Faculty Bibliography

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligand-binding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C betaII, ERK1/2, and c-Jun NH(2)-terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is a cell surface receptor implicated in tumor cell proliferation and migration. We hypothesized that RAGE signaling impacts tumorigenesis and metastatic tumor growth in murine models of colorectal carcinoma. MATERIALS AND METHODS: Tumorigenesis: Apc (1638N/+) mice were crossed with Rage (-/-) mice in the C57BL/6 background to generate Apc (1638N/+)/Rage (-/-) mice. Metastasis: BALB/c mice underwent portal vein injection with CT26 cells (syngeneic) and received daily soluble (s)RAGE or vehicle. Rage (-/-) mice and Rage (+/+) controls underwent portal vein injection with MC38 cells (syngeneic). Rage (+/+) mice underwent portal vein injection with MC38 cells after stable transfection with full-length RAGE or mock transfection control. RESULTS: Tumorigenesis: Apc (1638N/+)/Rage (-/-) mice had reduced tumor incidence, size, and histopathologic grade. Metastasis: Pharmacological blockade of RAGE with sRAGE or genetic deletion of Rage reduced hepatic tumor incidence, nodules, and burden. Gain of function by transfection with full-length RAGE increased hepatic tumor burden compared to vector control MC38 cells. CONCLUSION: RAGE signaling plays an important role in tumorigenesis and hepatic tumor growth in murine models of colorectal carcinoma. Further work is needed to target the ligand-RAGE axis for possible prophylaxis and treatment of primary and metastatic colorectal carcinoma

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

Cardiac tissue engineering will remain only a prospect unless large numbers of therapeutic cells can be provided, either from small samples of cardiac cells or from stem cell sources. In contrast to most adult cells, cardiomyocytes are terminally differentiated and cannot be expanded in culture. We explored the feasibility of enabling the in vitro expansion of primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then reverting the phenotype of the expanded cells back to the cardiomyocyte state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (alpha-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state. This strategy for controlled expansion of primary cardiomyocytes by gene transfer has potential for providing large amounts of a patient's own cardiomyocytes for cell therapy, and the cardiomyocytes derived by this method could be a useful cellular model by which to study cardiogenesis.

RATIONALE: S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products). OBJECTIVE: We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension. METHODS AND RESULTS: Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho-extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells. CONCLUSIONS: Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

OBJECTIVE: Rupture of abdominal aortic aneurysms (AAA) is a devastating event potentially preventable by therapies that inhibit growth of small aneurysms. Receptor of advanced glycation end products (RAGE) has been implicated in age related diseases including atherosclerosis and Alzheimer. Consequently, we explored whether RAGE may also contribute to the formation of AAAs. RESULTS: Implicating a role for RAGE in AAA, we found the expression of RAGE and its ligand AGE were highly elevated in human aneurysm specimens as compared with normal aortic tissue. In a mouse model of AAA, RAGE gene deletion (knockout) dramatically reduced the incidence of AAA to 1/3 of control (AAAs in 75.0% of controls vs. 25.0% knockouts). Moreover, aortic diameter was markedly reduced in RAGE knockout animals versus controls. As to mechanism, we found that RAGE was coexpressed in AAA macrophages with MMP-9, a promoter of matrix degradation, which is known to induce AAA. In vitro, AGE induced the production of MMP-9 in macrophages in a dose-dependent manner while blocking RAGE signaling with a soluble AGE inhibitor prevented MMP-9 expression. In vivo, RAGE gene deficiency eliminated MMP-9 activity that was prevalent in aneurismal wall of the wild-type mice. CONCLUSIONS: RAGE promotes the development of AAA by inducing MMP-9 expression. Blocking RAGE in a mouse aneurysm model has a dramatic inhibitory effect on the formation of aneurysms. These data suggest that larger animal and eventually human trials should be designed to test oral RAGE inhibitors and their potential to prevent progression of small aneurysms.

BACKGROUND: The receptor for advanced glycation end products (RAGE) mediates a variety of inflammatory responses. METHODS: To determine the role of RAGE in the innate immune response to abdominal sepsis caused by Escherichia coli, RAGE-deficient (RAGE(-/-)) and normal wild-type mice were intraperitoneally injected with E. coli. In a separate experiment, wild-type mice received either anti-RAGE immunoglobulin (Ig) G or control IgG. RESULTS: E. coli sepsis resulted in an up-regulation of RAGE in the liver but not in the lungs. RAGE-deficient mice demonstrated an enhanced bacterial outgrowth in their peritoneal cavity and increased dissemination of the infection, accompanied by increased hepatocellular injury and exaggerated systemic cytokine release and coagulation activation, 20 h after intraperitoneal administration of E. coli. Wild-type mice treated with anti-RAGE IgG also displayed a diminished defense against the growth and/or dissemination of E. coli. RAGE was important for the initiation of the host response, as reflected by a reduced immune and procoagulant response early after intraperitoneal injection of E. coli lipopolysaccharide. CONCLUSION: These data are the first to suggest that intact RAGE signaling contributes to an effective antibacterial defense during E. coli sepsis, thereby limiting the accompanying inflammatory and procoagulant response.

This review discusses current knowledge of the complex interactions between amyloid-beta (A beta) peptide, the receptor for advanced glycation endproducts (RAGE), and inflammatory mediators, focusing on the roles of such interactions in the pathogenesis of Alzheimer's disease. As a ubiquitous cell-surface receptor, RAGE demonstrates enhanced expression in an A beta-rich environment; the effects of RAGE on microglia, the blood-brain barrier and neurons are mediated through various signaling pathways. Relevant preclinical models illustrate that the A beta-RAGE interaction amplifies neuronal stress and the accumulation of A beta, impairs memory and learning, and exaggerates neuroinflammation. These findings suggest that RAGE may mediate a common proinflammatory pathway in neurodegenerative disorders.

The alternative splicing of pre-mRNAs is a critical mechanism in genomic complexity, disease, and development. Studies of the receptor for advanced glycation end-products (RAGE) indicate that this gene undergoes a variety of splice events in humans. However, no studies have extensively analyzed the tissue distribution in other species or compared evolutionary differences of RAGE isoforms. Because the majority of studies probing RAGE function have been performed in murine models, we therefore performed studies to identify and characterize the splice variants of the murine RAGE gene, and we compared these to human isoforms. Here, using mouse tissues, we identified numerous splice variants including changes in the extracellular domain or the removal of the transmembrane and cytoplasmic domains, which produce soluble splice isoforms. Comparison of splice variants between humans and mice revealed homologous regions in the RAGE gene that undergo splicing as well as key species-specific mechanisms of splicing. Further analysis of tissue splice variant distribution in mice revealed major differences between lung, kidney, heart, and brain. To probe the potential impact of disease-like pathological states, we studied diabetic mice and report that RAGE splice variation changed dramatically, resulting in an increase in production of soluble RAGE (sRAGE) splice variants, which were not associated with detectable levels of sRAGE in murine plasma. In conclusion, we have determined that the murine RAGE gene undergoes extensive splicing with distinct splice isoforms being uniquely distributed in different tissues. These differences in RAGE splicing in both physiological and pathogenic states further expand our understanding of the biological repertoire of this receptor in health and disease.