G(1) cells. These changes were accompanied by a reduction of apoptosis, the triggering of senescence, a decrease in telomerase activity, and the down-regulation of genes involved in maintaining stem cell properties. Senescence appeared to rely on impairment of DNA damage repair and seemed to occur through RB- and P53-related pathways. The effects of MECP2 silencing could be related to the modification of the DNA methylation status. Our results indicate that the silencing of MECP2 induces an increase in methylated cytosines in the genome. Nevertheless, MECP2 partial silencing did not change the methylation of promoters, whose expression is affected by MECP2 down-regulation.
on Clinical science (London, England : 1979)
by Forte A, Finicelli M, Grossi M, Vicchio M, Alessio N, Santé P, De Feo M, Cotrufo M, Berrino L, Rossi F, Galderisi U, Cipollaro M
Restenosis rate following vascular interventions still limits their long-term success. Oxidative stress plays a relevant role in this pathophysiological phenomenon, but less attention has been devoted to its effects on DNA damage and to the subsequent mechanisms of repair. We analysed in a model of arteriotomy-induced stenosis in rat carotids the time-dependent expression of DNA damage markers and of DNA repair genes, together with the assessment of proliferation and apoptosis indexes. The expression of the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine was increased at 3 and 7 days after arteriotomy, with immunostaining distributed in the injured vascular wall and in perivascular tissue. The expression of the DNA damage marker phospho-H2A.X was less relevant but increasing from 4 hrs to 7 days after arteriotomy, with immunostaining prevalently present in the adventitia and, to a lesser extent, in medial smooth muscle cells at the injury site. RT-PCR indicated a decrease of 8 out of 12 genes of the DNA repair machinery we selected from 4 hrs to 7 days after arteriotomy with the exception of increased Muyth and Slk genes (p<0.05). Western Blot revealed a decrease of p53 and catalase at 3 days after arteriotomy (p<0.05). A maximal 7% of BrdU-positive cells in endothelium and media occurred at 7 days after arteriotomy, while the apoptotic index peaked at 3 days after injury (p<0.05). Our results highlight a persistent DNA damage presumably related to a temporary decreased expression of the DNA repair machinery and of the antioxidant enzyme catalase, playing a role in stenosis progression.
on Analytical and bioanalytical chemistry
by Del Gaudio S, Cirillo A, Di Bernardo G, Galderisi U, Cipollaro M
DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.
on Cellular and molecular life sciences : CMLS
by Siniscalco D, Giordano C, Galderisi U, Luongo L, Alessio N, Di Bernardo G, de Novellis V, Rossi F, Maione S
Neuropathic pain is a very complex disease, involving several molecular pathways. Current available drugs are usually not acting on the several mechanisms underlying the generation and propagation of pain. We used spared nerve injury model of neuropathic pain to assess the possible use of human mesenchymal stem cells (hMSCs) as anti-neuropathic tool. Human MSCs were transplanted in the mouse lateral cerebral ventricle. Stem cells injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once transplanted in cerebral ventricle. Anti-nociceptive effect was detectable from day 10 after surgery (6 days post cell injection). Human MSCs reduced the mRNA levels of the pro-inflammatory interleukin IL-1beta mouse gene, as well as the neural beta-galactosidase over-activation in prefrontal cortex of SNI mice. Transplanted hMSCs were able to reduce astrocytic and microglial cell activation.