Faculty Bibliography

Objective. Receptor for advanced glycated endproducts (RAGE) plays an important role in atherogenesis in diabetes. We imaged RAGE to investigate the effect of glucose control to suppress RAGE and reduce atherosclerosis in apolipoprotein E null (apoE(-/-)) diabetic mice. Methods and Results. Thirty-three apoE(-/-) mice received streptozotocin and 6 weeks later 15 began treatment with insulin implants. Blood glucose measurements during study averaged: 140 +/- 23 mg/dL (treated) and 354 +/- 14 mg/dL (untreated). After 15 wk 30 mice were injected with (99m)Tc-anti-RAGE F(ab')2, 3 with (99m)Tc-nonimmune IgG F(ab')2, and all with CT contrast agent and underwent SPECT/CT imaging. At necropsy, the proximal aorta was weighed, counted, and sectioned and the % injected dose per gram (%ID/g) was calculated. From the merged SPECT/CT scans, tracer uptake localized to arteries was lower in the treated mice: 3.15 +/- 1.82 x 10(-3) versus 8.69 +/- 4.58 x 10(-3)%ID (P = 0.001). Percent cross-sectional lesion area was smaller in the treated (14.3 +/- 7.8% versus 29.5 +/- 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Lesion size as percent cross-sectional area was smaller in the treated (14.3 +/- 7.8% versus 29.5 +/- 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Conclusions. These results support the importance of suppressing RAGE to reduce atherosclerotic complications of diabetes and value of molecular imaging to assess treatment effect.

The receptor for glycation end products (RAGE) has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA)-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNgamma and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNgamma but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.

Abstract We investigated the pre-clinical utility of carbon monoxide form of PEGylated hemoglobin (PEG-Hb also named SANGUINATE()) in myocardial infarction (MI) and in particular the response of diabetic tissues to superimposed ischemia/reperfusion injury. SANGUINATE() was evaluated in diabetic and normal mice subjected to 30 min of coronary artery ligation followed by either 48 h or 28 days of reperfusion. Our results demonstrate that SANGUINATE() was effective in reducing infarct size when administered either prior to left anterior descending coronary artery (LAD) occlusion or during reperfusion. This finding is an important step in exploring the efficacy of a pharmacoinvasive strategy using SANGUINATE() in patients with acute coronary syndromes.

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of beta-sheet or alpha-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

BACKGROUND: Diabetic neuropathy and idiopathic neuropathy are among the most prevalent neuropathies in human patients. The molecular mechanism underlying pathological changes observed in the affected nerve remains unclear but one candidate molecule, the receptor for advanced glycation end-products (RAGE), has recently gained attention as a potential contributor to neuropathy. Our previous studies revealed that RAGE expression is higher in porcine and murine diabetic nerve, contributing to the inflammatory mechanisms leading to diabetic neuropathy. Here, for the first time, we focused on the expression of RAGE in human peripheral nerve. METHODS: Our study utilized de-identified human sural nerve surplus obtained from 5 non-neuropathic patients (control group), 6 patients with long-term mild-to-moderate diabetic neuropathy (diabetic group) and 5 patients with mild-to-moderate peripheral neuropathy of unknown etiology (idiopathic group). By using immunofluorescent staining and protein immunoblotting we studied the expression and colocalization patterns of RAGE and its ligands: carboxymethyllysine (CML), high mobility group box 1 (HMBG1) and mammalian Diaphanous 1 (mDia1) in control and neuropathic nerves. RESULTS: We found that in a normal, healthy human nerve, RAGE is expressed in almost 30% of all nerve fibers and that number is higher in pathological states such as peripheral neuropathy. We established that the levels of RAGE and its pro-inflammatory ligands, CML and HMBG1, are higher in both idiopathic and diabetic nerve, while the expression of the RAGE cytoplasmic domain-binding partner, mDia1 is similar among control, diabetic, and idiopathic nerve. The highest number of double stained nerve fibers was noted for RAGE and CML: approximately 76% (control), approximately 91% (idiopathic) and approximately 82% (diabetic) respectively. CONCLUSIONS: Our data suggest roles for RAGE and its inflammatory ligands in human peripheral neuropathies and lay the foundation for further, more detailed and clinically oriented investigation involving these proteins and their roles in disorders of the human peripheral nerve.

Oxidative stress is a central mechanism by which the receptor for advanced glycation endproducts (RAGE) mediates its pathological effects. Multiple experimental inquiries in RAGE-expressing cultured cells have demonstrated that ligand-RAGE interaction mediates generation of reactive oxygen species (ROS) and consequent downstream signal transduction and regulation of gene expression. The primary mechanism by which RAGE generates oxidative stress is via activation of NADPH oxidase; amplification mechanisms in the mitochondria may further drive ROS production. Recent studies indicating that the cytoplasmic domain of RAGE binds to the formin mDia1 provide further support for the critical roles of this pathway in oxidative stress; mDia1 was required for activation of rac1 and NADPH oxidase in primary murine aortic smooth muscle cells treated with RAGE ligand S100B. In vivo, in multiple distinct disease models in animals, RAGE action generates oxidative stress and modulates cellular/tissue fate in range of disorders, such as in myocardial ischemia, atherosclerosis, and aneurysm formation. Blockade or genetic deletion of RAGE was shown to be protective in these settings. Indeed, beyond cardiovascular disease, evidence is accruing in human subjects linking levels of RAGE ligands and soluble RAGE to oxidative stress in disorders such as doxorubicin toxicity, acetaminophen toxicity, neurodegeneration, hyperlipidemia, diabetes, preeclampsia, rheumatoid arthritis and pulmonary fibrosis. Blockade of RAGE signal transduction may be a key strategy for the prevention of the deleterious consequences of oxidative stress, particularly in chronic disease.

Increased accumulation of adipose tissue macrophages in obesity propagates chronic inflammation that is closely associated with insulin resistance and type 2 diabetes. Semaphorin 3E, a guidance molecule for neurons, takes on a new role in obesity by directing the recruitment of macrophages in visceral adipose tissue (Shimizu et al., 2013).

Diabetic peripheral nerve dysfunction is a common complication occurring in 30-50% of long-term diabetic patients. The pathogenesis of this dysfunction remains unclear but growing evidence suggests that it might be attributed, in part, to alteration in axonal transport. Our previous studies demonstrated that RAGE (Receptor for Advanced Glycation Endproducts) contributes to the pathogenesis of diabetic peripheral neuropathy and impairs nerve regeneration consequent to sciatic nerve crush, particularly in diabetes. We hypothesize that RAGE plays a role in axonal transport impairment via the interaction of its cytoplasmic domain with mammalian Diaphanous 1 (mDia1) - actin interacting molecule. Studies showed that mDia1-RAGE interaction is necessary for RAGE-ligand-dependent cellular migration, AKT phosphorylation, macrophage inflammatory response and smooth muscle migration. Here, we studied RAGE, mDia1 and markers of axonal transport rates in the peripheral nerves of wild-type C57BL/6 and RAGE null control and streptozotocin-injected diabetic mice at 1, 3 and 6 h after sciatic nerve crush. The results show that in both control and diabetic nerves, the amount of RAGE accumulated at the proximal and distal side of the crush area is similar, indicating that the recycling rate for RAGE is very high and that it is evenly transported from and towards the neuronal cell body. Furthermore, we show that slow axonal transport of proteins such as Neurofilament is affected by diabetes in a RAGE-independent manner. Finally, our study demonstrates that mDia1 axonal transport is impaired in diabetes, suggesting that diabetes-related changes affecting actin binding proteins occur early in the course of the disease.