Islet Vascularization Research Articles

Fetal Protein Restriction, Taurine and Islet Plasticity

The relative risk of type 2 diabetes is higher in individuals of low birth weight, and programming of the metabolic axis is influenced by the intrauterine nutritional environment. The β-cells of the pancreatic islets develop from precursor cells and islet development is controlled by the interactions of transcription factors and peptide growth factors. Both are modulated by fetal nutrition. Fetal programming of adult glucose intolerance has been studied using rodent models, including administration of a low protein (LP) diet. Long-term changes are found in insulin action within target tissues, as well as in the development and function of the endocrine pancreas. Intrauterine growth restriction in rat or man results in a reduced islet and β-cell mass at birth. A reduction of dietary protein to 8% throughout gestation caused relative growth restriction at birth with reduced β-cell mass and islet size. If LP diet is extended to weaning the changes are irreversible, and include an altered β-cell ontogeny, decreased cell replication but increased apoptosis, a reduced expression of insulin-like growth factors, and a reduction in islet vascularity and the number of endothelial precursor cells. The net result is a population of islets with limited plasticity and an inability to adapt to the associated insulin resistance. Studies with supplementation of selected amino acids in early life showed that the altered pancreatic morphology, and glucose intolerance, is reversible by taurine. Both LP diet and taurine are able to delay the onset of type 1 diabetes in the non-obese diabetic mouse.

Pancreatic Islet Production of Vascular Endothelial Growth Factor-A Is Essential for Islet Vascularization, Revascularization, and Function

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and beta-cell mass, mice with beta-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of beta-cell secretagogues to islets, we showed that reduced insulin output is not a result of beta-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Co-Expression of Vascular Endothelial Growth Factor and Interleukin-1 Receptor Antagonist Improves Human Islet Survival and Function

Ex vivo gene therapy approaches can improve the outcome of islet transplantation for treating type I diabetes. We have previously shown the improvement in islet function and vascularization following ex vivo transfection for human vascular endothelial growth factor (hVEGF) gene expression. In this study, we tested the hypothesis that co-expression of two genes, which target different challenges faced by islets post-transplantation, supplement each other to improve the survival and function of islets. We determined whether there is an additive effect of hVEGF and human interleukin-1 receptor antagonist (hIL-1Ra) gene expression in human islets. Human islets were co-infected with adenoviral vectors encoding hVEGF and hIL-1Ra. Islets were then incubated with a cocktail of inflammatory cytokines (IL-1beta+TNFalpha+IFNgamma), and islet viability and function were determined. In vivo function was evaluated by transplanting islets under the kidney capsules of streptozotocin-induced non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. Infection of human islets with Adv-hVEGF and/or Adv-hIL-1Ra inhibited expression of inducible nitric oxide synthase (iNOS), decreased the production of nitric oxide (NO), and prevented the loss of in vitro glucose-stimulated insulin response and viability. Moreover, co-expression of hVEGF and hIL-1Ra reduced the blood glucose level of mice, and increased the level of blood insulin and c-peptide upon glucose challenge. Our results indicated that co-expression of genes that target different insults to transplanted islets can improve the outcome of islet transplantation better than either gene alone.

Islet Inflammation and Fibrosis in a Spontaneous Model of Type 2 Diabetes, the GK Rat

The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II(+) and CD68(+)) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.

A Low Protein Diet Alters Gene Expression in Rat Pancreatic Islets

Insulin secretion is regulated mainly by circulating nutrients, particularly glucose, and is also modulated by hormonal and neuronal inputs. Nutritional alterations during fetal and early postnatal periods, induced by either low protein or energy-restricted diets, produce beta-cell dysfunction. As a consequence, insulin secretion in response to different secretagogues is reduced, as is the number of beta-cells and the size and vascularization of islets. In this study, we used a cDNA macroarray technique and RT-PCR to assess the pattern of gene expression in pancreatic islets from rats fed isocaloric low (6 g/100 g, LP) and normal (17 g/100 g, NP) protein diets, after weaning. Thirty-two genes related to metabolism, neurotransmitter receptors, protein trafficking and targeting, intracellular kinase network members and hormones had altered expression (up- or down-regulated). RT-PCR confirmed the macroarray results for five selected genes, i.e., clusterin, secretogranin II precursor, eukaryotic translation initiation factor 2, phospholipase A(2) and glucose transporter. Thus, cDNA macroarray analysis revealed significant changes in the gene expression pattern in rats fed a low protein diet after weaning. The range of proteins affected indicated that numerous mechanisms are involved in the intracellular alterations in the endocrine pancreas, including impaired glucose-induced insulin secretion.

Taurine Supplementation of a Low Protein Diet Fed to Rat Dams Normalizes the Vascularization of the Fetal Endocrine Pancreas

In rats, an isoenergetic low protein diet (LP) given throughout gestation perturbs the development of the endocrine pancreas by reducing beta-cell mass and islet vascularization at birth. Taurine, an important amino acid during development, has been found to be low in fetal and maternal plasma. When added to a LP diet, taurine normalizes beta-cell mass. Therefore, we investigated the ability of taurine to correct altered islet vascularization. Rats were given 20% [control (C)] or 8% (LP) protein in the diet with or without supplementation with 25 g/L taurine (T) in drinking water (C+T and LP+T) during gestation and lactation. Immunostaining for vascular endothelial growth factor (VEGF) and fetal liver kinase-1 (Flk-1), a VEGF receptor, was performed on fetal and neonatal pancreatic sections. Blood vessel density and blood vessel number were analyzed morphometrically on semi-thin sections. Taurine supplementation restored a normal volume and numerical density of vessels in fetal islets. The number of cells showing immunoreactivity for VEGF and Flk-1 was reduced by 33 and 45%, respectively, in islet cells from LP fetuses. In 1-mo-old pups, VEGF-positive cells remained decreased by nearly 22%. Both VEGF and Flk-1 were restored in pancreatic endocrine cells of fetuses and pups given taurine. The LP diet induced a threefold overexpression of Flk-1 in ductal cells, which contain precursors of beta cells. However, taurine supplementation was without effect. In conclusion, underexpression of VEGF and Flk-1 is associated with the lower fetal islet vascularization induced by the maternal malnutrition. The addition of taurine to the maternal diet prevents such damage and has a potential role in islet vasculogenesis.

Role of VEGF-A in Vascularization of Pancreatic Islets

Blood vessel endothelium has been recently shown to induce endocrine pancreatic development [1]. Because pancreatic endocrine cells or islets express high levels of vascular endothelial growth factors, VEGFs [2, 3], we investigated the role of a particular VEGF, VEGF-A, on islet vascularization and islet function. By deleting VEGF-A in the mouse pancreas, we show that endocrine cells signal back to the adjacent endothelial cells to induce the formation of a dense network of fenestrated capillaries in islets. Interestingly, VEGF-A is not required for the development of all islet capillaries. However, the few remaining capillaries found in the VEGF-A-deficient islets are not fenestrated and contain an unusual number of caveolae. In addition, glucose tolerance tests reveal that the VEGF-A-induced capillary network is not strictly required for blood glucose control but is essential for fine-tuning blood glucose regulation. In conclusion, we speculate that islet formation takes place in two sequential steps: in the first step, signals from blood vessel endothelium induce islet formation next to the vessels, and in the second step, the islets signal to the endothelium. The second step involves paracrine VEGF-A signaling to elaborate the interaction of islets with the circulatory system.

Heat shock preconditioning impairs revascularization of freely transplanted pancreatic islets.

Revascularization of freely transplanted pancreatic islets is essential for appropriate graft function and survival. During the first days after transplantation, however, islet transplants are avascular, and successful engraftment is believed to be markedly hampered by hypoxia-induced tissue injury. Because heat shock has been shown to induce cell resistance against hypoxia, it seems reasonable to stress pancreatic islets by heat before transplantation. In contrast, hypoxia is a major stimulus for angiogenesis, and thus heat shock preconditioning-induced resistance against hypoxia may decrease stimulation of angiogenesis. The authors therefore studied in vivo whether heat shock preconditioning of isolated islets affects angiogenesis and revascularization after free transplantation. After collagenase isolation, heat shock-preconditioned islets (42 degrees C for 30 min) were transplanted syngeneically into nontreated skinfold chambers of Syrian hamsters. In a second group of animals, nontreated islets were transplanted into heat shock-preconditioned chambers. Nontreated islets transplanted into nontreated chambers served as controls. Islet angiogenesis and revascularization were quantitatively analyzed during 14 days after transplantation using intravital fluorescence microscopy. Expression of heat shock proteins (HSP) was confirmed by immunohistochemistry and Western blotting. Immunohistochemistry revealed expression of HSP32 (heme oxygenase [HO]-1), HSP72, and also intracellular insulin in isolated and transplanted pancreatic islets. Western blot analysis showed enhanced HSP32 but slightly decreased HSP72 expression in heat shock-preconditioned islets when compared with controls. Intravital microscopy revealed appropriate vascularization of control islets within 14 days after transplantation. Heat shock preconditioning of the host tissue (i.e., the skinfold chambers) did not affect islet vascularization when compared with controls. In contrast, heat shock preconditioning of the isolated islets resulted in a significantly (P < 0.05) impaired take rate, a reduced (P < 0.05) size of the newly formed microvascular network, and thus a smaller area (P < 0.05) of microvascularly perfused endocrine tissue. These data suggest that heat shock preconditioning of isolated pancreatic islets before transplantation impairs the process of graft angiogenesis and revascularization. Therefore, transient exposure of isolated islets to heat may not be considered a promising tool to improve the outcome of islet transplantation.

A Low-Protein Isocaloric Diet During Gestation Affects Brain Development and Alters Permanently Cerebral Cortex Blood Vessels in Rat Offspring

In humans, low birth weight is associated with nonfatal stroke, cardiovascular disease and diabetes at adulthood. The aim of this study was to investigate in rats the effect of early protein restriction, inducing low birth weight, on brain and endocrine pancreas vascularization at birth and to study if such alterations lasted until adulthood. Pregnant rats were fed either 20 or 8% protein isocaloric diets. Control newborns were nursed by their dams fed the 20% protein diet and low protein (LP) pups by dams fed either the 8 or 20% protein diet. The diets given during lactation were maintained until adulthood. The blood vessel density of cerebral cortex analyzed by morphometry in 3-d-old pups from dams fed the 8% protein diet was lower than in control (C). It remained lower at adulthood whether a LP or a C diet was given postnatally. Reduction of vascularization at adulthood induced by the LP diet limited to fetal life seems characteristic for the brain since vascularization of islets of Langerhans was reduced in neonates but normalized at adulthood by a C diet postnatally. Body and brain weights were lower in LP pups and adults. DNA concentration was lower in forebrain and higher in cerebellum in LP pups. In brain of LP adults, DNA, protein, cholesterol and phospholipid concentrations were lower and were restored at adulthood by a normal diet after birth. In conclusion, cerebral cortex of offspring exposed to a LP isocaloric diet during fetal development showed reduced vascularization which remained throughout life.

Effects of cyclosporine A on the process of vascularization of freely transplanted islets of Langerhans.

We studied in vivo the effect of cyclosporine A (CsA) on both pancreatic islet vascularization and microvascular perfusion using intravital fluorescence microscopy and the dorsal skinfold chamber model in Syrian golden hamsters. Syngeneic transplantation was performed in order to exclude allograft- or xenograft-induced microvascular alterations. To study the effect of CsA on islet angiogenesis and vascularization, animals received 20 mg/kg CsA daily from day 0 until day 14 after transplantation (group A). To study toxic effects of CsA on islet microcirculation, the grafts were allowed to vascularize without immunosuppression, and 20 mg/kg CsA was given daily from day 10 until day 20 after transplantation (group B). Quantitative analysis of the process of islet vascularization in group A revealed a functional capillary density (FCD) of 515.6+/-72.7 cm(-1) at day 6 after transplantation without further increase until day 14 (504.3+/-16.7 cm(-1)). Islet transplants which were not treated with CsA during the process of angiogenesis/vascularization (group B) demonstrated a slightly but significantly (P<0.05) higher FCD (604.7+/-42.5 cm(-1)) at day 14 after transplantation, indicating slightly improved vascularization when compared to transplants of group A. Additional CsA treatment of these islet grafts until day 20 did not induce derangements of microvascular perfusion (601.2+/-67.0 cm(-1)), indicating that the immunosuppressive, drug has no toxic/detrimental effects on the transplants nutritional blood supply. We conclude that CsA only slightly alters the process of final vascularization of freely transplanted islets, and does not deteriorate nutritive perfusion of completely vascularized grafts.

Islet Function in Offspring of Mothers on Low-Protein Diet During Gestation

A low-protein diet (8 vs. 20%) administered during pregnancy affects the structure and function of the endocrine pancreas of the offspring. At 21.5 days of gestation, we reported a reduction of cell proliferation, islet size, islet vascularization, and pancreatic insulin content. In this study, we demonstrated an impairment of insulin secretion of these fetal islets when stimulated in vitro with amino acids such as arginine and leucine. If the offspring is kept on the same low-protein diet during suckling, weaning, and adulthood, fasting insulin levels remain low in the presence of normal blood glucose levels. Glucose tolerance at 70 days is impaired, with lower insulin response. In addition, permanent functional damage seems to be induced in utero by a low-protein diet, because a normal diet given from birth to adulthood does not restore normal insulin response after a glucose challenge. Our experimental results stress the impact of a balanced diet with qualitative and quantitative amino acid composition for the fetal endocrine pancreas to develop normally, without lasting functional and structural consequences in adulthood.

Effect of a Low Protein Diet during Pregnancy on the Fetal Rat Endocrine Pancreas

The administration of a low protein (LP, 8% protein/dry matter) but isocaloric diet to gestating rats did not affect their fertility, but slightly reduced the quantity of food intake as well as body weight gain. The LP diet also did not affect the placental weight, but the weight of the offspring was decreased. Accordingly the fetal endocrine pancreas was altered by the LP diet. Two different morphometric analyses showed that in the LP neonate B-cell proliferation and islet size were reduced in the head of the pancreas. In the pancreatic tail, these parameters were also decreased but to a lesser extend. Islet vascularization in the neonates was dramatically reduced in both parts of the pancreas when the mothers were fed with the LP diet.