Search

Oxidative and carbonyl stress leads to generation of N(epsilon)-carboxymethyllysine-modified proteins (CML-mps), which are known to bind the receptor for advanced glycation end products (RAGE) and induce nuclear factor (NF)-kappaB-dependent proinflammatory gene expression. To determine the impact of CML-mps in vivo, RAGE-dependent sustained NF-kappaB activation was studied in resection gut specimens from patients with inflammatory bowel disease. Inflamed gut biopsy tissue demonstrated a significant up-regulation of RAGE and increased NF-kappaB activation. Protein extracts from the inflamed zones, but not from noninflamed resection borders, caused perpetuated NF-kappaB activation in cultured endothelial cells, which was mediated by CML-mps including CML-modified S100 proteins. The resulting NF-kappaB activation, lasting 5 days, was primarily inhibited by either depletion of CML-mps or by the addition of sRAGE, p44/42 and p38 MAPKinase-specific inhibitors. Consistently, CML-mps isolated from inflamed gut areas and rectally applied into mice caused NF-kappaB activation, increased proinflammatory gene expression, and histologically detectable inflammation in wild-type mice, but not in RAGE-/- mice. A comparable up-regulation of NF-kappaB and inflammation on rectal application of CML-mps was observed in IL-10-/- mice. Thus, CML-mps generated in inflammatory lesions have the capacity to elicit a RAGE-dependent intestinal inflammatory response.

Most autoantigens implicated in multiple sclerosis (MS) are expressed not only in the central nervous system (CNS) but also in the thymus and the periphery. Nevertheless, these autoantigens might induce a strong autoimmune response leading to severe destruction within the CNS. To investigate the influence of a dominantly presented autoantigen on experimental autoimmune encephalomyelitis (EAE), we generated transgenic mice expressing the autoantigenic peptide MBP 1-10 covalently bound to the MHC class II molecule I-Au. These mice were crossed either with B10.PL or with TCR-transgenic Tg4 mice, specific for the transgenic peptide-MHC combination. In double transgenic mice we found strong thymic deletion and residual peripheral T cells were refractory to antigen stimulation in vitro. Residual peripheral CD4+ T cells expressed activation markers and a high proportion was CD25 positive. Transfer of both CD25-negative and CD25-positive CD4+ T cells from double transgenic animals into B10.PL mice strongly inhibited the progression of EAE. Despite this thorough tolerance induction, some double transgenic mice developed severe signs of EAE after an extended period of time. Our data show that in the circumstances where autoantigenic priming persists, and where the number of antigen-specific T cells is high enough, autoimmunity may prevail over very potent tolerance-inducing mechanisms.

Molecular events that result in loss of pain perception are poorly understood in diabetic neuropathy. Our results show that the receptor for advanced glycation end products (RAGE), a receptor associated with sustained NF-kappaB activation in the diabetic microenvironment, has a central role in sensory neuronal dysfunction. In sural nerve biopsies, ligands of RAGE, the receptor itself, activated NF-kappaBp65, and IL-6 colocalized in the microvasculature of patients with diabetic neuropathy. Activation of NF-kappaB and NF-kappaB-dependent gene expression was upregulated in peripheral nerves of diabetic mice, induced by advanced glycation end products, and prevented by RAGE blockade. NF-kappaB activation was blunted in RAGE-null (RAGE(-/-)) mice compared with robust enhancement in strain-matched controls, even 6 months after diabetes induction. Loss of pain perception, indicative of long-standing diabetic neuropathy, was reversed in WT mice treated with soluble RAGE. Most importantly, loss of pain perception was largely prevented in RAGE(-/-) mice, although they were not protected from diabetes-induced loss of PGP9.5-positive plantar nerve fibers. These data demonstrate, for the first time to our knowledge, that the RAGE-NF-kappaB axis operates in diabetic neuropathy, by mediating functional sensory deficits, and that its inhibition may provide new therapeutic approaches.

While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation.

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.

Diabetic nephropathy ensues from events involving earliest changes in the glomeruli and podocytes, followed by accumulation of extracellular matrix in the mesangium. Postulated mechanisms include roles for vascular endothelial growth factor (VEGF), produced by podocytes and contributing to enhanced excretion of urinary albumin and recruitment/activation of inflammatory cells, and transforming growth factor-beta (TGF-beta), elicited largely from mesangial cells and driving production of extracellular matrix. RAGE, a receptor for advanced glycation endproducts (AGEs) and S100/calgranulins, displays enhanced expression in podocytes of genetically diabetic db/db mice by age 13 weeks. RAGE-bearing podocytes express high levels of VEGF by this time, in parallel with enhanced recruitment of mononuclear phagocytes to the glomeruli; events prevented by blockade of RAGE. By age 27 weeks, soluble RAGE-treated db/db mice displayed diminished albuminuria and glomerulosclerosis, and improved renal function. Diabetic homozygous RAGE null mice failed to develop significantly increased mesangial matrix expansion or thickening of the glomerular basement membrane. We propose that activation of RAGE contributes to expression of VEGF and enhanced attraction/activation of inflammatory cells in the diabetic glomerulus, thereby setting the stage for mesangial activation and TGF-beta production; processes which converge to cause albuminuria and glomerulosclerosis.

Cellular proliferation, migration, and expression of extracellular matrix proteins and MMPs contribute to neointimal formation upon vascular injury. Wild-type mice undergoing arterial endothelial denudation displayed striking upregulation of receptor for advanced glycation end products (RAGE) in the injured vessel, particularly in activated smooth muscle cells of the expanding neointima. In parallel, two of RAGE's signal transducing ligands, advanced glycation end products (AGEs) and S100/calgranulins, demonstrated increased deposition/expression in the injured vessel wall. Blockade of RAGE, employing soluble truncated receptor or antibodies, or in homozygous RAGE null mice, resulted in significantly decreased neointimal expansion after arterial injury and decreased smooth muscle cell proliferation, migration, and expression of extracellular matrix proteins. A critical role for smooth muscle cell RAGE signaling was demonstrated in mice bearing a transgene encoding a RAGE cytosolic tail-deletion mutant, specifically in smooth muscle cells, driven by the SM22alpha promoter. Upon arterial injury, neointimal expansion was strikingly suppressed compared with that observed in wild-type littermates. Taken together, these data highlight key roles for RAGE in modulating smooth muscle cell properties after injury and suggest that RAGE is a logical target for suppression of untoward neointimal expansion consequent to arterial injury.

The use of the Cre/loxP system has greatly empowered the field of gene targeting. Here we describe the successful establishment of a novel knock-in EGFP reporter mouse line to monitor Cre-induced recombination in the vast majority of cell types. The value of this reporter mouse line is demonstrated by the use of a novel Tie2Cre transgenic mouse line that facilitates gene targeting in endothelial and hematopoietic cells. High efficiency of recombination was found in all endothelial cells and in the majority of hematopoietic cells but was absent in other tissues. Furthermore, in the second generation, the Tie2Cre mouse can be used to get 100% recombination of one allele, whilst allowing tissue specific in the second, therefore offering excellent efficiency., (Copyright 2001 Wiley-Liss, Inc.)

Endothelial cells coordinate the recruitment of inflammatory cells to sites of vascular injury. Endothelial cells produce and release cytokines and growth factors serving as communication signals to leukocytes as well as with organs and tissues. In addition, endothelial cells respond to inflammatory stimuli such as lipopolysaccharides (endotoxin, LPS), cytokines, or ligation of CD40. Functional changes in response to inflammatory stimuli are mediated by induction of signaling cascades leading to activation of transcription factors and alterations in endothelial gene expression. This article summarizes the current knowledge of molecular events resulting in cytokine-mediated endothelial dysfunction.

A spontaneously metastasizing, well-defined mouse lymphoma was chosen as an in vivo model to study the effect of tumor-host interaction on gene expression in liver sinusoidal endothelial cells. Forty-nine bovine aortic endothelial cell (BAEC) genes, recently isolated by a differential screening approach of a cDNA library enriched for tumor necrosis factor-alpha (TNF-alpha) suppressed genes, were investigated. Four of these genes were finally selected because they were affected differentially by host immuno-competence, TNF-alpha, and tumor cells. Sequence analysis showed them to encode the bovine polyubiquitin (A4), elongation factor 1alpha (B2), the acidic ribosomal phosphoprotein PO (C3), and the ribosomal protein S2 (E10). Gene expression was analyzed by dot-blot or Northern blot analysis. TNF-alpha and tumor cell conditioned supernatant suppressed the genes additive in BAEC but not in other endothelial cells except for bovine capillary endothelial cells. Ex vivo-isolated liver endothelial cells of tumor-bearing syngeneic DBA/2 mice showed strong downregulation of these four genes in comparison to normal control values. In contrast, endothelial cells of tumor-bearing immuno-incompetent Balb/c (nu/nu) mice showed no downregulation but upregulation of these genes. Consistently, all four genes were also downregulated when BAEC were incubated with supernatants derived from ex vivo-isolated liver metastases from immuno-competent but not from -incompetent mice. Thus, the expression of a group of genes involved in protein translation and processing was more profoundly altered in endothelial cells in vivo than in vitro, suggesting that microenviromental factors and cell-cell and cell-matrix interactions play an important role., (Copyright 1998 by The American Society of Hematology)

Background: The deposition of protein Z was investigated in atherosclerotic vascular lesions of patients with diabetes mellitus or atherosclerotic vascular disease without diabetes in comparison to controls., Patients and Methods: Protein Z antigen was evidenced by immunohistochemistry in arteries of 5 healthy control patients, 11 diabetic patients with arterio-occlusive disease and 7 patients suffering from arterio-occlusive disease without diabetes. For immunohistochemistry, a commercially available antibody was taken as first antibody, and immunopositivity was evaluated independently by two investigators (J.G.; I.K.) as negative (0), positive (+) and strongly positive (++). The results were assessed by the Whitney-Mann-Wilcoxon test., Results: Macrovascular endothelial cells were stained positive for protein Z in all arteries studied. Arteries of controls did not show significant immunopositivity in cells other than macrovascular endothelial cells, while the microvascular endothelial cells of control arteries were largely negative. The proliferating subendothelial space in atherosclerotic vascular lesions showed significant immunopositivity for protein Z. In contrast to control arteries, the microvascular endothelial cells of the proliferating areas stained positive. The staining pattern of the subendothelial space was similar in atherosclerotic vessels independent of the risk factor for atherosclerosis. Plaques were immunopositive for protein Z, too., Conclusions: Protein Z is present in atherosclerotic vascular lesions of diabetic and non-diabetic patients, but not in the subendothelial space and microvascular endothelial cells of healthy controls. Since protein Z-positivity was detected in microvascular endothelium as well as in extra-vascular deposits around plaques, it may play a role in the development of these lesions.

Objective: Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic-cytokine binding to and thereby stimulating vascular cells. TNF-alpha mediated intermediate stimulation of vascular cells is believed to play a pivotal role in the development of arteriosclerosis. While extensive information has recently become available on gene induction by TNF-alpha, less is known about gene suppression by TNF-alpha in vascular cells. Endothelial cells are the first cell layer within the vessel wall interacting with circulating, cytokine releasing cells. Therefore, they were selected as target for these study., Methods: A differential screening approach has been used to isolate cDNAs whose abundance was suppressed by incubating bovine aortic endothelial cells (BAEC) for 6 h with 1 nM TNF-alpha. The gene expression of 6 isolated cDNAs after TNF-alpha was investigated by dot blots and nuclear run-on analysis in BAEC. The investigated genes were partially or completely sequenced. Differential expression after TNF-alpha stimulation of BAEC, bovine fibroblasts and vascular smooth muscle cells (SMC) was studied by Northern blots. RNA transcripts of the clone C7 in aortic aneurysms were examined by in situ hybridization., Results: 49 independent cDNAs were isolated by the differential screening approach and 6 clones were further analyzed. These genes were downregulated in a time and dose dependent manner in BAEC. Sequence analysis revealed that 3 cDNAs encoded previously unidentified genes (C1, C5, C7), while 3 encoded known genes: connective tissue growth factor (CTGF; A1), fibronectin (A8) and the mitochondrial genome (B1). A1 and B1 were suppressed in BAEC, fibroblasts and SMC, whereas A8, C1, C5 and C7 were not uniformly downregulated in the investigated cells. C7 RNA transcripts were exclusively induced in the endothelium of an uninflamed aortic aneurysm. The transcripts were undetectable in an inflamed aortic aneurysm and control vessels., Conclusions: Gene suppression is a prominent feature of the intermediate effect of TNF-alpha on endothelial cells. Differences in the expression of the tested genes in endothelial cells, fibroblasts and vascular smooth muscle cells open possibilities for the study of cellular interactions in the vascular wall in disease situations with high local TNF-alpha concentrations.

The alpha-amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the culture fluid. On an SDS/polyacrylamide gel, the protein an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of the enzyme in Escherichia coli gave a product with the mass expected for the mature protein. The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and maltose. It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity. the pH and temperature optima were pH3 and 75 degrees C, respectively, characterizing the enzyme as being thermoacidophilic. Alignment of the sequence of the enzyme with that of its closest neutrophilic relatives and with that of alpha-1,4 or alpha-1,6 glycosidic-bond hydrolyzing enzymes of known three-dimensional structure showed that the acidophilic alpha-amylase contains approximately 30% less charged residues than do its closest relatives, that these residues are replaced by neutral polar residues, and that hot spots for these exchanges are likely to be located at the surface of the protein. Literature data show that similar effects are observed in three other acidophilic proteins. It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins.