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Vitreous fibrovascular membranes (FVMs), the hallmark of proliferative diabetic retinopathy (PDR), cause retinal hemorrhage, detachment, and eventually blindness. However, little is known about the pathophysiology of FVM. In this study, we used single-cell RNA sequencing on surgically harvested PDR-FVMs and generated a comprehensive cell atlas of FVM. Eight cellular compositions were identified, with microglia as the major cell population. We identified a GPNMB+ subpopulation of microglia, which presented both profibrotic and fibrogenic properties. Pseudotime analysis further revealed the profibrotic microglia was uniquely differentiated from retina-resident microglia and expanded in the PDR setting. Ligand-receptor interactions between the profibrotic microglia and cytokines upregulated in PDR vitreous implicated the involvement of several pathways, including CCR5, IFNGR1, and CD44 signaling, in the microglial activation within the PDR microenvironment. Collectively, our description of the novel microglia phenotypes in PDR-FVM may offer new insight into the cellular and molecular mechanism underlying the pathogenesis of DR, as well as potential signaling pathways amenable to disease-specific intervention. [ABSTRACT FROM AUTHOR]

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2, and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2, or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels, leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here, we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels, as observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels, even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of tumor cells but not most normal cells. Its roles in normal nontransformed tissues are not clear. To explore the potential roles of TRAIL in type 1 diabetes, we examined the consequences of TRAIL blockade or TRAIL deficiency in two animal models of autoimmune diabetes. In the first model, NOD mice received an injection of a soluble TRAIL receptor to block TRAIL function. This significantly accelerated the diabetes and increased the degree of autoimmune inflammation in both pancreatic islets and salivary glands. The GAD65-specific immune responses were also significantly enhanced in animals that received the soluble TRAIL receptor. In the second model, we treated normal and TRAIL-deficient C57BL/6 mice with multiple low-dose streptozotocin to induce diabetes. We found that both the incidence and the degree of islet inflammation were significantly enhanced in TRAIL-deficient animals. On the basis of these observations, we conclude that TRAIL deficiency accelerates autoimmune diabetes and enhances autoimmune responses. [ABSTRACT FROM AUTHOR]

Genes affecting autoimmune type 1 diabetes susceptibility in the nonobese diabetic (NOD) mouse (Idd loci) have been mapped using a congenic strain breeding strategy. In the present study, we used a combination of BAC clone contig construction, polymorphism analysis of DNA from congenic strains, and sequence mining of the human orthologous region to generate an integrated map of the Idd10 region on mouse chromosome 3. We found seven genes and one pseudogene in the 950-kb Idd10 region. Although all seven genes in the interval are Idd10 candidates, we suggest the gene encoding the EWI immunoglobulin subfamily member EWI-101 (Cd101) as the most likely Idd10 candidate because of the previously reported immune-associated properties of the human CD101 molecule. Additional support for the candidacy of Cd101 is the presence of 17 exonic single-nucleotide polymorphisms that differ between the NOD and B6 sequences, 10 causing amino acid substitutions in the predicted CD101 protein. Four of these 10 substitutions are nonconservative, 2 of which could potentially alter N-linked glycosylation. Considering our results together with those previous reports that antibodies recognizing human CD101 modulate human T-cell and dendritic cell function, there is now justification to test whether the alteration of CD101 function affects autoimmune islet destruction. [ABSTRACT FROM AUTHOR]

An association has recently been described between increased birth weight and increased risk of childhood-onset type 1 diabetes. Whether this relationship is explained by genes associated with both increased birth weight and increased risk of type 1 diabetes is unknown. In the present study, we tested the association between birth weight and HLA-DQ genotypes known to confer risk for type 1 diabetes among 969 nondiabetic children randomly selected from the Norwegian population. We found that HLA genotypes previously shown to confer risk for type 1 diabetes were associated with reduced birth weight (the mean difference in birth weight between the DQB1*0602/DQB1*0602 and DQ8/DQ2 genotypes was 354 g [95% CI 105-604]), which was opposite of that expected if HLA genes explained the birth weight-type 1 diabetes association. [ABSTRACT FROM AUTHOR]

Recent studies have shown that angiopoietins (Angs) and their receptor, Tie2, play a role in vascular integrity and neovascularization. The renin-angiotensin system has been hypothesized to contribute to the development of diabetic retinopathy. In this study, we investigated the effect of angiotensin II (AII) on Ang1 and Ang2 expression in cultured bovine retinal endothelial cells (BRECs). AII stimulated Ang2 but not Ang1 mRNA expression in a dose- and time-dependent manner. This response was inhibited completely by angiotensin type 1 receptor (AT1) antagonist. AII increased the transcription of Ang2 mRNA, but did not change the half-life. Protein kinase C (PKC) inhibitor completely inhibited AII-induced Ang2 expression, and the mitogen-activated protein kinase (MAPK) inhibitor also inhibited it by 69.4+/-15.6%. In addition, we confirmed the upregulation of Ang2 in an AII-induced in vivo rat corneal neovascularization model. These data suggest that AII stimulates Ang2 expression through AT1 receptor-mediated PKC and MAPK pathways in BREC, and AII may play a novel role in retinal neovascularization. [ABSTRACT FROM AUTHOR]

Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic beta-cells. The main factors directly implicated in beta-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse beta-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the beta-cells. Unexpectedly, we found that the resulting perforin-mediated killing of beta-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of beta-cells also requires a direct effect of gamma-interferon (IFN-gamma), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-gamma receptor on their beta-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most beta-cells occurs as cytokine-mediated death and requires IFN-gama in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes. [ABSTRACT FROM AUTHOR]

Islet transplantation for the treatment of autoimmune diabetes is more difficult because of the additional barrier presented by the autoimmunity. We tested the ability of hamster anti-rat CD154 to prevent recurrence of diabetes in renal subcapsular islet isografts in DR-BB (RT1uu) rats with established autoimmune diabetes. Experimental animals with established diabetes received intravenous injections of 15 mg/kg anti-CD154 on a specified schedule starting 2 days before renal subcapsular transplantation of an islet isograft. Control animals received either saline or hamster IgG. Plasma glucose levels >250 mg/dl over 3 days were used to indicate the recurrence of diabetes. Rats that received saline (n = 5) or control antibody (n = 3) had a recurrence of diabetes 6-11 days after transplantation. Histological examination of islet isografts from these rats showed complete destruction of the insulin-producing portion of the isograft with residual cells positive for glucagon. Recipient rats that received anti-CD154 at the 15-mg/kg dosage (n = 6) did not have a recurrence of diabetes for 308-461 days after transplantation. Islet isografts removed from the rats showed low levels of insulin immunoreactivity, high levels of insulin mRNA, and focal infiltration with lymphocytes but no evidence of islet destruction. Mean peak antibody concentration was 266 microg/ml and returned to undetectable levels by 67-88 days after transplantation. Rats that received anti-CD154 starting at 4-7 days after transplantation had a recurrence of diabetes within 11 days of the isotransplantation. Therefore, anti-CD154 as the sole immunomodulator prevented the recurrence of diabetes in islet isografts in rats with established autoimmune diabetes. This suggests that CD40/CD154 blockade is effective in preventing the insulitis or the effector phase of autoimmune diabetes. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is the result of destruction of the insulin-secreting beta-cells of the pancreas by a process in which T-cells play a central role. A tyrosine phosphatase-like protein, IA-2, is a major target for autoantibodies and T-cells in the disease. In this study, we have further characterized the T-cell response to IA-2 by the generation and characterization of T-cell lines. T-cell lines responsive to IA-2 antigen were generated from 17 of 32 patients and 3 of 10 control subjects. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. Five lines from diabetic patients responded to one of two peptides representing amino acids 831-850 and 841-860 of IA-2. The overlapping portion may therefore represent an immunodominant region of the molecule. The sixth patient-derived line responded to a peptide representing amino acids 751-770 of IA-2 presented by the DR 4 (DRB1*0401) allele that confers susceptibility to type 1 diabetes. Primary T-cell responses to peptides of the immunodominant region were detected in 9 of 19 (47%) type 1 diabetic patients and 16 of 22 (73%) nondiabetic siblings, consistent with this region having immunostimulatory properties. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule. [ABSTRACT FROM AUTHOR]

The type II transmembrane glycoprotein CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) has been proposed as a mediator of insulin secretion from pancreatic beta-cells and as a candidate for autoimmune reactions in type 2 diabetes. We evaluated the presence of anti-CD38 autoantibodies in Caucasian patients with diabetes and investigated the effect of these antibodies on insulin secretion from isolated human pancreatic islets. The presence of anti-CD38 autoantibodies was evaluated by using Western blot analysis in 236 patients with type 2 diabetes (mean age 63 years), in 160 patients with type 1 diabetes (mean age 38 years), and in 159 nondiabetic subjects. Anti-CD38 autoantibody titers at least 3 SD above the mean value of the control group were found in 9.7% of type 2 diabetic patients and in 13.1% of type 1 diabetic patients (chi2 = 15.9, P = 0.0003 vs. 1.3% of control subjects). No significant differences were observed in sex distribution, current age, age at diabetes onset, BMI, fasting serum glucose, or glycemic control between anti-CD38+ and anti-CD38-diabetic patients in either the type 2 or type 1 diabetic groups. The effect of 23 anti-CD38- and 13 anti-CD38+ sera on insulin secretion at low (3.3 mmol/l) or high (16.7 mmol/l) medium glucose concentrations was evaluated in isolated human pancreatic islets. Data are medians (interquartile range). The anti-CD38+ sera potentiated insulin release both at low [95 (64) vs. 23 (12) microU/ml of control incubations, respectively, P < 0.0001] and high [271 (336) vs. a control of 55 (37) microU/ml, respectively, P = 0.001] medium glucose concentrations, whereas the anti-CD38- sera did not. Furthermore, in the pooled data from all 36 tested sera, insulin levels in the islet incubation medium were directly related to the anti-CD38 antibody titer. We conclude that autoantibodies to CD38 are associated with both type 1 and type 2 diabetes in Caucasian subjects. These autoantibodies exert a stimulatory effect on insulin secretion by cultured human islets. The role of this autoimmune reaction in the pathogenesis of diabetes remains to be elucidated. [ABSTRACT FROM AUTHOR]

The genes responsible for insulin resistance are poorly defined. Plasma cell differentiation antigen (PC-1) glycoprotein inhibits insulin receptor signaling and is associated with insulin resistance. We describe here a novel polymorphism in exon 4 of the PC-1 gene (K121Q) and demonstrate that it is strongly associated with insulin resistance in 121 healthy nonobese (BMI <30 kg/m2) nondiabetic (by oral glucose tolerance test [OGTT]) Caucasians from Sicily. Compared with 80 KK subjects, Q allele carriers (n = 41, 39 KQ and 2 QQ) showed higher glucose and insulin levels during OGTT (P < 0.001 by two-way analysis of variance) and insulin resistance by euglycemic clamp (M value = 5.25 +/- 1.38 [n = 24] vs. 6.30 +/- 1.39 mg x kg(-1) x min(-1) [n = 49], P = 0.005). Q carriers had higher risk of being hyperinsulinemic and insulin resistant (odds ratio [CI]: 2.99 [1.28-7.0], P < 0.001). Insulin receptor autophosphorylation was reduced (P < 0.01) in cultured skin fibroblasts from KQ versus KK subjects. Skeletal muscle PC-1 content was not different in 11 KQ versus 32 KK subjects (33 +/- 16.1 vs. 17.5 +/- 15 ng/mg protein, P = 0.3). These results suggest a cause-effect relationship between the Q carrying genotype and the insulin resistance phenotype, and raise the possibility that PC-1 genotyping could identify individuals who are at risk of developing insulin resistance, a condition that predisposes to type 2 diabetes and coronary artery disease. [ABSTRACT FROM AUTHOR]

Membrane glycoprotein plasma cell 1 (PC-1) has been shown to be increased in type 2 diabetes and involved in insulin resistance through inhibiting the insulin receptor tyrosine kinase, which was demonstrated using cultured breast cancer cells. However, other reports have shown contradictory results in Chinese hamster ovary cells and in vitro kinase assay. Thus, we considered it necessary to investigate the effect of PC-1 using highly insulin-sensitive cells. Here, we used two of the following approaches: 1) investigating PC-1 expression levels in insulin-responsive tissues in rat models of diabetes and 2) overexpressing PC-1 in 3T3-L1 adipocytes. We found that PC-1 was highly expressed in insulin-responsive tissues, such as liver and adipose tissue, in normal rats. However, high-fat feeding or streptozotocin-induced diabetes did not change its expression levels in liver, adipose tissue, and skeletal muscle. Thus, PC-1 expression levels were not associated with high-fat-diet-induced insulin resistance or hyperglycemia. Although PC-1 was increased in adipose tissue in Zucker fatty rats (protein level, by 50%; mRNA level, by 90%), its expression levels in liver and skeletal muscle, tissues that are more responsible for whole body glucose metabolism than adipose tissue, did not significantly differ from those in normal rats. Next, we overexpressed PC-1 in 3T3-L1 adipocytes using an adenovirus transfection system. PC-1 expression was markedly increased to a level 16-fold greater than that in normal human adipose tissue, which is higher than the previously reported levels in diabetic patients. However, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1, activation of phosphatidylinositol 3-kinase, and glucose uptake were not affected by PC-1 overexpression. These results strongly suggest that increased PC-1 expression is not causally related to insulin resistance. [ABSTRACT FROM AUTHOR]

In diabetes-associated microangiopathies and atherosclerosis, there are alterations of the extracellular matrix (ECM) in the intima of small and large arteries. High levels of circulating nonesterified fatty acids (NEFAs) are present in insulin resistance and type 2 diabetes. High concentrations of NEFAs might alter the basement membrane composition of endothelial cells. In arteries, smooth muscle cells (SMCs) are the major producers of proteoglycans and glycoproteins in the intima, and this is the site of lipoprotein deposition and modification, key events in atherogenesis. We found that exposure of human arterial SMCs to 100-300 micromol/albumin-bound linoleic acid lowered their proliferation rate and altered cell morphology. SMCs expressed 2-10 times more mRNA for the core proteins of the proteoglycans versican, decorin, and syndecan 4 compared with control cells. There was no change in expression of fibronectin and perlecan. The decorin glycosaminoglycan chains increased in size after exposure to linoleic acid. The ECM produced by cells grown in the presence of linoleic acid bound 125I-labeled LDL more tightly than that of control cells. Darglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, neutralized the NEFA-mediated induction of the decorin gene. This suggests that some of the NEFA effects are mediated by PPAR-gamma. These actions of NEFAs, if present in vivo, could contribute to changes of the matrix of the arterial intima associated with micro- and macroangiopathies. [ABSTRACT FROM AUTHOR]

The related tyrosine phosphatase-like proteins, islet cell antigen 512 (ICA512) and phosphatase homologue in granules of insulinoma (phogrin), are major targets of autoantibodies in patients with type 1 diabetes. In the current study, we have examined the overlapping specificities and antigenic epitopes of autoantibodies to ICA512 and phogrin and determined whether intramolecular epitope spreading occurs during the development of diabetic autoimmunity. ICA512 autoantibodies and phogrin autoantibodies were detected in 65-70% (n = 110) of patients with new-onset type 1 diabetes and 60-65% (n = 42) of prediabetic relatives of patients with type 1 diabetes. Of the sera, 10% reacted with ICA512 but not phogrin, whereas only 1% of sera reacted with phogrin but not ICA512. The binding of phogrin autoantibodies in 88 dual (ICA512 and phogrin) autoantibody-positive sera could be completely blocked by excess recombinant ICA512, whereas the blocking of ICA512 autoantibodies with recombinant phogrin was only partial (mean inhibition of 58.9 +/- 3.7%, mean +/- SE). Binding and competition analysis using multiple chimeric ICA512/phogrin constructs demonstrated that a major unique epitope for ICA512 autoantibodies is localized to amino acids 762-887. A conformational epitope associated with the carboxy-terminal 31 amino acids of ICA512 was recognized by one-third of sera, and a minor epitope is located on amino acids 601-762 of ICA512. The major epitopes for phogrin-selective autoantibodies were localized to amino acids 640-922 of phogrin. Sequential serum samples were analyzed in 22 relatives who expressed ICA512/phogrin autoantibodies. Intramolecular epitope spreading was found for 5 of 13 relatives who have progressed to type 1 diabetes. Among nine relatives who have remained nondiabetic, three demonstrated a decrease in the number of epitopes recognized. These studies highlight the complexity of autoantibody recognition of ICA512/phogrin and are consistent with the hypothesis that ICA512/phogrin may be recognized as a consequence of beta-cell destruction. [ABSTRACT FROM AUTHOR]

We studied whether there is an association between the single nucleotide polymorphism c.533A>C (K121Q) in the glycoprotein PC-1 gene and features of the metabolic syndrome in case-control and intrafamily association studies in 922 subjects from Finland and Sweden. No difference was observed in the Q allele frequency between control subjects and type 2 diabetic subjects (12.9 vs. 15.1%). The QK genotype was associated with higher fasting plasma glucose (FPG) concentrations than the KK genotype in type 2 diabetic patients (P <0.001) and their relatives (P <0.05). A permutation test of siblings discordant for the QK and KK genotypes also showed that the nondiabetic siblings with the QK genotype had higher FPG (6.1 +/- 2.0 vs. 5.4 +/- 0.6 mmo/l, P <0.001) and fasting insulin (7.0 +/- 3.6 vs. 4.8 +/- 2.6 mU/l, P <0.05) concentrations than the carriers of the KK genotype. In addition, diabetic siblings with the QK genotype had higher systolic blood pressure (147.0 +/- 18.0 vs. 140.0 +/- 18.7 mmHg, P <0.05) and higher fasting (9.9 +/- 3.0 vs. 8.8 +/- 2.8 mmol/l, P <0.05) and 2-h plasma glucose (17.3 +/- 8.5 vs. 12.9 +/- 4.2 mmol/l, P < 0.05) concentrations than the diabetic carriers of the KK genotype. The present study shows that, although the Q allele of the human glycoprotein PC-1 gene is associated with surrogate measures of insulin resistance, it may not be enough to increase the susceptibility to type 2 diabetes. [ABSTRACT FROM AUTHOR]