Maria Elena Ferrero

Chromogranin A protects vessels against tumor necrosis factor alpha-induced vascular leakage

Elevated levels of circulating chromogranin A (CgA), a protein stored in the secretory granules of many neuroendocrine cells and neurons, have been detected in the blood of patients with neuroendocrine tumors or heart failure. The pathophysiological role of increased secretion of CgA is unknown. Using mice bearing subcutaneous tumors genetically engineered to secrete CgA in circulation, we have found that increased blood levels of this protein prevent vascular leakage induced by tumor necrosis factor‐α (TNF) in the liver venous system. Structure–activity studies, carried out with CgA fragments administered to normal mice, showed that an active site is located within residues 7–57 of CgA. Accordingly, an anti‐CgA antibody directed to residues 53–57 inhibited the effect of circulating CgA, either endogenously produced or exogenously administered, on liver vessels. Studies of the mechanism of action showed that CgA inhibits TNF‐induced VE‐cadherin down‐regulation and barrier alteration of cultured endothelial cells, in an indirect manner. Other effectors, such as thrombin and vascular endothelial growth factor were partially inhibited by CgA N‐terminal fragments in in vitro permeability assays. These findings suggest that circulating CgA could help regulate the endothelial barrier function and to protect vessels against TNF‐induced plasma leakage in pathological conditions characterized by increased production of TNF and CgA, such as cancer or heart failure.

Antinociceptive effect of a new P2Z/P2X7 antagonist, oxidized ATP, in arthritic rats

The neurotransmitter adenosine triphosphate (ATP) is released from sensory nerve endings during inflammation and acts at the level of P2X receptors. We used the irreversible inhibitor of P2z/P2X7 receptor, designated oxidized ATP (oATP), to test its possible antinociceptive activity in arthritic rats. We induced unilateral inflammation of the rat hind paw by local injection of Freunds complete adjuvant. Administration of the adjuvant resulted in a significant reduction of paw pressure threshold (PPT). Injection of oATP into inflamed paws significantly increased, in a dose-dependent manner, PPT values to levels comparable with or higher than those evaluated in control uninflamed paws. The data indicate that the P2z/P2X7 receptor system exerts a role in nociception and that oATP, by inhibiting such a receptor, reduces the nociceptive signal in the course of peripheral inflammation.

The platelet endothelial cell adhesion molecule-1 (PECAM1) contributes to endothelial barrier function

In this study we have analyzed the role of the platelet‐endothelial cell adhesion molecule‐1 (PECAM1) in vascular barrier function. PECAM1 is an immunoglobulin gene superfamily member expressed by endothelial cells at the cell boundaries. Macromolecule permeability assays performed on cell monolayers that express native or transfected PECAM1, indicated that the molecule participates in the establishment and maintenance of vascular barrier function in vitro. This hypothesis was confirmed by the finding that in vivo injection of the specific monoclonal antibody directed against the murine vascular PECAM1 led to a detectable leakage of hepatic and renal blood vessels.

The vasostatin-I fragment of chromogranin A inhibits VEGF-induced endothelial cell proliferation and migration

A growing body of evidence suggests that chromogranin A (CgA), a secretory protein released by many neuroendocrine cells and frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors, is a precursor of several bioactive fragments. This work was undertaken to assess whether the N‐terminal fragment CgA1–76 (called vasostatin I) can inhibit the proangiogenic activity of vascular endothelial growth factor (VEGF), a factor involved in tumor growth. The effect of recom‐binant human vasostatin I (VS‐1) on VEGF‐induced human umbilical endothelial cells (HUVEC) signaling, proliferation, migration, and organization has been investigated. We have found that VS‐1 (3 μg/ml;330 nM) can inhibit VEGF‐induced ERK phosphorylation, as well as cell migration, proliferation, morphogenesis, and invasion of collagen gels in various in vitro assays. In addition, VS‐1 could inhibit the formation of capillary‐like structures in Matrigel plugs in a rat model. VS‐1 could also inhibit basal ERK phosphorylation and motility of HUVEC, leading to a more quiescent state in the absence of VEGF, without inducing apoptotic or necrotic effects. Conclusion: These findings suggest that vasostatin I may play a novel role as a regulator of endothelial cell function and homeostasis.—Belloni, D., Scabini, S., Foglieni, C., Veschini, L., Giazzon, A., Colombo, B., Fulgenzi, A., Helle, K. B., Ferrero, M. E., Corti, A., Ferrero, E. The vasostatin‐I fragment of chromogranin A inhibits VEGF‐induced endothelial cell proliferation and migration. FASEB J. 21, 3052–3062 (2007)

Effect of the Purinergic Inhibitor Oxidized-ATP in a Model of Islet Allograft Rejection

The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2XRs) sense ATP released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2XRs during islet allograft rejection and to establish a novel anti-P2XRs strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet allograft-infiltrating cells, that only P2X7R is increasingly expressed during alloimmune response, and that P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP]) delays islet allograft rejection, reduces the frequency of Th1/Th17 cells, and induces hyporesponsiveness toward donor antigens. oATP-treated mice displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. Peripheral blood mononuclear cells obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R+CD4+ T cells compared with controls. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function.

Long-Term Heart Transplant Survival by Targeting the Ionotropic Purinergic Receptor P2X7

Background— Heart transplantation is a lifesaving procedure for patients with end-stage heart failure. Despite much effort and advances in the field, current immunosuppressive regimens are still associated with poor long-term cardiac allograft outcomes, and with the development of complications, including infections and malignancies, as well. The development of a novel, short-term, and effective immunomodulatory protocol will thus be an important achievement. The purine ATP, released during cell damage/activation, is sensed by the ionotropic purinergic receptor P2X7 (P2X7R) on lymphocytes and regulates T-cell activation. Novel clinical-grade P2X7R inhibitors are available, rendering the targeting of P2X7R a potential therapy in cardiac transplantation. Methods and Results— We analyzed P2X7R expression in patients and mice and P2X7R targeting in murine recipients in the context of cardiac transplantation. Our data demonstrate that P2X7R is specifically upregulated in graft-infiltrating lymphocytes in cardiac-transplanted humans and mice. Short-term P2X7R targeting with periodate-oxidized ATP promotes long-term cardiac transplant survival in 80% of murine recipients of a fully mismatched allograft. Long-term survival of cardiac transplants was associated with reduced T-cell activation, T-helper cell 1/T-helper cell 17 differentiation, and inhibition of STAT3 phosphorylation in T cells, thus leading to a reduced transplant infiltrate and coronaropathy. In vitro genetic upregulation of the P2X7R pathway was also shown to stimulate T-helper cell 1/T-helper cell 17 cell generation. Finally, P2X7R targeting halted the progression of coronaropathy in a murine model of chronic rejection as well. Conclusions— P2X7R targeting is a novel clinically relevant strategy to prolong cardiac transplant survival.

The effect of somatostatin on experimental inflammation in rats

The aim of the present study was to investigate the effect of somatostatin administration on experimentally induced inflammation in rats.Inflammation was induced by the intraplantar injection of carrageenan (50 micro L) into the hind paw of the rat. Animals were treated intraplantarly with somatostatin in a volume of 50 micro L at different doses (2.5, 25, and 250 ng, 10 micro g). The inflammatory response was studied 120, 180, and 240 min after drug administration. The antinociceptive effect of somatostatin was determined by using the Randall and Selitto test and by local production of beta-endorphin from lymphocytes obtained from popliteal lymph nodes. Data show that small doses of somatostatin were the most effective in reducing hyperalgesia. Moreover, our results show that somatostatin treatment significantly increased beta-endorphin in lymphocytes from popliteal lymph nodes. The secretion of opioid peptides, which enhance analgesia, could be stimulated by locally administered somatostatin. Implications: Acute pain because of intraplantar inflammation induced in rats by carrageenan injection was significantly reduced by small-dose, local administration of somatostatin, which possibly favors beta-endorphin release as a mechanism. These results may have implications regarding treatment of pain conditions associated with an inflammatory response. (Anesth Analg 1997;85:1112-5)

RANTES and MCP-1 chemokine plasma levels in chronic renal transplant dysfunction and chronic renal failure

OBJECTIVES Procedures to diagnose renal allograft rejection depend on detection of graft dysfunction due to the presence of mononuclear leukocytic infiltrates. DESIGN AND METHODS In our study, we pursued an immunodiagnostic approach utilizing an ELISA method on plasma samples to monitor patients waiting to undergo transplantation in order to evidence prognostic developments in renal transplantation and, at least, to diagnose renal chronic transplant dysfunction. We analyzed blood levels of two chemokines, RANTES and MCP-1, which are normally overexpressed locally in renal chronic rejection. RESULTS Our results showed that patients affected by chronic renal failure (and waiting for kidney transplant), as well as kidney-grafted patients affected by chronic transplant dysfunction, had plasma levels of RANTES significantly higher than those of controls (patients without acute or chronic pathologies). CONCLUSIONS Our data suggest a simple method to evaluate the plasmatic presence of RANTES, which could be involved in longterm kidney graft failure.

In vivo inhibition of TNFα-induced vascular permeability by resveratrol

RESVERATROL (3-49-5-trihydroxystilbene) is a natural phytoalexin present in grape juice and wine. It has been shown to have anti-inflammatory, anticancer, and antiaggregating properties. Resveratrol and other phenolic substances are able to inhibit oxidation of human LDL. Analogously, red wine consumption reduces the susceptibility of human LDL to lipid peroxidation, potentially reducing the risk of atherosclerosis.

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion

1 Leukocyte‐endothelial cell interactions play an important role during ischaemia‐reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2 Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia‐reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3 In basal conditions, defibrotide (1000 μg ml−1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% ± 3.6 (P < 0.05), and after endothelial cell stimulation (TNF‐α, 500 u ml−1) or after leukocyte stimulation (fMLP, 10−7 m), it inhibited leukocyte adhesion by 26.5% ± 3.4 and 32.4% ± 1.8, respectively (P < 0.05). 4 In adhesion blockage experiments, the use of the monoclonal anntibody anti‐CD31 (5 μg ml−1) did not demonstrate a significant inhibitory effect whereas use of the monoclonal antibody anti‐LFA‐1 (5 μg ml−1) significantly interfered with the effect of defibrotide. 5 This result was confirmed in NIH/3T3‐ICAM‐1 transfected cells. 6 We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM‐1/LFA‐1 adhesion system is involved in the defibrotide mechanism of action.