Velia D’Agata

Gene expression profiles of heme oxygenase isoforms in the rat brain.

In the last decade the heme oxygenase (HO) system has been strongly highlighted for its potential significance in maintaining cellular homeostasis. Nevertheless the physiological relevance of the three isoforms cloned to date, HO-1, HO-2 and HO-3, and their reciprocal interrelation have been poorly understood. In the brain the HO system has been reported to be very active and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. To discriminate the regional and cellular distribution of HO isoforms in the CNS, we have developed a real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) protocol. With this highly sensitive methodology we have assessed for the first time the expression of all known HO isoform mRNAs in different rat brain areas. Although they presented a highly dissimilar range of expression, with HO-2>HO-1>HO-3, all three HO isoform transcripts demonstrated high level of expression in the cerebellum and the hippocampus, showing in a different scale, a strikingly parallel distribution gradient. We have also quantified the expression of HO mRNAs in primary culture of cortical neurons and type I astrocytes. While HO-1 and HO-2 were detected in both cellular types, HO-3 transcript was uniquely found in astrocytes. To further investigate the regional brain expression of this elusive and poorly studied isoform, we have performed in situ hybridization using an HO-3 specific riboprobe. HO-3 mRNA was expressed mainly in hippocampus, cerebellum and cortex. The initial elucidation of HO isoforms distribution should facilitate further research on their pathophysiological role in the nervous system.

Potential role of probiotics on colorectal cancer prevention

BackgroundColorectal cancer represents the most common malignancy of the gastrointestinal tract. Owing to differences in dietary habits and lifestyle, this neoplasm is more common in industrialized countries than in developing ones. Evidence from a wide range of sources supports the assumption that the link between diet and colorectal cancer may be due to an imbalance of the intestinal microflora.DiscussionProbiotic bacteria are live microorganisms that, when administered in adequate amounts, confer a healthy benefit on the host, and they have been investigated for their protective anti-tumor effects. In vivo and molecular studies have displayed encouraging findings that support a role of probiotics in colorectal cancer prevention.SummarySeveral mechanisms could explain the preventive action of probiotics against colorectal cancer onset. They include: alteration of the intestinal microflora; inactivation of cancerogenic compounds; competition with putrefactive and pathogenic microbiota; improvement of the host’s immune response; anti-proliferative effects via regulation of apoptosis and cell differentiation; fermentation of undigested food; inhibition of tyrosine kinase signaling pathways.

Early changes in pituitary adenylate cyclase-activating peptide, vasoactive intestinal peptide and related receptors expression in retina of streptozotocin-induced diabetic rats.

The retinal expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) and their receptors was investigated in early streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection (60 mg/kg i.p.). PACAP, VIP and their receptors in nondiabetic control and diabetic retinas were assayed by quantitative real-time PCR and Western blot 1 and 3 weeks after STZ injection. Effects of intravitreal treatment with PACAP38 on the expression of the two apoptotic-related genes Bcl-2 and p53 were also evaluated. PACAP and VIP, as well as VPAC1 and VPAC2 receptors, but not PAC1 mRNA levels, were transiently induced in retinas 1 week following STZ. These findings were confirmed by immunoblot analyses. Three weeks after the induction of diabetes, significant decreases in the expression of peptides and their receptors were observed, Bcl-2 expression decreased and p53 expression increased. Intravitreal injection of PACAP38 restored STZ-induced changes in retinal Bcl-2 and p53 expression to nondiabetic levels. The initial upregulation of PACAP, VIP and related receptors and the subsequent downregulation in retina of diabetic rats along with the protective effects of PACAP38 treatment, suggest a role for both peptides in the pathogenesis of diabetic retinopathy.

Effects of PACAP and VIP on hyperglycemia-induced proliferation in murine microvascular endothelial cells.

Hyperglycemia is implicated both in micro- and macro-vascular complications in diabetes mellitus. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two known nonclassic regulators of angiogenesis, although their biological role on endothelial cell proliferation remains poorly defined. In the present study we hypothesized that either peptides might play an inhibitory role on hyperglycemia-induced cell growth. To this end, we investigated the effect of both PACAP and VIP on cell proliferation in murine microvascular endothelial cells (H5V) cultured both under euglycemic and hyperglycemic conditions (5 and 25 mM glucose, respectively) for 24, 48 h, 7 and 15 days. Results demonstrated that high glucose treatment induced a time-dependent increase in cell viability after 48 h (p<0.05), which was much more evident after 7 and 15 days (p<0.001). Similar effects were observed in cell proliferation, although significant changes were obtained after prolonged exposures to high glucose (7 and 15 days; p<0.001). The proliferative response to the glucose-enriched environment was correlated to changes in the expression of PAC1 and, to a minor extent, to VPAC2, but not VPAC1 receptors, as measured by quantitative real-time PCR. These results were further confirmed by Western blot and immunofluorescence analyses. Interestingly, 10⁻⁷ M PACAP or VIP treatment significantly attenuated hyperglycemia-induced increase in cell viability and proliferation after 7 and 15 days. Taken together, our findings demonstrate that both PACAP and VIP peptides exert an inhibitory activity on hyperglycemia-induced endothelial cell proliferation, thus suggesting that the effect might be mediated by PAC1 and VPAC2 receptors.

Ameliorative effect of PACAP and VIP against increased permeability in a model of outer blood retinal barrier dysfunction.

Breakdown of outer blood retinal barrier (BRB) due to the disruption of tight junctions (TJs) is one of the main factors accounting for diabetic macular edema (DME), a major complication of diabetic retinopathy. Previously it has been shown that PACAP and VIP are protective against several types of retinal injuries. However, their involvement in the maintenance of outer BRB function during DME remains uncovered. Here, using an in vitro model of DME, we explored the effects of both PACAP and VIP. Human retinal pigment epithelial cells (ARPE19) were cultured for 26 days either in normal glucose (5.5 mM, NG) or in high glucose (25 mM, HG). In addition, to mimic the inflammatory aspect of the diabetic milieu, cells were also treated with IL-1β (NG+IL-1β and HG+IL-1β). Effects of PACAP or VIP on cells permeability were evaluated by measuring both apical-to-basolateral movements of fluorescein isothyocyanate (FITC) dextran and transepithelial electrical resistance (TEER). Expression of TJ-related proteins was evaluated by immunoblot. Results demonstrated that NG+IL-1β and, to a greater extent, HG+IL-1β significantly increased FITC-dextran diffusion, paralleled by decreased TEER. PACAP or VIP reversed both of these effects. Furthermore, HG+IL-1β-induced reduction of claudin-1 and ZO-1 expression was reversed by PACAP and VIP. Occludin expression was not affected in any of the conditions tested. Altogether, these finding show that both peptides counteract HG+IL-1β-induced damage in ARPE19 cells, suggesting that they might be relevant to the maintenance of outer BRB function in DME.

Elevated serum levels of Chromogranin A in hepatocellular carcinoma

BackgroundDuring the past three decades, the incidence of hepatocellular carcinoma in the United States has tripled. The neuroendocrine character has been observed in some tumor cells within some hepatocellular carcinoma nodules and elevated serum chromogranin A also been reported in patients with hepatocellular carcinoma. The aim of this work was to investigate the role of serum concentration of chromogranin A in patients with hepatocellular carcinoma at different stages.MethodsThe study population consisted of 96 patients (63 males and 33 females age range 52-84) at their first hospital admission for hepatocellular carcinoma. The control group consisted of 35 volunteers (20 males and 15 females age range 50-80). The hepatocellular carcinoma patients were stratified according the Barcelona-Clinic Liver Cancer classification. Venous blood samples were collected before treatment from each patients before surgery, centrifuged to obtain serum samples and stored at -80° C until assayed.ResultsThe chromogranin A serum levels were elevated (> 100 ng/ml) in 72/96 patients with hepatocellular carcinoma. The serum levels of chromogranin A were significantly correlated (p<0.05) with alpha-fetoprotein. In comparison with controls, the hepatocellular carcinoma patients showed a significant increase (p<0.001) vs controls. The chromogranin A levels in the Barcelona staging of hepatocellular carcinoma was higher in stage D compared to stage C (p<0.01), to stage B (p<0.001), and to stage A (p<0.001).ConclusionsMolecular markers, such as chromogranin A, could be very useful tools for hepatocellular carcinoma diagnosis. However the molecular classification should be incorporated into a staging scheme, which effectively separated patients into groups with homogeneous prognosis and response to treatment, and thus serves to aid in the selection of appropriate therapy.

Antiproliferative Effects of PACAP and VIP in Serum-Starved Glioma Cells

Emerging evidence have suggested that calorie restriction (CR) is a reliable method to decrease cancer development since it produces changes in tumor microenvironment that interfere with cell proliferation, tissue invasion, and formation of metastases. Studies on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in cancer cells indicate that their influence on cell growth is either cell type specific or dependent on culture conditions. Evidence showing the effect of PACAP and VIP in glioma cells grown under conditions mimicking CR are currently unavailable. Therefore, we explored the effects of both PACAP and VIP in C6 glioma cells either grown in a normal growth medium or exposed to serum starvation, to resemble an acute condition of CR. Cell viability, expression of proteins related to cell proliferation (cyclin D1), apoptosis (Bcl2, p53, and cleaved caspase-3), and cell malignancy (GFAP and nestin) were assessed by MTT assay, immunoblot, and immunolocalization, respectively. Results demonstrated that CR significantly decreased cell proliferation, reduced levels of cyclin D1 and Bcl2, and increased the expression of p53 and cleaved caspase-3. Surprisingly, all of these CR-driven effects were further exacerbated by PACAP or VIP treatment. We also found that PACAP or VIP prevented GFAP decrease caused by CR and further reduced the expression of nestin, a prognostic marker of malignancy. In conclusion, these data demonstrate that PACAP and VIP possess antiproliferative properties against glioma cells that depend on the specific culture settings, further supporting the idea that CR might offer new avenues to improve peptide-oriented glioma cancer treatment.

Centenarians and supercentenarians: a black swan. Emerging social, medical and surgical problems

The Black Swan Theory was described by Nassim Nicholas Taleb in his book “The Black Swan”. This theory refers to “high-impact, hard-to-predict, and rare events beyond the realm of normal expectations”. According to Taleb’s criteria, a Black Swan Event is a surprise, it has a major impact and after the fact, the event is rationalized by hindsight, as if it had been expected. For most of human history centenarians were a rare and unpredictable phenomenon. The improvements of the social-environmental conditions, of medical care, and the quality of life caused a general improvement of the health status of the population and a consequent reduction of the overall morbidity and mortality, resulting in an overall increase of life expectancy. The study of centenarians and supercentenarians had the objective to consider this black swan and to evaluate the health, welfare, social and economic consequences of this phenomenon.

Different Retinal Expression Patterns of IL-1α, IL-1β, and Their Receptors in a Rat Model of Type 1 STZ-Induced Diabetes.

Diabetic retinopathy (DR), a common complication of diabetes, remains a major cause of blindness among population. Considerable amounts of evidences suggest the involvement of inflammatory process in this pathology. Increased levels of proinflammatory cytokines, including interleukin-1β (IL-1β), were found in the vitreous of diabetic patients and in the retina of diabetic rats. However, in this context, no attention has been given to the other main IL-1 family members: IL-1α, two transmembrane receptors IL-1RI and IL-1RII and the natural antagonist receptor IL-1Ra. Despite that they actively participate in the IL-1-mediated inflammation process, their implication in DR has not been described. Thus, we investigated by Western blot and confocal laser scanning microscopy analysis the effect of hyperglycemia on expression of IL-1 family members in retinal layers, using an in vivo model of type 1 diabetes. It was induced in adult rats by intraperitoneal injection of streptozotocin (STZ). Exposure to hyperglycemia induces a significant increase in the protein expression of IL-1β, IL-1RI, IL-RII and IL-1Ra but not of IL-1α. Moreover, high glucose alters their distribution pattern in the rat’s retinal layers. Among these latter, the most compromised are the photoreceptor, the inner plexiform and ganglion cell layers. These findings support previous data demonstrating the involvement of inflammation in DR and suggest new pharmacological approaches for the treatment of this pathology.

Increasing the Coding Potential of Genomes Through Alternative Splicing: The Case of PARK2 Gene

The completion of the Human Genome Project aroused renewed interest in alternative splicing, an efficient and widespread mechanism that generates multiple protein isoforms from individual genes. Although our knowledge about alternative splicing is growing exponentially, its real impact on cellular life is still to be clarified. Connecting all splicing features (genes, splice transcripts, isoforms, and relative functions) may be useful to resolve this tangle. Herein, we will start from the case of a single gene, Parkinson protein 2, E3 ubiquitin protein ligase (PARK2), one of the largest in our genome. This gene is implicated in the pathogenesis of autosomal recessive juvenile Parkinsonism and it has been recently linked to cancer, leprosy, autism, type 2 diabetes mellitus and Alzheimer’s disease. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification and protein diversity in tissues and cells. This review will provide an update of all human PARK2 alternative splice transcripts and isoforms presently known, and correlate them to those in rat and mouse, two common animal models for studying human disease genes. Alternative splicing relies upon a complex process that could be easily altered by both cis and trans-acting mutations. Although the contribution of PARK2 splicing in human disease remains to be fully explored, some evidences show disruption of this versatile form of genetic regulation may have pathological consequences.