Islet Endothelial Cells Research Articles

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

BackgroundInflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra).Methodology/Principal FindingsGene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia.Conclusions/SignificanceGK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.

Vascular niche of pancreatic islets

Pancreatic islets are highly vascularized micro-organs. Approximately 10% of an islet consists of blood vessels. The induction and maintenance of the islet vascular system depend on VEGF secreted from β-cells. VEGF is also critical for the phenotype of the islet vasculature by induction of a vast number of fenestrae. The islet vasculature serves the role of supplying the endocrine cells with oxygen and nutrients, but may also be important for proper glucose sensing of the cells, for paracrine support of endocrine function and growth, and for drainage of metabolites and secreted islet hormones into the systemic circulation. Emerging evidence suggests an important role of islet endothelial cells to maintain β-cell function and growth by secretion of molecules such as hepatocyte growth factor, thrombospondin-1 and laminins, thereby forming a vascular niche for the endocrine cells.

Endothelial cell signalling supports pancreatic beta cell function in the rat

The proximity of endothelial cells and beta cells in islets by necessity means that they are exposed to each other's products. Whereas islet endothelial cells require signals from beta cells to function properly, endothelin-1, thrombospondin-1 and laminins, among others, have been identified as endothelial-derived molecules, although their full effects on beta cells have not been explored. We tested the hypothesis that islet endothelial-derived products affect beta cell function. Endothelial cells from rat islets were proliferated and purified. Endothelium-conditioned culture medium (ECCM) was obtained by maintaining the endothelial cells in culture medium. Islet function was evaluated following exposure of cultured islets to standard culture medium or ECCM. Changes in mRNA levels for key beta cell metabolic enzymes were also measured in islets after ECCM exposure. Glucose-stimulated insulin release and islet insulin content were markedly enhanced by exposure to ECCM. This was at least partly explained by improved mitochondrial function, as assessed by glucose oxidation and an upregulation of the mitochondrial gene for glycerol-3-phosphate dehydrogenase (mGpdh [also known as Gpd2]), combined with upregulation of the rate-limiting enzyme in the glycolysis, glucokinase, in the islets. The intracellular degradation of insulin was also decreased in the islets. Islet endothelial cells produced laminins, and the positive effects of islet endothelial cells were prevented by addition of a neutralising antibody to the beta1-chain of laminin. Addition of exogenous laminin stimulated islet function. This study provides proof of principle that endothelial cells can affect the function of beta cells in their vicinity and that this is at least partially mediated by laminins.

Angiostatic factors normally restrict islet endothelial cell proliferation and migration: implications for islet transplantation

New blood vessel formation in transplanted islets occurs within 7-14 days post-transplantation through both the expansion of donor islet endothelium and ingrowth of blood vessels from the implantation organ. However, several studies indicate that although the islets attract recipient blood vessels, the formed intra-islet vascular network is insufficient, which affects islet post-transplant function. This study aimed to develop an in vitro model to investigate the migration and proliferation properties of isolated liver and islet endothelium.Rat islet or liver endothelium was purified using Bandeiraea simplicifolia(BS-1)-coated Dynabeads. The liver endothelium displayed an increased migration and proliferation to islet-conditioned medium. These effects were fully prevented by adding a neutralizing vascular endothelial growth factor (VEGF)-antibody. In contrast, islet-produced VEGF failed to induce islet endothelial cell migration and only had marginal effects on islet endothelial cell proliferation.These properties could, however, be activated through blocking the effects of either endostatin, thrombospondin-1 or alpha(1)-antitrypsin. In conclusion, VEGF may attract recipient blood vessels towards intrahepatically transplanted islets,but intra-islet vascular expansion is hampered by angiostatic factors present within the islets and the islet endothelium. Inhibition of angiostatic factors early after transplantation may provide a strategy to restore the islet vascular network and improve islet graft function.

Angiomotin-Like Protein 1 Controls Endothelial Polarity and Junction Stability During Sprouting Angiogenesis

We have previously shown that angiomotin (Amot) is essential for endothelial cell migration during mouse embryogenesis. However, approximately 5% of Amot knockout mice survived without any detectable vascular defects. Angiomotin-like protein 1 (AmotL1) potentially compensates for the absence of Amot as it is 62% homologous to Amot and exhibits similar expression pattern in endothelial cells. Here, we report the identification of a novel isoform of AmotL1 that controls endothelial cell polarization and directional migration. Small interfering RNA-mediated silencing of AmotL1 in mouse aortic endothelial cells caused a significant reduction in migration. In confluent mouse pancreatic islet endothelial cells (MS-1), AmotL1 colocalized with Amot to tight junctions. Small interfering RNA knockdown of both Amot and AmotL1 in MS-1 cells exhibited an additive effect on increasing paracellular permeability compared to that of knocking down either Amot or AmotL1, indicating both proteins were required for proper tight junction activity. Moreover, as visualized using high-resolution 2-photon microscopy, the morpholino-mediated knockdown of amotl1 during zebrafish embryogenesis resulted in vascular migratory defect of intersegmental vessels with strikingly decreased junction stability between the stalk cells and the aorta. However, the phenotype was quite distinct from that of amot knockdown which affected polarization of the tip cells of intersegmental vessels. Double knockdown resulted in an additive phenotype of depolarized tip cells with no or decreased connection of the stalk cells to the dorsal aorta. These results cumulatively validate that Amot and AmotL1 have similar effects on endothelial migration and tight junction formation in vitro. However, in vivo Amot appears to control the polarity of vascular tip cells whereas AmotL1 mainly affects the stability of cell-cell junctions of the stalk cells.

Genetic Deficiency of Itgb2 or ItgaL Prevents Autoimmune Diabetes Through Distinctly Different Mechanisms in NOD/LtJ Mice

OBJECTIVEInsulitis is an important pathological feature of autoimmune diabetes; however, mechanisms governing the recruitment of diabetogenic T-cells into pancreatic islets are poorly understood. Here, we determined the importance of leukocyte integrins β2(Itgb2) and αL (ItgaL) in developing insulitis and frank diabetes.RESEARCH DESIGN AND METHODSGene-targeted mutations of either Itgb2 or ItgaL were established on the NOD/LtJ mouse strain. Experiments were performed to measure insulitis and diabetes development. Studies were also performed measuring mutant T-cell adhesion to islet microvascular endothelial cells under hydrodynamic flow conditions. T-cell adhesion molecule profiles and adoptive transfer studies were also performed.RESULTSGenetic deficiency of either Itgb2 or ItgaL completely prevented the development of hyperglycemia and frank diabetes in NOD mice. Loss of Itgb2 or ItgaL prevented insulitis with Itgb2 deficiency conferring complete protection. In vitro hydrodynamic flow adhesion studies also showed that loss of Itgb2 completely abrogated T-cell adhesion. However, ItgaL deficiency did not alter NOD T-cell adhesion to or transmigration across islet endothelial cells. Adoptive transfer of ItgaL-deficient splenocytes into NOD/Rag-1 mice did not result in development of diabetes, suggesting a role for ItgaL in NOD/LtJ T-cell activation.CONCLUSIONSTogether, these data demonstrate that genetic deficiency of Itgb2 or ItgaL confers protection against autoimmune diabetes through distinctly different mechanisms.

Beneficial Role of Pancreatic Microenvironment for Angiogenesis in Transplanted Pancreatic Islets

Pancreatic islets implanted heterotopically (i.e., into the kidney, spleen, or liver) become poorly revascularized following transplantation. We hypothesized that islets implanted into the pancreas would become better revascularized. Islets isolated from transgenic mice expressing enhanced yellow fluorescent protein (EYFP) in all somatic cells were cultured before they were implanted into the pancreas or beneath the renal capsule of athymic mice. Vascular density was evaluated in histological sections 1 month posttransplantation. EYFP was used as reporter for the transgene to identify the transplanted islets. Islet endothelial cells were visualized by staining with the lectin Bandeiraea simplicifolia (BS-1). Capillary numbers in intrapancreatically implanted islets were only slightly lower than those counted in endogenous islets, whereas islets implanted beneath the renal capsule had a markedly lower vascular density. In order to determine if this high graft vascular density at the intrapancreatic site reflected expansion of remnant donor endothelial cells or increased ingrowth of blood vessels from the host, also islets from Tie2-green fluorescent protein (GFP) mice (i.e., islets with fluorescent endothelial cells) were transplanted into the pancreas or beneath the renal capsule of athymic mice. These islet grafts revealed that the new vascular structures formed in the islet grafts contained very few GFP-positive cells, and thus mainly were of recipient origin. The reason(s) for the much better ingrowth of blood vessels at the intrapancreatic site merits further studies, because this may help us form strategies to overcome the barrier for ingrowth of host vessels also into islets in heterotopic implantation sites.

Expression of CD86 on Human Islet Endothelial Cells Facilitates T Cell Adhesion and Migration

Pancreatic islet endothelial cells (ECs) form the barrier across which autoreactive T cells transmigrate during the development of islet inflammation in type 1 diabetes. Little is known about the immune phenotype of islet ECs that might shape their molecular interaction with autoreactive T cells before and during the development of islet inflammation. In this study we examined the expression and functional significance of costimulatory molecules by human islet ECs. Freshly isolated human islet ECs constitutively expressed CD86 (B7-2) and ICOS ligand but not CD80 (B7-1) or CD40 costimulatory molecules. The functional activity of islet EC-expressed CD86 was examined by coculture of resting islet ECs with CD4 T cells stimulated by CD3 ligation alone. Marked T cell proliferation in the coculture was completely abrogated by mAb blockade of CD86, confirming that costimulatory properties are conferred on ECs by CD86 expression. In view of its location on the vasculature, we hypothesized a role for CD86 in T cell adhesion/transmigration. In keeping with this, adhesion/transmigration of activated (CD3 ligated) memory (CD45R0(+)) CD4 T cells across islet ECs was completely inhibited in the presence of CD86 blocking mAb. Identical results were obtained for T cell adhesion using either CTLA-4 blocking mAb or CTLA-4Ig (abatacept), indicating CTLA-4 as the T cell ligand for these CD86-mediated effects. These data suggest a novel role for CD86 expression on the microvasculature, whereby ligation of CTLA-4 on CD4 T cells by CD86 on islet ECs is key to the adhesion of recently activated T cells.

Endothelial glutathione- S-transferase A4-4 protects against oxidative stress and modulates iNOS expression through NF-κB translocation

Our recent work in endothelial cells and human atherosclerotic plaque showed that overexpression of glutathione- S-tranferases (GSTs) in endothelium protects against oxidative damage from aldehydes such as 4-HNE. Nuclear factor (NF)-κB plays a crucial role during inflammation and immune responses by regulating the expression of inducible genes such as inducible nitric oxide synthase (iNOS). 4-HNE induces apoptosis and affects NF-κB mediated gene expression, but conflicting results on 4-HNE's effect on NF-κB have been reported. We compared the effect of 4-HNE on iNOS and the NF-κB pathway in control mouse pancreatic islet endothelial (MS1) cells and those transfected with mGSTA4, a α-class GST with highest activity toward 4-HNE. When treated with 4-HNE, mGSTA4-transfected cells showed significant upregulation of iNOS and nitric oxide (NO) through (NF)-κB (p65) translocation in comparison with wild-type or vector-transfected cells. Immunohistochemical studies of early human plaques showed lower 4-HNE content and upregulation of iNOS, which we take to indicate that GSTA4-4 induction acts as an enzymatic defense against high levels of 4-HNE, since 4-HNE accumulated in more advanced plaques, when detoxification and exocytotic mechanisms are likely to be overwhelmed. These studies suggest that GSTA4-4 may play an important defensive role against atherogenesis through detoxification of 4-HNE and upregulation of iNOS.

From Endothelial to β Cells: Insights into Pancreatic Islet Microendothelium

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.

The Fate of Extracellular Matrix and Intra- Islet Endothelial Cells in Encapsulated Islets

The Fate of Extracellular Matrix and Intra- Islet Endothelial Cells in Encapsulated Islets

Effects of external ATP on Ca2+ signalling in endothelial cells isolated from mouse islets

External ATP is believed to initiate and propagate Ca(2+) signals co-ordinating the insulin release pulses within and among the different islets in the pancreas. The possibility that islet endothelial cells participate in this process was evaluated by comparing the effects on [Ca(2+)](i) of purinoceptor activation in these cells with those in beta-cells. beta-Cell-rich pancreatic islets were isolated from ob/ob mice and dispersed into single cells/aggregates. After culture with or without endothelial cell growth supplement (ECGS), the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was measured with ratiometric fura-2 technique. Presence of ECGS or prolongation of culture (>5 days) resulted in proliferation of endothelial cells and altered their phenotype from rounded to elongated. Endothelial cells, preliminarily identified by attachment of Dynabeads coated with the Bandeiraea simplicifolia 1 lectin (BS-1), responded in a similar way as those stained with CD31 antibodies after measurements of [Ca(2+)](i). Spontaneous transients and oscillations of [Ca(2+)](i )were seen in beta-cells, but not in endothelial cells exposed to 20 mM glucose. Addition of ATP (10 microM) resulted in pronounced and more extended rise of [Ca(2+)](i) in endothelial cells than in beta-cells. The endothelial cells differed from the beta-cells by also responding with a rise of [Ca(2+)](i) to 10 microM UTP, but not to equimolar ADP and acetylcholine. The results support the idea of mutual interactions between islet endothelium and beta-cells based on ATP-induced Ca(2+) signals. It is suggested that the endothelial cells have a tonic inhibitory action on beta-cell P2 purinoceptors resulting in impaired synchronization of the insulin release pulses.

Atherogenic vascular and lipid phenotypes in young patients with Type 1 diabetes are associated with diabetes high-risk HLA genotype

Expression of human leukocyte antigen (HLA) class II molecules on islet endothelial cells is a central vascular event in the pathogenesis of Type 1 diabetes. Previous studies demonstrated the ability of other vascular endothelial cells to express HLA and thereby to process islet autoantigens on their surface. We investigated whether the HLA-DQ2/8 genotype, which confers the highest risk for Type 1 diabetes, is associated with early atherosclerosis in youths with this disease. Brachial artery endothelium-dependent, flow-mediated dilation (BA-FMD) and carotid artery intima-media thickness (CA-IMT), as well as markers of systemic inflammation [C-reactive protein (CRP), fibrinogen, and orosomucoid], HbA(1C), LDL, HDL, and total cholesterol, were assessed in 86 children and adolescents with Type 1 diabetes (mean age and diabetes duration, 15 and 7 yr, respectively) between 2004 and 2006. HLA genotypes were determined in dried blood spots by an oligoblot hybridization method. As a result, HLA-DQ2/8 was detected in 34 patients (DQ2/8). When this group was compared with the remaining patients (non-DQ2/8, n = 52), there were no differences in age, diabetes duration, HbA(1C), body mass index, inflammatory markers, and IMT (P > or = 0.4). In the DQ2/8 group, LDL-to-HDL ratio was elevated compared with that in the non-DQ2/8 group (1.8 vs. 1.3, respectively; P = 0.001), whereas FMD did not significantly differ between the groups (5.3% vs. 6.7%, respectively; P = 0.08). When patients were further categorized in relation to CRP (cut-off value, 1 mg/l), BA-FMD was significantly lower (3%, P < 0.01), whereas LDL-to-HDL ratio increased further (2.2, P < 0.001) in the subgroup of DQ2/8 and CRP > or = 1 patients compared with the remaining three subgroups. These associations remained significant after adjustment for age, diabetes duration, and HbA(1C) by analysis of covariance. The brachial artery responses to nitroglycerine were similar in all subgroups. In conclusion, the diabetes-predisposing HLA-DQ2/8 genotype in children and adolescents with Type 1 diabetes interferes with endothelial and lipid-related mechanisms of early atherosclerosis, possibly in part through inflammatory pathways.

Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection

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.

Age- and Sex-Related Differences in Spontaneous Hemorrhage and Fibrosis of the Pancreatic Islets in Sprague–Dawley Rats

To elucidate the spontaneous occurrence of hemorrhage in the pancreatic islet, naïve Crj:CD(SD)IGS rats were given a commercially available standard diet ad libitum over 20 weeks, and were sequentially examined. Islet hemorrhage was morphologically observed from 12 weeks of age, and its incidence was significantly higher in males than in females, with a wide distribution in all pancreatic lobes. The incidence (%) of affected islets to examined islets was increased with age. Hemorrhage was accompanied by brownish pigmentation, and reacted positively for iron by Prussian/Berlin blue staining. In 26 weeks old, most of the islets were dissected by dense fibrous tissue into small nests, and disarranged beta cells were detected by insulin immunostaining. Ultrastructurally, no apparent morphological change was seen in any islet endothelial cell, although blood leakage with migrated macrophages and dense collagen fibers was observed around the capillaries. In serum biochemistry of rats aged 26 weeks, the estradiol level in males with hemorrhage was significantly lower than that in males with non-hemorrhage, presumably suggesting the lack of capillary protective ability. Next, when rats were given a high fat/protein diet over 20 weeks to clarify whether it accelerated the frequency or timing of hemorrhage, its occurrence was essentially identical to that of the animals fed the standard diet. In conclusion, the onset of spontaneous islet hemorrhage was observed predominantly in aged males, resulting from the low estradiol level in serum.

Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species‐mediated activation of ERK

Sphingosylphosphorylcholine (SPC) produces reactive oxygen species (ROS) in MS1 pancreatic islet endothelial cells. In the present study, we explored the physiological significance of the SPC-induced ROS generation in endothelial cells. SPC induced cell death of MS1 cells at higher than 10 microM concentration through a caspase-3-dependent pathway. SPC treatment induced sustained activation of an extracellular signal-regulated kinase (ERK), in contrast to transient activation of ERK in response to platelet-derived growth factor (PDGF)-BB, which stimulated proliferation of MS1 cells. Both the SPC-induced cell death and ERK activation were abolished by pretreatment of the cells with the MEK inhibitor U0126 or by overexpression of a dominant negative mutant of MEK1 (DN-MEK1). Pretreatment of the cells with N-acetylcysteine, an antioxidant, completely prevented the SPC-induced ROS generation, apoptosis, and ERK activation, whereas the ROS generation was not abrogated by treatment with U0126. Consistent with these results, SPC induced cell death of human umbilical vein endothelial cells (HUVECs) through ROS-mediated activation of ERK. These results suggest that the SPC-induced generation of ROS plays a crucial role in the cell death of endothelial cells through ERK-dependent pathway.

Beneficial effects of candesartan, an angiotensin II type 1 receptor blocker, on β-cell function and morphology in db/db mice

Several epidemiological studies suggested that treatment with angiotensin II type 1 receptor blocker (ARB) provided a risk reduction of developing type 2 diabetes. In this study, we investigated whether and how ARB treatment can improve abnormalities of pancreatic islets in diabetes state. We randomized db/db mice, a model of type 2 diabetes with obesity, at the age of 8 weeks to receive candesartan, an ARB, for 6 weeks. We also studied age-matched db/misty mice as control. Glucose tolerance test revealed that candesartan treatment improved glucose tolerance with the modest increase in serum insulin level in db/db mice. Concurrently, candesartan increased β-cell mass, increased staining intensity of insulin, and decreased staining intensity of components of NAD(P)H oxidase, p22phox and gp91phox, and those of oxidative stress markers in β-cells. These changes were accompanied by reduction of mitochondrial volume. Treatment with candesartan also reduced fibrosis in and around the islets and prevented the loss of endothelial cells in islets. Our results showed that candesartan partially prevented deterioration of glucose tolerance by providing protection against progressive β-cell damage in diabetes.

Endothelial Cells in Endogenous and Transplanted Pancreatic Islets: Differences in the Expression of Angiogenic Peptides and Receptors

Background/Aims: An important reason for the large amount of islets required for successful islet transplantation is likely to be inadequate engraftment of the transplanted islets. Thus, the revascularization is of major importance for graft survival. In order to study the expression of angiogenic peptides and receptors on islet endothelial cells (EC), we needed methods giving access to such endothelium. Therefore, we developed methods to isolate EC from islets transplanted intraportally or beneath the kidney capsule. Methods: Pancreatic islets were isolated, cultured and syngeneically transplanted into the liver or beneath the kidney capsule of C57BL/6 mice. One month post-transplantation, the islets were retrieved and EC from these islets were explanted. EC were also collected from freshly isolated and cultured non-transplanted islets. The EC were purified with Dynabeads and identified with immunocytochemistry. Angiogenesis GEArray technology was used to study angiogenic gene expression. Results: Several angiogenic genes were expressed in EC; e.g. endostatin, pigment-epithelial derived factor, vascular endothelial growth factor and angiopoietin-2, and their expression were affected by culture. Conclusion: The expression of angiogenesis-related genes in islet EC from non-transplanted islets is affected by culture. Moreover, we also describe a technique, which makes it possible to obtain EC from transplanted islets.

Platelet-Activating Factor Synthesis and Response on Pancreatic Islet Endothelial Cells: Relevance for Islet Transplantation

Recent data suggest that donor intraislet endothelial cells may survive islet transplantation and participate to the events that influence islet engraftment. However, the mechanisms that regulate islet endothelial behavior in this setting are poorly known. We obtained immortalized human (hIECs) and mouse (mIECs) islet endothelial cells by transfection with SV40-T-large antigen and studied the synthesis and response to Platelet-activating factor (PAF), a multipotent phospholipid that acts as endothelial mediator of both inflammation and angiogenesis. HIECs showed typical endothelial markers such as expression of vWF, CD31, and CD105, uptake of acetylated-LDL and binding to ULE-A lectin. Moreover, they expressed nestin, the PAF-receptor and possess surface fenestrations and in vitro angiogenic ability of forming tubular structures on Matrigel. Likewise, mIECs showed expression of vWF, CD31, nestin, PAF-receptor and CD105, and uptake of acetylated-LDL. HIECs and mIECs rapidly produced PAF under stimulation with thrombin in a dose-dependent way. Exogenous PAF or thrombin-induced PAF synthesis increased leukocyte adhesion to hIECS and mIECs and cell motility of both endothelial cell lines. Moreover, PAF or thrombin-induced PAF synthesis accelerated in vitro formation of vessel-like tubular structures when hIECs are seeded on Matrigel. Notably, gene-microarray analysis detected up-regulation of beta3 integrin gene on hIECs stimulated with PAF, that was confirmed at the protein level. Based on the novel development of immortalized islet endothelium, these results suggest that PAF may have a dual role that links inflammation to angiogenesis in the early events of islet transplantation.

Islet Endothelial Cells and Pancreatic β-Cell Proliferation: Studiesin Vitroand during Pregnancy in Adult Rats

The growth of both tumors and nonneoplastic tissues may be influenced by signals from the vascular endothelium. In the present investigation we show that purified proliferating endothelial cells from pancreatic islets can stimulate beta-cell proliferation through secretion of hepatocyte growth factor (HGF). This secretion could be induced by soluble signals from the islets, such as vascular endothelial growth factor-A (VEGF-A) and insulin. During pregnancy, the pancreatic beta-cells display a highly reproducible physiological proliferation. We show that islet endothelial cell proliferation precedes beta-cell proliferation in pregnant animals. Vascular growth was closely associated with endocrine cell proliferation, and prominent expression of HGF was observed in islet endothelium on d 15 of pregnancy, i.e. coinciding with the peak of beta-cell proliferation. In summary, our results suggest the existence of an endothelial-endocrine axis within adult pancreatic islets, which is of importance for adult beta-cell proliferation.