Ilaria Miceli

The ghrelin gene products and exendin-4 promote survival of human pancreatic islet endothelial cells in hyperglycaemic conditions, through phosphoinositide 3-kinase/Akt, extracellular signal-related kinase (ERK)1/2 and cAMP/protein kinase A (PKA) signalling pathways.

Aims/hypothesisPancreatic 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.MethodsIslet 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.ResultsIslet 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/interpretationThe 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.

Stretch reduces nephrin expression via an angiotensin II-AT1-dependent mechanism in human podocytes: effect of rosiglitazone

Increased glomerular permeability to proteins is a characteristic feature of diabetic nephropathy (DN). The slit diaphragm is the major restriction site to protein filtration, and the loss of nephrin, a key component of the slit diaphragm, has been demonstrated in both human and experimental DN. Both systemic and glomerular hypertension are believed to be important in the pathogenesis of DN. Human immortalized podocytes were subjected to repeated stretch-relaxation cycles by mechanical deformation with the use of a stress unit (10% elongation, 60 cycles/min) in the presence or absence of candesartan (1 microM), PD-123319 (1 microM), and rosiglitazone (0.1 microM). Nephrin mRNA and protein expression were assessed using quantitative real-time PCR, immunoblotting, and immunofluorescence, and the protein expression of AT(1) receptor and angiotensin II secretion were evaluated. Exposure to stretch induced a significant approximately 50% decrease in both nephrin mRNA and protein expression. This effect was mediated by an angiotensin II-AT(1) mechanism. Indeed, podocyte stretching induced both angiotensin II secretion and AT(1) receptor overexpression, podocyte exposure to angiotensin II reduced nephrin protein expression, and both the AT-1 receptor antagonist candesartan and a specific anti-angiotensin II antibody completely abolished stretch-induced nephrin downregulation. Similar to candesartan, the peroxisome proliferator-activated receptor (PPAR)-gamma agonist, rosiglitazone, also inhibited stretch-induced nephrin downregulation, suggesting interference with stretch-induced activation of the angiotensin II-AT(1) receptor system. Accordingly, rosiglitazone did not alter stretch-induced angiotensin II secretion, but it prevented AT(1) upregulation in response to stretch. These results suggest a role for hemodynamic stress in loss of nephrin expression and allude to a role of PPAR-gamma agonists in the prevention of this loss.

Hyperglycemia Induces Apoptosis of Human Pancreatic Islet Endothelial Cells : Effects of Pravastatin on the Akt Survival Pathway

Pancreatic islet microendothelium and beta 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-1beta, 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 mumol/L) decreased apoptosis induced by high glucose or oxidized LDL and increased Akt phosphorylation. Hyperglycemia significantly increased the production of the proinflammatory cytokine interleukin-1beta and stimulated the expression of inducible nitric oxide synthase and the production of nitric oxide, possibly relevant to beta 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.

Human mesenchymal stem cells modulate cellular immune response to islet antigen glutamic acid decarboxylase in type 1 diabetes.

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-gamma, 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-gamma, 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-gamma 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-gamma 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.

Association of cytomegalovirus infections with recurrence of humoral and cellular autoimmunity to islet autoantigens and of type 1 diabetes in a pancreas transplanted patient.

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‐γ 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.

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections

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).