Faculty Bibliography

BACKGROUND: Advanced glycation end-products (AGEs) have been implicated in diverse pathological settings including diabetes, inflammation and acute ischemia/reperfusion injury in the heart. AGEs interact with the receptor for AGEs (RAGE) and transduce signals through activation of MAPKs and proapoptotic pathways. In the current study, adult cardiomyocytes were studied in an in vitro ischemia/reperfusion (I/R) injury model to delineate the molecular mechanisms underlying RAGE-mediated injury due to hypoxia/reoxygenation (H/R). METHODOLOGY/PRINCIPAL FINDINGS: Cardiomyocytes isolated from adult wild-type (WT), homozygous RAGE-null (RKO), and WT mice treated with soluble RAGE (sRAGE) were subjected to hypoxia for 30 minutes alone or followed by reoxygenation for 1 hour. In specific experiments, RAGE ligand carboxymethyllysine (CML)-AGE (termed 'CML' in this manuscript) was evaluated in vitro. LDH, a marker of cellular injury, was assayed in the supernatant in the presence or absence of signaling inhibitor-treated cardiomyocytes. Cardiomyocyte levels of heterogeneous AGEs were measured using ELISA. A pronounced increase in RAGE expression along with AGEs was observed in H/R vs. normoxia in WT cardiomyocytes. WT cardiomyocytes after H/R displayed increased LDH release compared to RKO or sRAGE-treated cardiomyocytes. Our results revealed significant increases in phospho-JNK in WT cardiomyocytes after H/R. In contrast, neither RKO nor sRAGE-treated cardiomyocytes exhibited increased phosphorylation of JNK after H/R stress. The impact of RAGE deletion on GSK-3beta phosphorylation in the cardiomyocytes subjected to H/R revealed significantly higher levels of phospho-GSK-3beta/total GSK-3beta in RKO, as well as in sRAGE-treated cardiomyocytes versus WT cardiomyocytes after H/R. Further investigation established a key role for Akt, which functions upstream of GSK-3beta, in modulating H/R injury in adult cardiomyocytes. CONCLUSIONS/SIGNIFICANCE: These data illustrate key roles for RAGE-ligand interaction in the pathogenesis of cardiomyocyte injury induced by hypoxia/reoxygenation and indicate that the effects of RAGE are mediated by JNK activation and dephosphorylation of GSK-3beta. The outcome in this study lends further support to the potential use of RAGE blockade as an adjunctive therapy for protection of the ischemic heart

RATIONALE: The multiligand RAGE (receptor for advanced glycation end products) contributes to atherosclerosis in apolipoprotein (Apo)E-null mice. OBJECTIVE: To delineate the specific mechanisms by which RAGE accelerated atherosclerosis, we performed Affymetrix gene expression arrays on aortas of nondiabetic and diabetic ApoE-null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but that we would be able to identify the genes likely involved in diabetes- and RAGE-dependent atherogenesis. METHODS AND RESULTS: We report that there is very little overlap of the genes that are differentially expressed both in the onset of diabetes in ApoE-null mice, and in the effect of RAGE deletion in diabetic ApoE-null mice. Pathway-Express analysis revealed that the transforming growth factor-beta pathway and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE-null mice, and the mechanism by which deletion of RAGE ameliorates this effect. Quantitative polymerase chain reaction studies, Western blotting, and confocal microscopy in aortic tissue and in primary cultures of murine aortic smooth muscle cells supported these findings. CONCLUSIONS: Taken together, our work suggests that RAGE-dependent acceleration of atherosclerosis in ApoE-null mice is dependent, at least in part, on the action of the ROCK1 (rho-associated protein kinase 1) branch of the transforming growth factor-beta pathway

Microglia are critical for amyloid-beta peptide (Abeta)-mediated neuronal perturbation relevant to Alzheimer's disease (AD) pathogenesis. We demonstrate that overexpression of receptor for advanced glycation end products (RAGE) in imbroglio exaggerates neuroinflammation, as evidenced by increased proinflammatory mediator production, Abeta accumulation, impaired learning/memory, and neurotoxicity in an Abeta-rich environment. Transgenic (Tg) mice expressing human mutant APP (mAPP) in neurons and RAGE in microglia displayed enhanced IL-1beta and TNF-alpha production, increased infiltration of microglia and astrocytes, accumulation of Abeta, reduced acetylcholine esterase (AChE) activity, and accelerated deterioration of spatial learning/memory. Notably, introduction of a signal transduction-defective mutant RAGE (DN-RAGE) to microglia attenuates deterioration induced by Abeta. These findings indicate that RAGE signaling in microglia contributes to the pathogenesis of an inflammatory response that ultimately impairs neuronal function and directly affects amyloid accumulation. We conclude that blockade of microglial RAGE may have a beneficial effect on Abeta-mediated neuronal perturbation relevant to AD pathogenesis.-Fang, F., Lue, L.-F., Yan, S., Xu, H., Luddy, J. S., Chen, D., Walker, D. G., Stern, D. M., Yan, S., Schmidt, A. M., Chen, J. X., Yan, S. S. RAGE-dependent signaling in microglia contributes to neuroinflammation, Abeta accumulation, and impaired learning/memory in a mouse model of Alzheimer's disease

The immunoglobulin superfamily molecule RAGE (receptor for advanced glycation end product) transduces the effects of multiple ligands, including AGEs (advanced glycation end products), advanced oxidation protein products, S100/calgranulins, high-mobility group box-1, amyloid-beta peptide, and beta-sheet fibrils. In diabetes, hyperglycemia likely stimulates the initial burst of production of ligands that interact with RAGE and activate signaling mechanisms. Consequently, increased generation of proinflammatory and prothrombotic molecules and reactive oxygen species trigger further cycles of oxidative stress via RAGE, thus setting the stage for augmented damage to diabetic tissues in the face of further insults. Many of the ligand families of RAGE have been identified in atherosclerotic plaques and in the infarcted heart. Together with increased expression of RAGE in diabetic settings, we propose that release and accumulation of RAGE ligands contribute to exaggerated cellular damage. Stopping the vicious cycle of AGE-RAGE and RAGE axis signaling in the vulnerable heart and great vessels may be essential in controlling and preventing the consequences of diabetes

BACKGROUND: The cardioprotective effects of soluble receptor for advanced glycation end-products (sRAGE) have not been evaluated in large animals and the underlying mechanisms are not fully understood. This study aimed to evaluate the effects of intra-coronary administration of sRAGE on left ventricular function and myocardial remodeling in a porcine model of ischemia-reperfusion (I/R) injury. METHODS: Ten male minipigs with I/R injury were randomly allocated to receive intra-coronary administration of sRAGE (sRAGE group, n = 5) or saline (control group, n = 5). Echocardiography was performed before and 2 months after infarction. Myocardial expression of transforming growth factor (TGF)-beta1 was determined by immunohistochemistry and fibrosis was evaluated by Sirius red staining. RESULTS: As compared with the baseline values in the control animals, left ventricular end-diastolic volume (from (19.5 +/- 5.1) to (32.3 +/- 5.6) ml, P < 0.05) and end-systolic volume (from (8.3 +/- 3.2) to (15.2 +/- 4.1) ml, P< 0.05) were significantly increased, whereas ejection fraction was decreased (from (61.6 +/- 13.3)% to (50.2 +/- 11.9)%, P < 0.05). No obvious change in these parameters was observed in the sRAGE group. Myocardial expression of TGF-beta1 was significantly elevated in the infarct and non-infarct regions in the control group, as compared with sRAGE group (both P< 0.01). Fibrotic lesions were consistently more prominent in the infarct region of the myocardium in the control animals (P < 0.05). CONCLUSION: Intra-coronary sRAGE administration attenuates RAGE-mediated myocardial fibrosis and I/R injury through a TGF-beta1-dependent mechanism, suggesting a clinical potential in treating RAGE/ligand-associated cardiovascular diseases.

PURPOSE: To assess retinal capillary basement membrane thickening (BMT) in a swine model of type 1 diabetes. MATERIALS AND METHODS: Yorkshire pigs were rendered diabetic with streptozotocin and dyslipidemic with a high fat and cholesterol diet. At 18, 26, and 32 weeks of diabetes, the retina sections within 3 disc diameters from the optic disc were examined under transmission electron microscopy to evaluate the ultrastructural features of the capillary BM. Digital morphometric analysis was performed to measure BMT. RESULTS: Diabetic swine had significantly thicker retinal capillary BMs compared to controls. Pigs that sustained diabetes for longer periods or experienced severe diabetes tended to have more BMT. Those pigs that did not sustain glucose levels above 200 mg/dL did not demonstrate thicker retinal capillary BMs. Characteristic ultrastructural features of diabetic vasculopathy observed included rarefaction as an early stage of Swiss cheese cavitation, lamellation with multiplication of electron dense layers, and fibrillar materials within capillary BM. CONCLUSIONS: Diabetic Yorkshire pigs develop characteristic features of an early retinal microvasculopathy fairly rapidly and may serve as a higher-order animal model for studies of type 1 diabetes.

The multi-ligand Receptor for Advanced Glycation Endproducts (RAGE) is expressed in podocytes and endothelial cells in the human and murine glomerulus. Although present at low levels in homeostasis, RAGE expression is increased during disease. Pharmacological antagonism of RAGE or its genetic deletion imparts marked protection from podocyte effacement, albuminuria and glomerular sclerosis in disease models. In human subjects, associations between specific genetic polymorphisms of RAGE and levels of soluble forms of RAGE are linked to disease states in the kidney. In this review, we summarize the evidence from mouse to man, linking RAGE to the pathogenesis of nephropathy

Receptor for advanced glycation end products (RAGE) binds advanced glycation end products and other inflammatory ligands and is expressed in atherosclerotic plaques in diabetic and nondiabetic subjects. The higher expression in diabetes mellitus corresponds to the accelerated course of the atherosclerosis. This study was designed to test the hypothesis that the level of RAGE expression in atherosclerosis can be detected by quantitative in vivo SPECT and that counts in the target will correlate with the strength of the biologic signal. METHODS: A monoclonal murine antibody was developed against the V-domain of RAGE, fragmented into F(ab')(2) and labeled with (99m)Tc, and injected at a dose of 15.14 +/- 1.23 MBq into 24-wk-old male apolipoprotein E null (ApoE(-/-)) mice (n = 22), including mice with streptozotocin-induced diabetes mellitus (n = 8), nondiabetic mice (n = 8), and control ApoE(-/-)/RAGE(-/-) double-knock-out mice (n = 6). Four hours later (allowing for blood-pool clearance), the mice were imaged and sacrificed, and the proximal aorta was removed and counted to calculate the percentage injected dose of RAGE per gram of tissue, followed by histologic and immunohistochemical characterization. RESULTS: Radiotracer uptake in the aortic lesions was clearly visualized noninvasively by SPECT. RAGE uptake as percentage injected dose in diabetic ApoE(-/-) mice (1.39 +/- 0.16 x 10(-2)) was significantly higher than that in nondiabetic ApoE(-/-) mice (0.48 +/- 0.27 x 10(-2)) (P < 0.0001). The radiotracer uptake was highly correlated with RAGE expression by quantitative immunohistomorphometry (r = 0.82, P = 0.002) and with percentage of macrophages (r = 0.86, P < 0.0001). CONCLUSION: In this study, (99m)Tc-labeled anti-RAGE F(ab')(2) SPECT successfully identified early accelerated disease in diabetes mellitus for age-matched ApoE(-/-) mice and quantified RAGE expression over a range of lesion severities.

Exaggerated inflammatory responses and the resultant increases in alveolar-capillary permeability underlie the pathogenesis of acute lung injury during sepsis. This study examined the functions of aldose reductase (AR) in mediating acute lung inflammation. Transgenic mice expressing human AR (ARTg) were used to study the functions of AR since mice have low intrinsic AR activity. In a mild cecal ligation and puncture model, ARTg mice demonstrated an enhanced AR activity and a greater inflammatory response as evaluated by circulating cytokine levels, neutrophil accumulation in the lungs, and activation of Rho kinase in lung endothelial cells (ECs). Compared with WT lung cells, ARTg lung cells produced more IL-6 and showed augmented JNK activation in response to LPS stimulation ex vivo. In human neutrophils, AR activity was required for fMLP-included CD11b activation and up-regulation, respiratory burst, and shape changes. In human pulmonary microvascular ECs, AR activity was required for TNF-alpha-induced activation of the Rho kinase/MKK4/JNK pathway and IL-6 production, but not p38 activation or ICAM-1 expression. Importantly, AR activity in both human neutrophils and ECs was required for neutrophil adhesion to TNF-alpha-stimulated ECs. These data demonstrate a novel role for AR in regulating the signaling pathways leading to neutrophil-EC adhesion during acute lung inflammation