Faculty Bibliography

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall

Myocardial infarction, stroke, and venous thromboembolism are characterized by oxygen deprivation. In hypoxia, biological responses are activated that evoke tissue damage. Rapid activation of early growth response-1 in hypoxia upregulates fundamental inflammatory and prothrombotic stress genes. We probed the mechanisms mediating regulation of early growth response-1 and demonstrate that hypoxia stimulates brisk generation of advanced glycation end products (AGEs) by endothelial cells. Via AGE interaction with their chief signaling receptor, RAGE, membrane translocation of protein kinase C-betaII occurs, provoking phosphorylation of c-Jun NH(2)-terminal kinase and increased transcription of early growth response-1 and its downstream target genes. These findings identify RAGE as a master regulator of tissue stress elicited by hypoxia and highlight this receptor as a central therapeutic target to suppress the tissue injury-provoking effects of oxygen deprivation

Soluble amyloid-beta (Abeta) peptide is likely to play a key role during early stages of Alzheimer's disease (AD) by perturbing synaptic function and cognitive processes. Receptor for advanced glycation end products (RAGE) has been identified as a receptor involved in Abeta-induced neuronal dysfunction. We investigated the role of neuronal RAGE in Abeta-induced synaptic dysfunction in the entorhinal cortex, an area of the brain important in memory processes that is affected early in AD. We found that soluble oligomeric Abeta peptide (Abeta42) blocked long-term potentiation (LTP), but did not affect long-term depression, paired-pulse facilitation, or basal synaptic transmission. In contrast, Abeta did not inhibit LTP in slices from RAGE-null mutant mice or in slices from wild-type mice treated with anti-RAGE IgG. Similarly, transgenic mice expressing a dominant-negative form of RAGE targeted to neurons showed normal LTP in the presence of Abeta, suggesting that neuronal RAGE functions as a signal transducer for Abeta-mediated LTP impairment. To investigate intracellular pathway transducing RAGE activation by Abeta, we used inhibitors of stress activated kinases. We found that inhibiting p38 mitogen-activated protein kinase (p38 MAPK), but not blocking c-Jun N-terminal kinase activation, was capable of maintaining LTP in Abeta-treated slices. Moreover, Abeta-mediated enhancement of p38 MAPK phosphorylation in cortical neurons was reduced by blocking antibodies to RAGE. Together, our results indicate that Abeta impairs LTP in the entorhinal cortex through neuronal RAGE-mediated activation of p38 MAPK

RAGE, the receptor for advanced glycation endproducts (AGEs), is a multiligand signal transduction receptor of the immunoglobulin superfamily of cell surface molecules that has been implicated in the pathogenesis of diabetic complications, neurodegenerative diseases, inflammatory disorders, and cancer. These diverse biologic disorders reflect the multiplicity of ligands capable of cellular interaction via RAGE that include, in addition to AGEs, amyloid-beta (Abeta) peptide, the S100/calgranulin family of proinflammatory cytokines, and amphoterin, a member of the High Mobility Group Box (HMGB) DNA-binding proteins. In the retina, RAGE expression is present in neural cells, the vasculature, and RPE cells, and it has also been detected in pathologic cellular retinal responses including epiretinal and neovascular membrane formation. Ligands for RAGE, in particular AGEs, have emerged as relevant to the pathogenesis of diabetic retinopathy and age-related macular disease. While the understanding of RAGE and its role in retinal dysfunction with aging, diabetes mellitus, and/or activation of pro-inflammatory pathways is less complete compared to other organ systems, increasing evidence indicates that RAGE can initiate and sustain significant cellular perturbations in the inner and outer retina. For these reasons, antagonism of RAGE interactions with its ligands may be a worthwhile therapeutic target in such seemingly disparate, visually threatening retinal diseases as diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinopathy.

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.

This review focuses on the current findings regarding interaction between amyloid beta peptide (Abeta) and receptor for advanced glycation endproducts (RAGE) and its roles in the pathogenesis of Alzheimer's disease (AD). As a ubiquitously expressed cell surface receptor, RAGE mediates the effects of Abeta on microglia, blood-brain barrier (BBB) and neurons through activating different signaling pathways. Data from autopsy brain tissues, in vitro cell cultures and transgenic mouse models suggest that Abeta-RAGE interaction exaggerates neuronal stress, accumulation of Abeta, impaired learning memory, and neuroinflammation. Blockade of RAGE protects against Abeta-mediated cellular perturbation. These findings may have an important therapeutic implication for neurodegenerative disorders relevant to AD.

BACKGROUND: Limited access to endothelial tissue is a major constraint when investigating the cellular mechanisms of vascular inflammation in patients with cardiovascular and metabolic diseases. We introduce venous endothelial sampling coupled to quantitative analysis of gene transcripts by real-time PCR, as a novel approach to study endothelial gene expression in human subjects. METHODS: Endothelial cells were collected from a superficial forearm vein using five guide wires sequentially inserted through a 20-gauge angiocatheter in seven patients with history of cardiovascular events related to advanced vascular disease and in 17 healthy subjects. Endothelial cells were purified using magnetic beads coated with endothelial specific antibodies. Endothelial mRNA was amplified using RiboAmp HS RNA Amplification kit (Molecular Devices, Sunnyvale, CA). Amplified RNA was analyzed by real-time PCR. RESULTS: Linearity of RNA amplification was validated by real-time PCR using RNA from 1,000 human umbilical endothelial cells (HUVECs) before and after amplification. In human subjects, vascular disease was associated with significant induction of proatherosclerotic genes: early growth response gene product (Egr-1) and monocyte chemoattractant protein-1 (MCP-1). CONCLUSION: Venous endothelial sampling coupled to real-time PCR analysis is a minimally invasive, safe, and reliable technique to monitor vascular inflammation in human subjects. Expression of genes implicated in the atherosclerotic process is increased in the venous endothelium of patients with arterial vascular disease. Venous endothelial sampling and quantitative analysis of gene expression may help develop new vascular-targeted biomarkers to identify and track the impact of disease states and therapeutic interventions in vascular diseases.

PURPOSE: Colon cancer is the third most prevalent cancer in the United States. However, the molecular mechanisms involved in the development and progression of colon cancer are incompletely understood. This study was initiated to explore the potential role of the receptor for advanced glycation end-products and S100P in modulation of key properties of human colon cancer cells. METHODS: Western blot, reverse transcription-polymerase chain reaction, and quantitative polymerase chain reaction were performed for detection of the receptor for advanced glycation end-products and S100P in colon cancer and matched normal colon. The influence of exogenously added S100P was analyzed on SW480 colon cancer cell line proliferation, migration, phosphorylation of mitogen activated protein kinases, and NFkappaB activation. To identify the mechanisms involved in these responses, coimmunoprecipitation examining the S100P/Receptor for advanced glycation end-products interaction and the effects of receptor for advanced glycation end-products inhibition in this interaction were analyzed. RESULTS: Although the receptor for advanced glycation end-products was present in normal and malignant colon specimens, only the malignant specimens expressed S100P. Treatment of SW480 cells with S100P increased proliferation and cell migration. Addition of exogenous S100P stimulated both ERK1/2 phosphorylation and NFkappaB activity. The interaction between S100P and the receptor for advanced glycation end-products was demonstrated by coimmunoprecipitation of these molecules from SW480 cells. Antagonism of the receptor for advanced glycation end-products blocked this interaction and the biologic effects of S100P on these cells. CONCLUSIONS: These data indicate that S100P is expressed at greater levels in colon cancer than matched normal tissue and that S100P stimulates colon cancer cell growth, migration, Erk phosphorylation, and NFkappaB activation in vitro, suggesting that this ligand/receptor pair may be targeted for the development of new therapies.

Porphyromonas gingivalis is a periodontal pathogen that promotes a proatherogenic response in endothelial cells. Cell death responses of human aortic endothelial cells to P. gingivalis at various multiplicities of infection (MOI) were investigated by assessment of cell detachment, histone-associated DNA fragmentation, lactate dehydrogenase release and ADP:ATP ratio. Porphyromonas gingivalis at MOI 1:10-1:100 did not have a cytotoxic effect, but induced apoptotic cell death at MOI 1:500 and 1:1000. Monocyte chemoattractant protein-1 production was significantly enhanced by P. gingivalis at MOI 1:100. At higher MOI, at least in vitro, P. gingivalis mediates endothelial apoptosis, thereby potentially amplifying proatherogenic mechanisms in the perturbed vasculature.