Faculty Bibliography

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.

Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules and engages diverse ligands relevant to distinct pathological processes. One class of RAGE ligands includes glycoxidation products, termed advanced glycation end products, which occur in diabetes, at sites of oxidant stress in tissues, and in renal failure and amyloidoses. RAGE also functions as a signal transduction receptor for amyloid beta peptide, known to accumulate in Alzheimer disease in both affected brain parenchyma and cerebral vasculature. Interaction of RAGE with these ligands enhances receptor expression and initiates a positive feedback loop whereby receptor occupancy triggers increased RAGE expression, thereby perpetuating another wave of cellular activation. Sustained expression of RAGE by critical target cells, including endothelium, smooth muscle cells, mononuclear phagocytes, and neurons, in proximity to these ligands, sets the stage for chronic cellular activation and tissue damage. In a model of accelerated atherosclerosis associated with diabetes in genetically manipulated mice, blockade of cell surface RAGE by infusion of a soluble, truncated form of the receptor completely suppressed enhanced formation of vascular lesions. Amelioration of atherosclerosis in these diabetic/atherosclerotic animals by soluble RAGE occurred in the absence of changes in plasma lipids or glycemia, emphasizing the contribution of a lipid- and glycemia-independent mechanism(s) to atherogenesis, which we postulate to be interaction of RAGE with its ligands. Future studies using mice in which RAGE expression has been genetically manipulated and with selective low molecular weight RAGE inhibitors will be required to definitively assign a critical role for RAGE activation in diabetic vasculopathy. However, sustained receptor expression in a microenvironment with a plethora of ligand makes possible prolonged receptor stimulation, suggesting that interaction of cellular RAGE with its ligands could be a factor contributing to a range of important chronic disorders

Accelerated atherosclerosis in patients with diabetes is a major cause of their morbidity and mortality, and it is unresponsive to therapy aimed at restoring relative euglycemia. In hyperglycemia, nonenzymatic glycation and oxidation of proteins and lipids results in the accumulation of irreversibly formed advanced glycation endproducts. These advanced glycation endproducts engage their receptor in cells of the blood vessel wall, thereby activating mechanisms linked to the development of vascular lesions. We report here a model of accelerated and advanced atherosclerosis in diabetic mice deficient for apolipoprotein E. Treatment of these mice with the soluble extracellular domain of the receptor for advanced glycation endproducts completely suppressed diabetic atherosclerosis in a glycemia- and lipid-independent manner. These findings indicate interaction between the advanced glycation endproducts and their receptor is involved in the development of accelerated atherosclerosis in diabetes, and identify this receptor as a new therapeutic target in diabetic macrovascular disease

A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Abeta) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Abeta, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappaB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Abeta deposits, and in cerebrospinal fluid from AD patients there was approximately 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Abeta-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Abeta, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Abeta on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death

Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail