Overexpression of Plg-RKT protects against adipose dysfunction and dysregulation of glucose homeostasis in diet-induced obese mice.

The plasminogen receptor, Plg-RKT, is a unique cell surface receptor that is broadly expressed in cells and tissues throughout the body. Plg-RKT localizes plasminogen on cell surfaces and promotes its activation to the broad-spectrum serine protease, plasmin. In this study, we show that overexpression of Plg-RKT protects mice from high fat diet (HFD)-induced adipose and metabolic dysfunction. During the first 10 weeks on the HFD, the body weights of mice that overexpressed Plg-RKT (Plg-RKT-OEX) were lower than those of control mice (CagRosaPlgRKT). After 10 weeks on the HFD, CagRosaPlgRKT and Plg-RKT-OEX mice had similar body weights. However, Plg-RKT-OEX mice showed a more metabolically favourable body composition phenotype. Plg-RKT-OEX mice also showed improved glucose tolerance and increased insulin sensitivity. We found that the improved metabolic functions of Plg-RKT-OEX mice were mechanistically associated with increased energy expenditure and activity, decreased proinflammatory adipose macrophages and decreased inflammation, elevated brown fat thermogenesis, and higher expression of adipose PPARγ and adiponectin. These findings suggest that Plg-RKT signalling promotes healthy adipose function via multiple mechanisms to defend against obesity-associated adverse metabolic phenotypes.

S1P/S1PR3 signalling axis protects against obesity-induced metabolic dysfunction.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that interacts via 5 G-protein coupled receptors, S1PR1-5, to regulate signalling pathways critical to biological processes including cell growth, immune cell trafficking, and inflammation.We demonstrate that in Type 2 diabetic (T2D) subjects, plasma S1P levels significantly increased in response to the anti-diabetic drug, rosiglitazone, and, S1P levels correlated positively with measures of improved glucose homeostasis. In HFD-induced obese C57BL/6 J mice S1PR3 gene expression was increased in adipose tissues (AT) and liver compared with low fat diet (LFD)-fed counterparts. On a HFD, weight gain was similar in both S1PR3-/- mice and WT littermates; however, HFD-fed S1PR3-/- mice exhibited a phenotype of partial lipodystrophy, exacerbated insulin resistance and glucose intolerance. This worsened metabolic phenotype of HFD-fed S1PR3-/- mice was mechanistically linked with increased adipose inflammation, adipose macrophage and T-cell accumulation, hepatic inflammation and hepatic steatosis. In 3T3-L1 preadipocytes S1P increased adipogenesis and S1P-S1PR3 signalling regulated the expression of PPARγ, suggesting a novel role for this signalling pathway in the adipogenic program. These results reveal an anti-diabetic role for S1P, and, that S1P-S1PR3 signalling in the adipose and liver defends against excessive inflammation and steatosis to maintain metabolic homeostasis at key regulatory pathways.

The plasminogen receptor Plg-RKT regulates adipose function and metabolic homeostasis.

BACKGROUND: Plg-RKT , a unique transmembrane plasminogen receptor, enhances the activation of plasminogen to plasmin, and localizes the proteolytic activity of plasmin on the cell surface. OBJECTIVES: We investigated the role of Plg-RKT in adipose function, metabolic homeostasis, and obesity. METHODS: We used adipose tissue (AT) sections from bariatric surgery patients and from high fat diet (HFD)-induced obese mice together with immunofluorescence and real-time polymerase chain reaction to study adipose expression of Plg-RKT . Mice genetically deficient in Plg-RKT and littermate controls fed a HFD or control low fat diet (LFD) were used to determine the role of Plg-RKT in insulin resistance, glucose tolerance, type 2 diabetes, and associated mechanisms including adipose inflammation, fibrosis, and ectopic lipid storage. The role of Plg-RKT in adipogenesis was determined using 3T3-L1 preadipocytes and primary cultures established from Plg-RKT -deficient and littermate control mice. RESULTS: Plg-RKT was highly expressed in both human and mouse AT, and its levels dramatically increased during adipogenesis. Plg-RKT -deficient mice, when fed a HFD, gained more weight, developed more hepatic steatosis, and were more insulin resistant/glucose intolerant than HFD-fed wild-type littermates. Mechanistically, these metabolic defects were linked with increased AT inflammation, AT macrophage and T-cell accumulation, adipose and hepatic fibrosis, and decreased insulin signaling in the AT and liver. Moreover, Plg-RKT regulated the expression of PPARγ and other adipogenic molecules, suggesting a novel role for Plg-RKT in the adipogenic program. CONCLUSIONS: Plg-RKT coordinately regulates multiple aspects of adipose function that are important to maintain efficient metabolic homeostasis.

Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling.

The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin ß1. A point mutation in this motif markedly reduces TF-FVIIa association with integrins, attenuates integrin translocation into early endosomes, and reduces delayed mitogen-activated protein kinase phosphorylation required for the induction of proangiogenic cytokines. Pharmacologic or genetic blockade of the small GTPase ADP-ribosylation factor 6 (arf6) that regulates integrin trafficking increases availability of TF-FVIIa with procoagulant activity on the cell surface, while inhibiting TF-FVIIa signaling that leads to proangiogenic cytokine expression and tumor cell migration. These experiments delineate the structural basis for the crosstalk of the TF-FVIIa complex with integrin trafficking and suggest a crucial role for endosomal PAR2 signaling in pathways of tissue repair and tumor biology.

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells.

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor-positive (EPCR(+)) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α-converting enzyme (TACE), enhanced CXCL12-CXCR4-induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR(+) LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NO(low) EPCR(+) LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR(+) LT-HSCs, with potential clinical relevance for stem cell transplantation.

Hematopoietic tissue factor-protease-activated receptor 2 signaling promotes hepatic inflammation and contributes to pathways of gluconeogenesis and steatosis in obese mice.

Failure to inhibit hepatic gluconeogenesis is a major mechanism contributing to fasting hyperglycemia in type 2 diabetes and, along with steatosis, is the hallmark of hepatic insulin resistance. Obesity is associated with chronic inflammation in multiple tissues, and hepatic inflammation is mechanistically linked to both steatosis and hepatic insulin resistance. Here, we delineate a role for coagulation signaling via tissue factor (TF) and proteinase-activated receptor 2 (PAR2) in obesity-mediated hepatic inflammation, steatosis, and gluconeogenesis. In diet-induced obese mice, TF tail signaling independent of PAR2 drives CD11b(+)CD11c(+) hepatic macrophage recruitment, and TF-PAR2 signaling contributes to the accumulation of hepatic CD8(+) T cells. Transcripts of key pathways of gluconeogenesis, lipogenesis, and inflammatory cytokines were reduced in high-fat diet-fed mice that lack the cytoplasmic domain of TF (F3) (TF(ΔCT)) or that are deficient in PAR2 (F2rl1), as well as by pharmacological inhibition of TF-PAR2 signaling in diet-induced obese mice. These gluconeogenic, lipogenic, and inflammatory pathway transcripts were similarly reduced in response to genetic ablation or pharmacological inhibition of TF-PAR2 signaling in hematopoietic cells and were mechanistically associated with activation of AMP-activated protein kinase (AMPK). These findings indicate that hematopoietic TF-PAR2 signaling plays a pivotal role in the hepatic inflammatory responses, steatosis, and hepatic insulin resistance that lead to systemic insulin resistance and type 2 diabetes in obesity.

Hypomethylation of the IL17RC promoter associates with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis.

Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration.

BACKGROUND: Age related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD. METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500. RESULTS: We show that C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1ß and IL-6 expression from monocytes. We have also found that C5a could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls. CONCLUSIONS: Our results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.

Human CD14hi monocytes and myeloid dendritic cells provide a cell contact-dependent costimulatory signal for early CD40 ligand expression.

CD40L on CD4(+) T cells plays a vital role in the activation of antigen-presenting cells, thus catalyzing a positive feedback loop for T-cell activation. Despite the pivotal juxtaposition of CD40L between antigen-presenting cells and T-cell activation, only a T-cell receptor stimulus is thought to be required for early CD40L surface expression. We show, for the first time, that CD40L expression on peripheral blood CD4(+) T cells is highly dependent on a cell-cell interaction with CD14(hi)CD16(-) monocytes. Interactions with ICAM-1, LFA-3, and to a lesser extent CD80/CD86 contribute to this enhancement of CD40L expression but are not themselves sufficient. The contact-mediated increase in CD40L expression is dependent on new mRNA and protein synthesis. Circulating myeloid dendritic cells also possess this costimulatory activity. By contrast, CD14(lo)CD16(+) monocytes, plasmacytoid dendritic cells, B-cell lymphoma lines, and resting, activated, and Epstein-Barr virus-immortalized primary B cells all lack the capacity to up-regulate early CD40L. The latter indicates that a human B cell cannot activate its cognate T cell to deliver CD40L-mediated help. This finding has functional implications for the role of biphasic CD40L expression, suggesting that the early phase is associated with antigen-presenting cell activation, whereas the late phase is related to B-cell activation.

PDGF-CC blockade inhibits pathological angiogenesis by acting on multiple cellular and molecular targets.

The importance of identifying VEGF-independent pathways in pathological angiogenesis is increasingly recognized as a result of the emerging drug resistance to anti-VEGF therapies. PDGF-CC is the third member of the PDGF family discovered after more than two decades of studies on PDGF-AA and PDGF-BB. The biological function of PDGF-CC and the underlying cellular and molecular mechanisms remain largely unexplored. Here, using different animal models, we report that PDGF-CC inhibition by neutralizing antibody, shRNA, or genetic deletion suppressed both choroidal and retinal neovascularization. Importantly, we revealed that PDGF-CC targeting acted not only on multiple cell types important for pathological angiogenesis, such as vascular mural and endothelial cells, macrophages, choroidal fibroblasts and retinal pigment epithelial cells, but also on the expression of other important angiogenic genes, such as PDGF-BB and PDGF receptors. At a molecular level, we found that PDGF-CC regulated glycogen synthase kinase (GSK)-3beta phosphorylation and expression both in vitro and in vivo. Activation of GSK3beta impaired PDGF-CC-induced angiogenesis, and inhibition of GSK3beta abolished the antiangiogenic effect of PDGF-CC blockade. Thus, we identified PDGF-CC as an important candidate target gene for antiangiogenic therapy, and PDGF-CC inhibition may be of therapeutic value in treating neovascular diseases.

Dynamic changes in pancreatic endocrine cell abundance, distribution, and function in antigen-induced and spontaneous autoimmune diabetes.

OBJECTIVE: Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by beta-cell-specific killing by autoreactive T-cells. Less is known about beta-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents. RESEARCH DESIGN AND METHODS: We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell-mediated beta-cell-destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse. RESULTS: The proportion of pancreatic insulin-positive beta-cells to glucagon-positive alpha-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe beta-cell depletion accompanied by phenotypic beta-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of alpha-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non-insulin-induced hypoglycemia. CONCLUSIONS: Systematically applying multicolor flow cytometry and immunohistochemistry to track declining beta-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet alpha-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub-islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.

Cytotoxic T cell-mediated diabetes in RIP-CD80 transgenic mice: autoantigen peptide sensitivity and fine specificity.

Rodent immune-mediated diabetes model studies have advanced understanding of beta cell-specific T cell responses, and the testing of therapeutic approaches. We have used an inducible diabetes model based on rat insulin promotor (RIP)-driven expression of CD80 (B7-1) on pancreatic beta cells. Using these mice, we have established that immunizing with a single autoantigen can promote progressive islet inflammation and eventually T cell-mediated diabetes. We now describe a potent immunization protocol using peptide-pulsed mature dendritic cells (DCs) to examine peptide epitopes derived from endogenous (preproinsulin) and transgenically expressed beta cell antigens, namely lymphocytic choriomeningitis virus glycoprotein (LCMV-GP). LCMV-GP epitopes efficiently promote beta cell destruction, and the autoantigenic peptide concentration used to load the DCs correlates directly with diabetes onset. The system allowed us to assess cytotoxic T cell (CTL) fine specificity by immunizing with DCs presenting altered peptide ligands (APLs) of the dominant LCMV-GP epitope, gp33. Finally, using an adoptive transfer system, we tested alternative in vitro T cell activation conditions, including APLs and mitogens, for their impact on T cell effector function and diabetes onset. Our studies revealed a marked discrepancy between (inflammatory) effector functions and diabetes progression, thus emphasizing the importance of structural identity between sensitizing and target epitope and the context of initial T cell activation.

Detection of GAD65-reactive T-Cells in type 1 diabetes by immunoglobulin-free ELISPOT assays.

OBJECTIVE: To investigate the prevalence of beta-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELISPOT) assay and assessed its usefulness for diagnosing this disease. RESEARCH DESIGN AND METHODS: Cellular immune responses to beta -cell autoantigens were studied both by immunoglobulin-free proliferation assays and ELISPOT assays in 33 patients with type 1 diabetes and 15 patients with type 2 diabetes, compared with 23 healthy control subjects. Autoantibodies against GAD65 and IA-2 were measured by radioimmunoassay. RESULTS: Significant proliferative responses to GAD65 were observed in 10 of 31 (32.3%) type 1 diabetic patients (P < 0.05), whereas GAD65-reactive gamma-interferon (IFN-gamma)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay (P < 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-gamma positivity in ELISPOT and two of five (40.0%) showed significant proliferation against GAD65. CONCLUSIONS: Using a newly developed ELISPOT assay, GAD-reactive T-helper 1 cells in PBMC of type 1 diabetic patients could be identified at a higher frequency than by the proliferation assay. Therefore, the immunoglobulin-free ELISPOT assay is an excellent tool for detecting T-cell reactivity to autoantigens with greater specificity and, in combination with beta-cell autoantibody determination, will improve the diagnosis of type 1 diabetes.

Fas/Fas ligand interactions play an essential role in the initiation of murine autoimmune diabetes.

Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. To elucidate the role of Fas/FasL interactions in NOD diabetes, the effects of neutralizing anti-FasL antibody on autoimmune responses were evaluated. Islet-specific CD8(+) and CD4(+) T-cells expressed FasL upon activation and mediated FasL-dependent cytotoxicity against Fas-expressing target cells in vitro, although their cytotoxicity against islet cells was not blocked by anti-FasL antibody. Moreover, administration of anti-FasL antibody failed to inhibit diabetes in vivo in the CD8(+) T-cell adoptive transfer model. On the other hand, blockade of Fas/FasL interactions significantly inhibited CD4(+) T-cell-dependent diabetes in adoptive transfer models. These results suggest a substantial contribution of Fas/FasL interactions to CD4(+), but not CD8(+), T-cell-mediated destruction of pancreatic beta-cells. When anti-FasL antibody was administered to NOD mice between 5 and 15 weeks of age, the onset of diabetes was slightly delayed but the incidence was not decreased. However, administration of anti-FasL antibody at 2-4 weeks of age completely prevented insulitis and diabetes. These results suggest that Fas/FasL interactions contribute to CD4(+) T-cell-mediated beta-cell destruction and play an essential role in the initiation of autoimmune NOD diabetes.

Tolerance mechanisms in murine autoimmune diabetes induced by anti-ICAM-1/LFA-1 mAb and anti-CD8 mAb.

A short-term administration of antibodies against ICAM-1/LFA-1 or CD8 molecules during a critical period of younger age resulted in complete protection of autoimmune diabetes in NOD mice. In this study, we attempted to elucidate the tolerance mechanisms. Transfer of splenocytes from both antibody-treated NOD mice to NOD-SCID mice failed to develop diabetes. On the other hand, when splenocytes from diabetic mice were transferred to the antibody-treated mice, 40% of both mAb-treated recipients became diabetic. In vitro response of T cells from these protected mice exhibited strong proliferation against syngeneic islet cells or ConA. Furthermore, semiquantitative RT-PCR analysis of cytokines showed that T cells from anti-CD8-treated mice could express IFN-gamma, IL-4, IL-10 and TGF-beta1 in response to islet antigen. In contrast, T cells from anti-ICAM-1/LFA-1-treated mice expressed IFN-gamma, IL-10 and TGF-beta1 but not IL-4. These results suggest that tolerance mechanisms like clonal deletion, anergy, immunoregulatory T cells or Th1 to Th2/Th3 cytokine shifting are not responsible for the tolerance induction, indicating the presence of other unrevealed mechanism responsible for the loss of capability of autoreactive T cells to infiltrate and destroy the pancreatic beta-cells in vivo.