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Background Breast Cancer (BC) is a leading public-health issue affecting women on a global scale, accounting for about 500,000 new cases every year in Europe. Thanks to the widespread implementation of screening programs and the improvement in therapies, women with BC live longer but they also are more likely to experience an increased risk of other diseases, including second primary cancer, cardiovascular, and metabolic diseases. Reasons for this increased risk include genetics, shared risk factors, and adverse effects from BC treatment. Methods To analyse the risk of myocardial infarction (MI) and stroke in women with BC considering the potential side effects of treatments, we used data from the Piedmont Longitudinal Study, an administrative cohort based on the record-linkage among census data and several health-administrative databases to include than 4 million inhabitants of an Italian region. Results Among 1,342,333 women ranging from 30 to 75 years old 19,203 had a BC diagnosis in the follow-up period, of whom 206 experienced a subsequent MI and 203 a stroke. Women with BC showed an increased risk for MI (HR: 1.20; 95%CI: 1.05–1.38) and for stroke (HR: 1.58; 95%CI: 1.38–1.82). Chemotherapy seemed to be the major risk factor for MI in BC women, while no different risk by therapy was found for stroke. Conclusion The results of this study supported the hypothesis about the toxic effect of therapies, suggesting both clinicians to routinely and actively screen for these treatment-related toxicities in women with BC and researchers to prioritize personalized treatments in order to minimize potentially devastating side effects.

Antibody-based anti-cancer therapy is considered a successful approach to impair tumour progression. This study aimed to investigate the clinical impact of targeting the IL-3 signalling in the microenvironment of solid tumours. We intended to investigate whether the IL-3Rα blockade on tumour-derived endothelial cells (TEC) can modulate PD-L1 expression in tumour cells and peripheral blood mononuclear cells (PBMC) to reshape the anti-tumour immune response. Extracellular vesicles released by TEC after IL-3Rα blockade (aTEV) were used as the ultimate effectors of the antibody-based approach, while naive TEC-derived extracellular vesicles (nTEV) served as control. Firstly, we demonstrated that, either directly or indirectly via nTEV, IL-3 controls the expression of its receptor on TEC and PBMC respectively. Moreover, we found that nTEV, moulded by the autocrine secretion of IL-3, increased PD-L1 expression in myeloid cells both in vitro and in vivo. In addition, we found that nTEV-primed PBMC favour tumour cell growth (TEC and MDA-MB-231 cells), whereas PBMC-primed with aTEV still retain their anti-tumour properties. Isolated T-cells pre-conditioned with nTEV or aTEV and co-cultured with TEC or MDA-MB-231 cells have no effects, thereby sustaining the key role of myeloid cells in tumour immune editing. In vivo nTEV, but not aTEV, increased the expression of PD-L1 in primary tumours, lung and liver metastases. Finally, we demonstrated that the enrichment of miR-214 in aTEV impacts on PD-L1 expression in vivo. Overall, these data indicate that an approach based on IL-3Rα blockade in TEC rearranges EV cargo and may reshape the anti-tumour immune response.

Extracellular vesicles (EVs) are implicated in the crosstalk between adipocytes and other metabolic organs, and an altered biological cargo has been observed in EVs from human obese adipose tissue (AT). Yet, the role of adipocyte-derived EVs in pancreatic β cells remains to be determined. Here, we explored the effects of EVs released from adipocytes isolated from both rodents and humans and human AT explants on survival and function of pancreatic β cells and human pancreatic islets. EVs from healthy 3T3-L1 adipocytes increased survival and proliferation and promoted insulin secretion in INS-1E β cells and human pancreatic islets, both those untreated or exposed to cytokines or glucolipotoxicity, whereas EVs from inflamed adipocytes caused β cell death and dysfunction. Human lean adipocyte-derived EVs produced similar beneficial effects, whereas EVs from obese AT explants were harmful for human EndoC-βH3 β cells. We observed differential expression of miRNAs in EVs from healthy and inflamed adipocytes, as well as alteration in signaling pathways and expression of β cell genes, adipokines, and cytokines in recipient β cells. These in vitro results suggest that, depending on the physiopathological state of AT, adipocyte-derived EVs may influence β cell fate and function.

The extracellular vesicles (EVs) are membrane vesicles carrying proteins, nucleic acids, and bioactive lipids of the cell of origin. These vesicles released within the extracellular space and entering into the circulation may transfer their cargo to neighboring or distant cells and induce phenotypical and functional changes that may be relevant in several physiopathological conditions. In an attempt to define the biological properties of EVs, several investigations have focused on their cargo and on the effects elicited in recipient cells. EVs have been involved in modulation of tumor microenvironment and behavior, as well as in the immune and inflammatory response. In the present review, we address the paracrine action of EVs released by stem cells and their potential involvement in the activation of regenerative programs in injured cells.

Advanced therapies which combine cells with biomaterial-based carriers are recognized as an emerging and powerful method to treat challenging diseases, such as spinal cord injury (SCI). By enhancing transplanted cell survival and grafting, biomimetic hydrogels can be properly engineered to encapsulate cells and locate them at the injured site in a minimally invasive way. In this work, chitosan (CS) based hydrogels were developed to host mesenchymal stem cells (MSCs), since their paracrine action can therapeutically enhance the SC regeneration, limiting the formation of a glial scar and reducing cell death at the injured site. An injectable and highly permeable CS-based hydrogel was fabricated having a rapid gelation upon temperature increase from 0 to 37 °C. CS was selected as former material both for its high biocompatibility that guarantees the proper environment for MSCs survival and for its ability to provide anti-inflammatory and anti-oxidant cues. MSCs were mixed with the hydrogel solution prior to gelation. MSC viability was not affected by the CS hydrogel and encapsulated MSCs were able to release MSC-vesicles as well as to maintain their anti-oxidant features. Finally, preliminary in vivo tests on SCI mice revealed good handling of the CS solution loading MSCs during implantation and high encapsulated MSCs survival after 7 days.

Prostate cancer (PCa) is one of the most common cancer in men. Although hormone-sensitive PCa responds to androgen-deprivation, there are no effective therapies for castration-resistant PCa. It has been recently suggested that proton pump inhibitors (PPIs) may increase the risk of certain cancers; however, association with PCa remains elusive. Here, we evaluated the tumorigenic activities of PPIs in vitro, in PCa cell lines and epithelial cells from benign prostatic hyperplasia (BPH) and in vivo, in PCa mice xenografts. PPIs increased survival and proliferation, and inhibited apoptosis in LNCaP cells. These effects were attenuated or absent in androgen-insensitive DU-145 and PC3 cells, respectively. Specifically, omeprazole (OME) promoted cell cycle progression, increased c-Myc expression, ErbB2 activity and PSA secretion. Furthermore, OME induced the phosphorylation of MAPK-ERK1/2, PI3K/Akt and GSK-3β, and blunted the expression and activity of cellular prostatic acid phosphatase. OME also increased survival, proliferation and PSA levels in BPH cells. In vivo, OME promoted tumor growth in mice bearing LNCaP xenografts. Our results indicate that PPIs display tumorigenic activities in PCa cells, suggesting that their long-term administration in patients should be carefully monitored.

We previously have shown that platelet-derived growth factor (PDGF) modulates the biological activity of extracellular vesicles released by adipose-derived mesenchymal stem cells (ASC-EVs). ASC-EVs may interact with blood and vessel cells by transferring proteins and nucleic acids and regulate their functions. In this study, we investigated immunomodulatory activity and protection from acute hindlimb ischemia of EVs released by PDGF-stimulated ASC (PDGF-EVs). PDGF treatment of ASC changed protein and RNA composition of released EVs by enhancing the expression of anti-inflammatory and immunomodulatory factors. In vitro, control EVs (cEVs) derived from non-stimulated ASC increased the secretion of both the IL-1b, IL-17, IFNγ, TNFα pro-inflammatory factors and the IL-10 anti-inflammatory factor, and enhanced the in vitro peripheral blood mononuclear cell (PBMC) adhesion on endothelium. In contrast, PDGF-EVs enhanced IL-10 secretion and induced TGF-β1 secretion by PBMC. Moreover, PDGF-EVs stimulated the formation of T regulatory cells. In vivo, PDGF-EVs protected muscle tissue from acute ischemia, reduced infiltration of inflammatory cells and increased T regulatory cell infiltration in respect to cEVs. Our results suggest that PDGF-EVs are enriched in anti-inflammatory and immunomodulatory factors and induced in PBMC an enhanced production of IL-10 and TGF-β1 resulting in protection of muscle from acute ischemia in vivo.

Mesenchymal stem cells (MSCs) can exert an immunosuppressive effect on any component of the immune system, including dendritic cells (DCs), by direct contact, the release of soluble markers and extracellular vesicles (EVs). We evaluated whether MSCs and MSC-derived EVs have an immunomodulatory effect on monocyte-derived DCs in type 1 diabetes.Bone marrow derived MSCs were characterised and EVs were obtained by ultracentrifugation. DCs were differentiated from CD14(+) cells, obtained from nine type 1 diabetic patients at disease onset, pulsed with antigen GAD65 and cultured with MSCs or EVs. Levels of DC maturation and activation markers were evaluated by flow cytometry. GAD65-pulsed DCs and autologous CD14(-) cell were co-cultured and IFN-γ enzyme-linked immunosorbent spot responses were assayed. Secreted cytokine levels were measured and Th17 and regulatory T cells were analysed.MSC- and EV-conditioned DCs acquired an immature phenotype with reduced levels of activation markers and increased IL-10 and IL-6 production. Conditioned DC plus T cell co-cultures showed significantly decreased IFN-γ spots and secretion levels. Moreover, higher levels of TGF-β, IL-10 and IL-6 were detected compared with unconditioned DC plus T cell co-cultures. Conditioned DCs decreased Th17 cell numbers and IL-17 levels, and increased FOXP3(+) regulatory T cell numbers. EVs were internalised by DCs and EV-conditioned DCs exhibited a similar effect.In type 1 diabetes, MSCs induce immature IL-10-secreting DCs in vitro, thus potentially intercepting the priming and amplification of autoreactive T cells in tissue inflammation. These DCs can contribute to the inhibition of inflammatory T cell responses to islet antigens and the promotion of the anti-inflammatory, regulatory responses exerted by MSCs.

Obestatin is a 23 amino acid amidated peptide, member of the preproghrelin gene-derived peptides. Initially, obestatin was reported to exert opposite effects to those of ghrelin on food intake and body weight gain, through interaction with GPR39; however, these findings are still strongly debated and obestatin biological role remains largely unknown. Interestingly, binding of obestatin to the glucagon-like peptide 1 receptor has been recently suggested. Despite being a controversial peptide, recent findings have clearly indicated that obestatin is indeed a multifunctional peptide, exerting a variety of effects, such as stimulation of cell proliferation, survival and differentiation, influence on glucose and lipid metabolism, as well as anti-inflammatory and cardioprotective actions. Its positive effects on glucose and lipid metabolism candidate this peptide as a potential therapeutic tool in pathological conditions such as insulin resistance and diabetes. © 2013 IUBMB Life, 65(12):976–982, 2013.

Context: Mesenchymal stem cells (MSCs) exert an immunosuppressive effect on the immune system. However, studies on the immunomodulatory potential of MSCs in type 1 diabetes are lacking. Objective: We aimed to evaluate whether human MSCs may inhibit in vitro pancreatic islet antigen-specific T cell activation in type 1 diabetes. Design: Human MSCs were isolated and characterized. Peripheral blood mononuclear cells (PBMCs) were obtained from nine type 1 diabetic patients at disease onset and 13 healthy control subjects. IFN-γ, IL-10, and IL-4 enzyme-linked immunospot responses of lymphocytes incubated with glutamic acid decarboxylase 65 (GAD65) were investigated in PBMC cultures and PBMC/MSC cocultures. Levels of prostaglandin E2 (PGE2), IFN-γ, IL-4, and IL-10 in supernatants were measured by ELISA. PGE2 inhibition experiments with NS-398 and indomethacin were also performed. Results: Five diabetic patients were identified with a positive PBMC IFN-γ response to GAD65 and negative IL-10 and IL-4 response. PBMC/MSC cocultures resulted in a significant decrease in the number of spots and in detection of IL-4-secreting cells. PGE2 inhibitors abrogated the immune-suppressive effect, indicating an involvement of PGE2 production, and the constitutive production of PGE2 by MSCs was enhanced in PBMC/MSC coculture. Moreover, in GAD-responder patients, GAD-stimulated PBMC/MSC cocultures significantly decreased secretion of IFN-γ and IL-10 and increased secretion of IL-4. Conclusions: These results provide evidence that human MSCs abrogate in vitro a proinflammatory T helper type 1 response to an islet antigenic stimulus in type 1 diabetes. MSCs induce IL-4-producing cells, suggesting a possible switch to an antiinflammatory T helper type 2 signaling of T cells.

Pancreatic islet microendothelium and β cells exhibit an interdependent physical and functional relationship. In this study, we analyzed the effect of chronic hyperglycemia on human pancreatic islet microendothelial cells as well as the involvement of the phosphatidylinositol 3-kinase/Akt and nephrin pathways, interleukin-1β, and nitric oxide production. In addition, whether 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors can reverse the response to high-glucose conditions was investigated. Proliferation of purified islet microendothelial cells cultured under hyperglycemic conditions (28 mmol/L glucose) decreased compared to that of normoglycemic cells (from 12.7% after 2 days to 47.7% after 30 days, P < 0.05). In parallel, apoptosis progressively increased from 7% after 2 days to 79% after 30 days in high glucose (P < 0.05) concomitant with an early increase of caspase-3 activity. Intermittent hyperglycemia induced greater apoptosis than sustained hyperglycemia. Apoptosis was accompanied by a reduced p-Akt/Akt ratio and inhibition of nephrin tyrosine phosphorylation. Pravastatin (1 μmol/L) decreased apoptosis induced by high glucose or oxidized LDL and increased Akt phosphorylation. Hyperglycemia significantly increased the production of the proinflammatory cytokine interleukin-1β and stimulated the expression of inducible nitric oxide synthase and the production of nitric oxide, possibly relevant to β cell mass and function. Thus, chronic hyperglycemia reduces islet microendothelial cell survival by inhibiting the serine-threonine kinase Akt pathway, and the effect of pravastatin on this pathway represents a potential tool to improve islet vascularization and, indirectly, islet function.

The microvasculature is a key interface between blood and tissues and participates in numerous pathophysiological processes. The heterogeneity of microvascular endothelial cells derived from different organs, suggests that these cells have specialised functions at different anatomical sites. Pancreatic islet microcirculation exhibits distinctive features, with an islet capillary network showing five times higher density than the capillary network of the exocrine counterpart and high permeability. Moreover, the islet microvascular endothelial cells show about 10 times more fenestrations than those of the exocrine tissue. In an interdependent physical and functional relationship with beta cells, islet endothelial cells are involved not only in the delivery of oxygen and nutrients to endocrine cells, but induce insulin gene expression during islet development, affect adult beta cell function, promote beta cell proliferation, and produce a number of vasoactive, angiogenic substances and growth factors. Specific markers of islet microvasculature are alpha-1 proteinase inhibitor and nephrin, a highly specific barrier protein with adhesion and signalling function. The islet microendothelium also appears to have a role in fine-tuning blood glucose sensing and regulation, and to behave as an active "gatekeeper" in the control of leukocyte recruitment into the islets, adopting an activated phenotype during autoimmune insulitis in type 1 diabetes. This dense vasculature is therefore likely to play a role in the physiology as well as in the disease of the pancreatic islets. In this review we will describe the phenotypic and functional characteristics of islet microendothelium and its possible involvement in type 1 and 2 diabetes, and islet revascularisation in transplantation settings.

Syncope is a frequent symptom characterizing a wide group of pathologies with very different prognosis. By this reason, it is necessary a careful risk stratification for a better patient management. For this aim medical history and physical examination are fundamental. Diagnostic exams complete and confirm diagnostic suspiciousness formulated by medical history and examination, but rarely give elements for unexpected diagnosis. Correct identification of pathological mechanism of syncope and determination of associated clinical risk allow the best patient management avoiding dangerous discharge but also, in relation to increasingly limited resources, inappropriate hospitalizations.

OBJECTIVE We prospectively evaluated the association between autoimmunity to autonomic nervous structures and autonomic neuropathy in type 1 diabetes in relation to clinical variables. RESEARCH DESIGN AND METHODS A cohort of 112 patients with type 1 diabetes was prospectively followed from adolescence (T0) to approximately 4 (T4) and 16 (T16) years later. Standard cardiovascular (CV) tests and neurological examination were performed and related to the presence of circulating antibodies (Ab) to autonomic nervous structures detected at T0 and T4. Quality of life was assessed by a diabetes-specific questionnaire. RESULTS Sixty-six patients (59% of the cohort) were reexamined at T16 (age 31.4 ± 2 years; disease duration 23.4 ± 3.7 years). Nineteen had circulating Ab to autonomic structures. Prevalence of abnormal tests and autonomic symptoms were higher in Ab-positive (68 and 26%, respectively) than Ab-negative (32 and 4%) patients (P < 0.05). Among Ab-positive patients, the relative risk (RR) of having at least one altered CV test was 5.77 (95% CI 1.56–21.33), and an altered deep breathing (DB) test ( CONCLUSIONS Circulating Ab to autonomic structures are associated with the development of autonomic dysfunction in young diabetic patients independent of glycemic control.

Mesenchymal stem cells (MSCs) have been shown to abrogate in vitro the proinflammatory response in type 1 diabetes. The mechanism involves paracrine factors, which may include microvesicles (MVs). We evaluated whether MVs derived from heterologous bone-marrow MSCs exert an immunomodulatory effect on T cell responses against GAD (glutamic acid decarboxylase) antigen in type 1 diabetes. MVs were purified from heterologous human MSCs by differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with type 1 diabetes at disease onset, and responses to GAD65 stimulation were assessed by IFN-γ enzyme-linked immunosorbent spot analysis. Levels of cytokines and prostaglandin E2 (PGE2) were measured in the supernatant fraction, and T helper 17 (Th17) and regulatory T cell analysis was performed. MVs were internalised by PBMCs, as assessed by confocal microscopy and flow cytometry analyses. MVs significantly decreased IFN-γ spots and levels in GAD65-stimulated PBMCs, and significantly increased transforming growth factor-β (TGF-β), IL-10, IL-6 and PGE2 levels. Furthermore, MVs decreased the number of Th17 cells and the levels of IL-17, and increased FoxP3+ regulatory T cells in GAD65-stimulated PBMCs. These results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-β signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

The heterogeneity of microvascular endothelial cells derived from different organs, suggests that these cells have specialised functions at different anatomical sites. The microvasculature is, in fact, a key interface between blood and tissues and participates in numerous pathophysiological processes. Pancreatic islet microcirculation exhibits distinctive features, in an interdependent physical and functional relationship with ┚ cells, from organogenesis to adult life. The islet microendothelium behaves as an active “gatekeeper” in the control of leukocyte recruitment into the islets during autoimmune insulitis in type 1 diabetes. Furthermore, microvascular endothelial cells, forming the key lining between the vascular space and organ parenchyma, have been shown to influence organ and tissue specific susceptibility to viral infection, and to modulate the pathological expression of virusinduced diseases, which potentially includes type 1 diabetes. Endothelial cells expressing appropriate receptors would fail to act as effective barrier to infections, allowing viral particles to pass through, and replicate in, the vascular endothelium. Human Enteroviruses (EV), especially those of the Coxsackievirus B (CVB) group, are associated with a wide variety of clinical syndromes and have long been considered possible culprits of inflammatory conditions and immune-mediated pathological processes, such as chronic dilated cardiomyopathy, chronic myositis and type 1 diabetes mellitus (Rose et al., 1993; Luppi et al., 1998; Hyoty & Taylor, 2002). Several mechanisms, including molecular mimicry, bystander activation of autoreactive T cells, superantigenic activity of viral proteins, not mutually exclusive, have been proposed to explain the relationship between EV infections and induction of autoimmune diseases (Varela-Calvino & Peakman, 2003; Horwitz et al., 1998; Wucherpfennig, 2001). Evidences of a link between viral infections and initiation or acceleration of pancreatic islet autoimmunity have been under investigation for almost 30 years, and EVs, especially those of the Coxsackievirus B (CVB) group, are historically the prime suspects as important aetiological determinants in type 1 diabetes (Hyoty & Taylor, 2002; Varela-Calvino & Peakman, 2003). Endothelial cells derived from different organs show distinct susceptibility to CVB infections, and the behaviour against a viral challenge of endothelial cells in large vessels and microvessels may differ (Friedman et al. 1981; Huber et al., 1990; Conaldi et al. 1997; Zanone et al., 2003; Saijets et al., 2003).

Type 1 diabetes is an autoimmune disease leading to the destruction of pancreatic cells. The reduction of cell mass results in insulin deficiency that leads to a failure of glucose homeostasis with increased levels of glucose in blood. Hyperglycemia which per se is detrimental for the organism and may be life-threatening, may in the long term associate with chronic complications involving blood vessels and nerves. The gold standard treatment for diabetes patients aimed to reach a tight control of glycemia, relies on intensive insulin therapy based on multiple daily injections or continuous subcutaneous infusion of insulin. A tight control of glycemia reached with such regimens was shown to significantly reduce the incidence of microvascular complications in respect to the conventional insulin therapy. Nevertheless, to reach an optimal control of blood levels may prove to be difficult when compared to the physiological condition where this is guaranteed by the pancreatic  cells (Suckale, 2008). Therefore, preservation of cell mass could be an important therapeutic target to reduce microvascular complications and to improve the glycemia control (Gonez & Knight, 2010). It is generally accepted that the endocrine pancreas has some regenerative capabilities, although it is still debated which cells are involved in cell turnover. In rodents the capability of adult pancreas to increase cell mass has been documented in physiological conditions and after injury. The understanding of mechanisms involved in  cell turnover may therefore be relevant to design new therapeutic strategies aimed to maintain a  cell mass or to favour regeneration of  cells. These strategies however, should take to account the problem of recurrent autoimmunity that in type 1 diabetes not only impairs the original cell mass, but may also limit the regenerative process. Indeed, autoimmune T lymphocytes may kill the cells newly formed in response to injury (Fan & Rudert, 2009).

Association of type 1 diabetes and cytomegalovirus (CMV) is suspected and CMV infections have also been linked to increased risk of new onset post-transplantation diabetes. We monitored response to islet autoantigens, pancreatic endocrine function, and CMV infections in one type 1 diabetic patient receiving pancreas allograft. Time course analyses of levels of islet autoantibodies (Abs), IFN-gamma ELISPOT response, analysis of T cell function, levels of C peptide together with CMV pp65 antigenaemia and viraemia and graft biopsy histopathology were performed in comparison with a cohort of diabetic recipients. Evidence of autoimmune activation to GAD and IA2, modification of CD4(+) CD25hi T cells, loss of pancreatic function, concomitantly with multiple CMV infections and allograft rejection with peri-insulitis is provided. The parallel between metabolic outcome, initiation and progression of autoreactivity to islet autoantigens and early CMV infections after transplantation, suggests that persistent CMV infections may be relevant to the pathogenesis of type 1 diabetes.

Human amniotic fluid (HAF) contains multipotent stem cells [amniotic fluid-derived stem cells (AFSC)] which can differentiate into a variety of different cell types. Recently, we demonstrated that obestatin, a peptide encoded by the ghrelin gene, exerts anti-apoptotic effects in pancreatic beta-cells and human islets and increases the expression of genes involved in beta-cells differentiation. We investigated whether: 1) AFSC would differentiate into pancreatic beta-cells and 2) obestatin would increase beta-cells differentiation from AFSC. Fluorescence-activated cell sorting analysis and immunocytochemical staining showed the presence of mesenchymal and endothelial markers in AFSC. Real-time PCR evidenced the expression of Octamer binding transcription factor 4 (OCT-4), a marker of pluripotency, during the early differentiation phase. However, the beta-cells differentiation marker duodenal homeobox factor-1 (PDX-1) could not be detected. Obestatin increased OCT-4 expression but had no effect on beta-cells differentiation. These results suggest that, at least under the experimental conditions used in this study, AFSC do not differentiate into beta-cells and obestatin has no additional effect.

The microvasculature is a key interface between blood and tissues and participates in numerous pathophysiological processes. The heterogeneity of microvascular endothelial cells derived from different organs, suggests that these cells have specialised functions at different anatomical sites. Pancreatic islet microcirculation exhibits distinctive features, with an islet capillary network showing five times higher density than the capillary network of the exocrine counterpart and high permeability. Moreover, the islet microvascular endothelial cells show about 10 times more fenestrations than those of the exocrine tissue. In an interdependent physical and functional relationship with beta cells, islet endothelial cells are involved not only in the delivery of oxygen and nutrients to endocrine cells, but induce insulin gene expression during islet development, affect adult beta cell function, promote beta cell proliferation, and produce a number of vasoactive, angiogenic substances and growth factors. Specific markers of islet microvasculature are alpha-1 proteinase inhibitor and nephrin, a highly specific barrier protein with adhesion and signalling function. The islet microendothelium also appears to have a role in fine-tuning blood glucose sensing and regulation, and to behave as an active "gatekeeper" in the control of leukocyte recruitment into the islets, adopting an activated phenotype during autoimmune insulitis in type 1 diabetes. This dense vasculature is therefore likely to play a role in the physiology as well as in the disease of the pancreatic islets. In this review we will describe the phenotypic and functional characteristics of islet microendothelium and its possible involvement in type 1 and 2 diabetes, and islet revascularisation in transplantation settings.

Enteroviruses, such as the coxsackievirus (CV) group, have been linked to the induction of inflammatory and autoimmune diseases. Virus tropism and tissue access are modulated by endothelial cells. To examine the susceptibility of microvascular endothelial cells (MECs) derived from pancreatic islets to infection with CV group B (CVB), purified cultured human islet MECs were infected with CVB-4 strain, and the immunological phenotype of the infected cells was analyzed. CVB-4 persistently infected the islet MECs, which expressed the CV receptors human coxsackievirus and adenovirus receptor (HCAR) and decay accelerating factor (DAF) and maintained EC characteristics, without overt cytopathic effects. CVB-4 infection transiently up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1 and increased production of the proinflammatory cytokines IL-1beta and IL-6, and chemokines IL-8 and lymphotactin, as well as IFN-alpha. Mononuclear cell adhesion to CVB infected monolayers was increased, compared to uninfected monolayers. Moreover, infection up-regulated the viral receptors HCAR and DAF and coreceptor alpha(v)beta3 integrin on islet MECs, while down-regulating expression of HCAR on human aortic endothelial cells, indicating potential tissue-specific influence on the pathological outcome of infection. These results provide evidence that islet MECs are natural targets and reservoirs for persistent CVB infection resulting in acute endothelial cell activation by virus, which may contribute to selective recruitment of subsets of leukocytes during inflammatory immune responses, such as insulitis in type 1 diabetes.

Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures.Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus.Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-alpha) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-gamma) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration.The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.

The islet microcirculation has morphological characteristics resembling those of renal glomeruli. Transcription of the nephrin gene, a highly specific barrier protein of the slit diaphragm of podocyte foot processes, has been reported in the pancreas, although its cellular localisation and function remain to be defined. In this study, we purified and characterised microvascular endothelial cells (MECs) isolated from human islets and investigated the expression and distribution of nephrin on these cells.Human islet MECs were extracted and purified using anti-CD105-coated immunomagnetic beads and their endothelial characteristics were confirmed by expression of classical endothelial markers and basal high-level expression of intercellular adhesion molecule-1 and TNF-alpha-inducible vascular cell adhesion molecule-1. Nephrin expression was assessed by immunofluorescence, flow cytometric analysis and western blotting on cell lysates, as well as by RT-PCR.Immunofluorescence studies detected nephrin in a fine, punctate, diffuse pattern on cultured islet MECs, and also in human pancreatic islet sections. In both cases nephrin colocalised with endothelial markers. TNF-alpha treatment induced a marked reduction and redistribution of the protein in one or multiple aggregates. Nephrin expression was confirmed by flow cytometry, western blotting and RT-PCR studies. In contrast, nephrin could not be detected at the protein or mRNA level in human macro- and microvascular cells from other sites.Nephrin is expressed at protein and mRNA levels in islet microendothelium, supporting the hypothesis that islet MECs exhibit distinctive morphological characteristics that indicate functional specialisation of potential pathophysiological importance.

Recent studies have linked autoimmunity to nervous tissue structures and diabetic autonomic neuropathy. To evaluate prospectively the early stage of type 1 diabetes and the natural history of this association, we monitored the autonomic function and the presence of autoantibodies (Ab) to sympathetic and parasympathetic nervous structures in a cohort of 92 diabetic adolescent patients, recruited and followed-up after 40+/-3 months. The presence of circulating Ab and their ability to activate complement was also assessed using a human adrenergic neuroblastoma cell line, and the effect of patients' sera on these cells was evaluated by different methods assessing cytotoxic effects and apoptosis. Thirty-nine percent of the Ab-positive patients had one abnormal test (p=0.07 vs. Ab-negative patients). Serum from four patients positive for anti-cervical ganglia Ab showed positive staining of neuroblastoma cells and displayed ability to activate complement. Serum from two adolescent patients with anti-cervical ganglia and anti-neuroblastoma cells Ab, induced cytotoxic effects and damaged the plasma membrane of the neuroblastoma cells. Moreover, sera from two adult patients with overt autonomic neuropathy, used as positive controls, induced apoptosis of these cells, assessed by TUNEL. Our data indicate that symptomatic autonomic neuropathy is not characteristic of young diabetic patients, but that autoantibodies to autonomic structures are present and persist in the first 20 years of disease, possibly associated with subtle autonomic dysfunction and cytotoxic effect on sympathetic cells.

Numerous studies indicate that enteroviruses, such as the Coxsackievirus (CV) group, are linked to autoimmune diseases. Virus tropism and tissue access are modulated by vascular endothelial cells (ECs), mainly at the level of the microvasculature. Data on the permissiveness of ECs to CV are, however, scanty and derived from studies on large vessel ECs. To examine the susceptibility of microvascular ECs to infection of group B CV (CVB), human dermal microvascular ECs (HMEC-1) were infected with three CVB strains, and the immunological phenotype of the infected cells was analyzed. All CVB persistently infected the EC cultures without producing overt cytopathic effects. Infected ECs retained endothelial characteristics. Release of infectious particles in cell supernatants persisted for up to 3 mo of culture. Infection up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1, with the highest values detected during the first 30 days of infection (p < 0.05 vs uninfected HMEC-1). CVB infection increased production of the proinflammatory cytokines, IL-6, IL-8, and TNF-α, which may account for the enhanced expression of adhesion molecules. Parallel infection of macrovascular HUVEC had less evident effects on induction of ICAM-1 and did not significantly increase expression of VCAM-1. Moreover, mononuclear cell adhesion to CVB-infected HMEC-1 monolayers was increased, compared with uninfected monolayers. These results provide evidence that small vessel ECs can harbor a persistent viral infection, resulting in quantitative modification of adhesion molecule expression, which may contribute to the selective recruitment of subsets of leukocytes during inflammatory immune responses. Furthermore, our data confirm that the behavior against a viral challenge of ECs in large vessels and microvessels may differ.

Aims/hypothesis Pancreatic islet microendothelium exhibits unique features in interdependent relationship with beta cells. Gastrointestinal products of the ghrelin gene, acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (Ob), and the incretin, glucagon-like peptide-1 (GLP-1), prevent apoptosis of pancreatic beta cells. We investigated whether the ghrelin gene products and the GLP-1 receptor agonist exendin-4 (Ex-4) display survival effects in human pancreatic islet microendothelial cells (MECs) exposed to chronic hyperglycaemia. Methods Islet MECs were cultured in high glucose concentration and treated with AG, UAG, Ob or Ex-4. Apoptosis was assessed by DNA fragmentation, Hoechst staining of the nuclei and caspase-3 activity. Western blot analyses and pharmacological inhibition of protein kinase B (Akt) and extracellular signal-related kinase (ERK)1/2 pathways, detection of intracellular cAMP levels and blockade of adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) signalling were performed. Levels of NO, IL-1β and vascular endothelial growth factor (VEGF)-A in cell culture supernatant fractions were measured. Results Islet MECs express the ghrelin receptor GHS-R1A as well as GLP-1R. Treatment with AG, UAG, Ob and Ex-4 promoted cell survival and significantly inhibited glucose-induced apoptosis, through activation of PI3K/Akt, ERK1/2 phosphorylation and intracellular cAMP increase. Moreover, peptides upregulated B cell lymphoma 2 (BCL-2) and downregulated BCL-2-associated X protein (BAX) and CD40 ligand (CD40L) production, and significantly reduced the secretion of NO, IL-1β and VEGF-A. Conclusions/interpretation The ghrelin gene-derived peptides and Ex-4 exert cytoprotective effects in islet MECs. The anti-apoptotic effects involve phosphoinositide 3-kinase (PI3K)/Akt, ERK1/2 and cAMP/PKA pathways. These peptides could therefore represent a potential tool to improve islet vascularisation and, indirectly, islet cell function. Electronic supplementary material The online version of this article (doi:10.1007/s00125-011-2423-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.