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.

Amplification of innate immune responses by endogenous danger-associated molecular patterns (DAMPs) promotes inflammation. The involvement of S100A8 and S100A9, DAMPs belonging to the S100 calgranulin family, in the pathogenesis of cardiovascular disease is attracting an increasing amount of interest. S100A8 and S100A9 (also termed MRP8 and MRP14) preferentially form the S100A8/A9 heterodimer (MRP8/14 or calprotectin) and are constitutively expressed in myeloid cells. The levels of circulating S100A8/A9 in humans strongly correlate to blood neutrophil counts and are increased by traditional cardiovascular risk factors such as smoking, obesity, hyperglycemia, and dyslipidemia. S100A8/A9 is an endogenous ligand of toll-like receptor 4 (TLR4) and of the receptor for advanced glycation end products (RAGE) and has been shown to promote atherogenesis in mice. In humans, S100A8/A9 correlates with the extent of coronary and carotid atherosclerosis and with a vulnerable plaque phenotype. S100A8/A9 is locally released following myocardial infarction and amplifies the inflammatory responses associated with myocardial ischemia/reperfusion injury. Elevated plasma levels of S100A8/A9 are associated with increased risk of future coronary events in healthy individuals and in myocardial infarction survivors. Thus, S100A8/A9 might represent a useful biomarker and therapeutic target in cardiovascular disease. Importantly, S100A8/A9 blockers have been developed and are approved for clinical testing.

OBJECTIVE

Previous studies showed that genetic deletion or pharmacological blockade of the receptor for advanced glycation end products (RAGE) prevents the early structural changes in the glomerulus associated with diabetic nephropathy. To overcome limitations of mouse models that lack the progressive glomerulosclerosis observed in humans, we studied the contribution of RAGE to diabetic nephropathy in the OVE26 type 1 mouse, a model of progressive glomerulosclerosis and decline of renal function.

METHODS

We bred OVE26 mice with homozygous RAGE knockout (RKO) mice and examined structural changes associated with diabetic nephropathy and used inulin clearance studies and albumin:creatinine measurements to assess renal function. Transcriptional changes in the Tgf-beta1 and plasminogen activator inhibitor 1 gene products were measured to investigate mechanisms underlying accumulation of mesangial matrix in OVE26 mice.

RESULTS

Deletion of RAGE in OVE26 mice reduced nephromegaly, mesangial sclerosis, cast formation, glomerular basement membrane thickening, podocyte effacement, and albuminuria. The significant 29% reduction in glomerular filtration rate observed in OVE26 mice was completely prevented by deletion of RAGE. Increased transcription of the genes for plasminogen activator inhibitor 1, Tgf-beta1, Tgf-beta-induced, and alpha1-(IV) collagen observed in OVE26 renal cortex was significantly reduced in OVE26 RKO kidney cortex. ROCK1 activity was significantly lower in OVE26 RKO compared with OVE26 kidney cortex.

CONCLUSIONS

These data provide compelling evidence for critical roles for RAGE in the pathogenesis of diabetic nephropathy and suggest that strategies targeting RAGE in long-term diabetes may prevent loss of renal function.

OBJECTIVE

Infiltration of synovial tissue by neutrophils is crucial in rheumatoid arthritis (RA), psoriatic arthritis (PsA) and seronegative arthritis (SA). Altered vascular function and endothelial activation are important in PsA. S100A12 (EN-RAGE) is secreted by activated granulocytes and binds to the receptor for advanced glycation end products, which induces nuclear factor (NF)-kappaB-dependent activation of endothelium.

METHODS

Immunohistochemical studies were performed to detect synovial S100A12 expression. We analysed serum and synovial fluid of 42 patients for S100A12 levels.

RESULTS

S100A12 was strongly expressed in inflamed synovial tissue whereas it was nearly undetectable in synovia of controls or patients after successful treatment. Serum levels of S100A12 correlated with disease activity.

CONCLUSIONS

Local expression of S100A12 in inflamed tissue suggests a role in synovitis, especially in PsA. High serum concentrations of S100A12 in patients with active arthritis compared with healthy controls or patients in remission point to its usefulness as a serum marker.

Vascular complications are an important cause of morbidity and mortality in patients with diabetes. The extent of vascular complications has been linked statistically to enhanced adherence of diabetic erythrocytes to endothelial cells (ECs) and to the accumulation of a class of glycated proteins termed advanced glycation end products (AGEs). We hypothesized that formation of AGEs on the surface of diabetic erythrocytes could mediate their interaction with ECs leading to binding and induction of vascular dysfunction. Enhanced binding of diabetic erythrocytes to ECs was blocked by preincubation of erythrocytes with anti-AGE IgG or preincubation of ECs with antibodies to the receptor for AGE (RAGE). Immunoblotting of cultured human ECs and immunostaining of normal/diabetic human tissue confirmed the presence of RAGE in the vessel wall. Binding of diabetic erythrocytes to endothelium generated an oxidant stress, as measured by production of thiobarbituric acid-reactive substances (TBARS) and activation of the transcription factor NF-kappa B, both of which were blocked by probucol or anti-RAGE IgG. Erythrocytes from diabetic rats infused into normal rats had an accelerated, early phase of clearance that was prevented, in part, by antibody to RAGE. Liver tissue from rats infused with diabetic erythrocytes showed elevated levels of TBARS, which was prevented by pretreatment with anti-RAGE IgG or probucol. Thus, erythrocyte surface AGEs can function as ligands that interact with RAGE on endothelium. The extensive contact of diabetic erythrocytes bearing surface-associated AGEs with vessel wall RAGE could be important in the development of vascular complications.

We hypothesized that impaired nitric oxide (NO)-dependent dilation (endothelial dysfunction) in type 2 diabetes results, in part, from elevated production of superoxide (O(2)(*-)) induced by the interaction of advanced glycation end products (AGE)/receptor for AGE (RAGE) and TNF-alpha signaling. We assessed the role of AGE/RAGE and TNF-alpha signaling in endothelial dysfunction in type 2 diabetic (Lepr(db)) mice by evaluation of endothelial function in isolated coronary resistance vessels of normal control (nondiabetic, m Lepr(db)) and diabetic mice. Although dilation of vessels to the endothelium-independent vasodilator sodium nitroprusside (SNP) was not different between diabetic and control mice, dilation to the endothelium-dependent agonist acetylcholine (ACh) was reduced in diabetic vs. control mice. The activation of RAGE with RAGE agonist S100b eliminated SNP-potentiated dilation to ACh in Lepr(db) mice. Administration of a soluble form of RAGE (sRAGE) partially restored dilation in diabetic mice but did not affect dilation in control mice. The expression of RAGE in coronary arterioles was markedly increased in diabetic vs. control mice. We also observed in diabetic mice that augmented RAGE signaling augmented expression of TNF-alpha, because this increase was attenuated by sRAGE or NF-kappaB inhibitor MG132. Protein and mRNA expression of NAD(P)H oxidase subunits including NOX-2, p22(phox), and p40(phox) increased in diabetic compared with control mice. sRAGE significantly inhibited the expression of NAD(P)H oxidase in diabetic mice. These results indicate that AGE/RAGE signaling plays a pivotal role in regulating the production/expression of TNF-alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes.

Oxidative stress is a central mechanism by which the receptor for advanced glycation endproducts (RAGE) mediates its pathological effects. Multiple experimental inquiries in RAGE-expressing cultured cells have demonstrated that ligand-RAGE interaction mediates generation of reactive oxygen species (ROS) and consequent downstream signal transduction and regulation of gene expression. The primary mechanism by which RAGE generates oxidative stress is via activation of NADPH oxidase; amplification mechanisms in the mitochondria may further drive ROS production. Recent studies indicating that the cytoplasmic domain of RAGE binds to the formin mDia1 provide further support for the critical roles of this pathway in oxidative stress; mDia1 was required for activation of rac1 and NADPH oxidase in primary murine aortic smooth muscle cells treated with RAGE ligand S100B. In vivo, in multiple distinct disease models in animals, RAGE action generates oxidative stress and modulates cellular/tissue fate in range of disorders, such as in myocardial ischemia, atherosclerosis, and aneurysm formation. Blockade or genetic deletion of RAGE was shown to be protective in these settings. Indeed, beyond cardiovascular disease, evidence is accruing in human subjects linking levels of RAGE ligands and soluble RAGE to oxidative stress in disorders such as doxorubicin toxicity, acetaminophen toxicity, neurodegeneration, hyperlipidemia, diabetes, preeclampsia, rheumatoid arthritis and pulmonary fibrosis. Blockade of RAGE signal transduction may be a key strategy for the prevention of the deleterious consequences of oxidative stress, particularly in chronic disease.

OBJECTIVE

High mobility group box-1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. In this study, we examined the involvement of HMGB1 in traumatic brain injury (TBI) and evaluated the ability of intravenously administered neutralizing anti-HMGB1 monoclonal antibody (mAb) to attenuate brain injury.

METHODS

Traumatic brain injury was induced in rats or mice by fluid percussion. Anti-HMGB1 mAb or control mAb was administered intravenously after TBI.

RESULTS

Anti-HMGB1 mAb remarkably inhibited fluid percussion-induced brain edema in rats, as detected by T2-weighted magnetic resonance imaging; this was associated with inhibition of HMGB1 translocation, protection of blood-brain barrier (BBB) integrity, suppression of inflammatory molecule expression, and improvement of motor function. In contrast, intravenous injection of recombinant HMGB1 dose-dependently produced the opposite effects. Experiments using receptor for advanced glycation end product (RAGE)(-/-) , toll-like receptor-4 (TLR4)(-/-) , and TLR2(-/-) mice suggested the involvement of RAGE as the predominant receptor for HMGB1.

CONCLUSIONS

Anti-HMGB1 mAb may provide a novel and effective therapy for TBI by protecting against BBB disruption and reducing the inflammatory responses induced by HMGB1.

OBJECTIVE

Circulating levels of soluble receptor for advanced glycation end products (sRAGE) likely comprise both a secreted isoform (esRAGE) and wild-type RAGE cleaved from the cell membrane. Both sRAGE and esRAGE have been proposed as biomarkers of cardiovascular disease (CVD), but prospective data are limited. We examined the relationship of sRAGE and esRAGE to incident coronary heart disease (CHD) and stroke in type 2 diabetic patients followed for 3.9 years in a trial of atorvastatin: the Collaborative Atorvastatin Diabetes Study (CARDS).

METHODS

We used a nested case-control design sampling all incident cases of CVD with available plasma and randomly selecting three control subjects, who were free of CVD throughout follow-up, per case. Analysis was by Cox regression with adjustment for treatment allocation and relevant covariates.

RESULTS

sRAGE and esRAGE were strongly correlated (ρ = 0.88) and were both higher in those with lower BMI (P < 0.001), higher adiponectin (P < 0.001), lower estimated glomerular filtration rate (P = 0.009), and white ethnicity (P < 0.001). Both sRAGE and esRAGE were associated with incident CHD events, independently of treatment allocation and the above factors; hazard ratio (HR) = 1.74 (95% CI 1.25-2.41; P = 0.002) for a doubling of the sRAGE level; HR = 1.45 (1.11-1.89; P = 0.006) for a doubling of the esRAGE level. There was no significant association with stroke; HR for sRAGE = 0.66 (0.38-1.14). Atorvastatin, 10 mg daily, did not alter sRAGE.

CONCLUSIONS

Higher levels of sRAGE and esRAGE are associated with incident CHD but not stroke in type 2 diabetes.

Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet-induced obesity and diabetes. Obese LGALS3(-/-) mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3(-/-) mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c(+)CD11b(+) macrophages and decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3(-/-) animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3(-/-) peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1-dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3(-/-) macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes.

Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.

The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia.

Macrophages can be activated and regulated by high-mobility group box 1 (HMGB1), a highly conserved nuclear protein. Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors, including the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, TLR4, and TLR9. Pyroptosis is a caspase-1-dependent programmed cell death, which features rapid plasma membrane rupture, DNA fragmentation, and release of proinflammatory intracellular contents. Pyroptosis can be triggered by various stimuli, however, the mechanism underlying pyroptosis remains unclear. In this study, we identify a novel pathway of HMGB1-induced macrophage pyroptosis. We demonstrate that HMGB1, acting through RAGE and dynamin-dependent signaling, initiates HMGB1endocytosis, which in turn induces cell pyroptosis. The endocytosis of HMGB1 triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. We further confirm that HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for this form of pyroptosis in the development of inflammation. These findings shed light on the regulatory role of ligand-receptor internalization in directing cell fate, which may have an important role in the progress of inflammation following infection and injury.

It has been extensively reported that diabetes mellitus (DM) patients have a higher risk of developing Alzheimer's disease (AD), but a mechanistic connection between both pathologies has not been provided so far. Carbohydrate-derived advanced glycation endproducts (AGEs) have been implicated in the chronic complications of DM and have been reported to play an important role in the pathogenesis of AD. The earliest histopathological manifestation of AD is the apparition of extracellular aggregates of the amyloid beta peptide (Abeta). To investigate possible correlations between AGEs and Abeta aggregates with both pathologies, we have performed an immuhistochemical study in human post-mortem samples of AD, AD with diabetes (ADD), diabetic and nondemented controls. ADD brains showed increased number of Abeta dense plaques and receptor for AGEs (RAGE)-positive and Tau-positive cells, higher AGEs levels and major microglial activation, compared to AD brain. Our results indicate that ADD patients present a significant increase of cell damage through a RAGE-dependent mechanism, suggesting that AGEs may promote the generation of an oxidative stress vicious cycle, which can explain the severe progression of patients with both pathologies.

Although epidemiological data associate hypertension with a strong predisposition to develop Alzheimer disease, no mechanistic explanation exists so far. We developed a model of hypertension, obtained by transverse aortic constriction, leading to alterations typical of Alzheimer disease, such as amyloid plaques, neuroinflammation, blood-brain barrier dysfunction, and cognitive impairment, shown here for the first time. The aim of this work was to investigate the mechanisms involved in Alzheimer disease of hypertensive mice. We focused on receptor for advanced glycation end products (RAGE) that critically regulates Aβ transport at the blood-brain barrier and could be influenced by vascular factors. The hypertensive challenge had an early and sustained effect on RAGE upregulation in brain vessels of the cortex and hippocampus. Interestingly, RAGE inhibition protected from hypertension-induced Alzheimer pathology, as showed by rescue from cognitive impairment and parenchymal Aβ deposition. The increased RAGE expression in transverse aortic coarctation mice was induced by increased circulating advanced glycation end products and sustained by their later deposition in brain vessels. Interestingly, a daily treatment with an advanced glycation end product inhibitor or antioxidant prevented the development of Alzheimer traits. So far, Alzheimer pathology in experimental animal models has been recognized using only transgenic mice overexpressing amyloid precursor. This is the first study demonstrating that a chronic vascular insult can activate brain vascular RAGE, favoring parenchymal Aβ deposition and the onset of cognitive deterioration. Overall we demonstrate that RAGE activation in brain vessels is a crucial pathogenetic event in hypertension-induced Alzheimer disease, suggesting that inhibiting this target can limit the onset of vascular-related Alzheimer disease.

Functional MRI is based on changes in cerebral microvasculature triggered by increased neuronal oxidative metabolism. This change in blood flow follows a pattern known as the hemodynamic response function (HRF), which typically peaks 4-6 s following stimulus delivery. However, in the presence of cerebrovascular disease the HRF may not follow this normal pattern, due to either the temporal signal to noise (tSNR) ratio or delays in the HRF, which may result in misinterpretation or underestimation of fMRI signal. The present study examined the HRF and SNR in five individuals with aphasia resulting from stroke and four unimpaired participants using a lexical decision task and a long trial event-related design. T1-weighted images were acquired using an MP-RAGE sequence and BOLD T2*-weighted images were acquired using Echo Planar Imaging to measure time to peak (TTP) in the HRF. Data were analyzed using Brain Voyager in four anatomic regions known to be involved in language processing: Broca's area and the posterior perisylvian network (PPN) (including Wernicke's area, the angular and supramarginal gyri) and right hemisphere homologues of these regions. The occipital area also was examined as a control region. Analyses showed that the TTP in three out of five patients in the left perisylvian area was increased significantly as compared to normal individuals and the left primary visual cortex in the same patients. In two other patients no significant delays were detected. We also found that the SNR for BOLD signal detection may by insufficient in damaged areas. These findings indicate that obtaining physiologic (TTP) and quality assurance (tSNR) information is essential for studying activation patterns in brain-damaged patients in order to avoid errors in interpretation of the data. An example of one such misinterpretation and the need for alternative data analysis strategies is discussed.

BACKGROUND

We previously found that S100P, a member of the S100 protein family, is expressed in more than 90% of pancreatic tumors and is associated with tumor growth and invasion. In the current study, we investigated the ability of the antiallergy drug, cromolyn, to block S100P function.

METHODS

Interactions between cromolyn and S100P were investigated using a drug affinity column and by examining cromolyn's effects on coimmunoprecipitation of S100P and receptor for advanced glycation end-products (RAGE). The effects of cromolyn on cell growth, invasion, and nuclear factor-kappaB (NFkappaB) activity of pancreatic cancer cells with (BxPC-3 and MPanc-96) and without (Panc-1) endogenous S100P were investigated by cell proliferation assay, by cell invasion assay, and by luciferase reporter gene assay, respectively. The effects of cromolyn on tumor growth in vivo were investigated in three orthotopic models (n = 20 mice per model) by administration of cromolyn (5 mg/kg body weight, daily) with and without gemcitabine (125 mg/kg body weight, biweekly), the drug currently used to treat pancreatic cancer. Tumor growth was assayed by reporter gene expression. All statistical tests were two-sided.

RESULTS

S100P was retained on a cromolyn affinity column. Cromolyn blocked the coimmunoprecipitation of S100P and RAGE. In vitro, cromolyn (100 microM) inhibited S100P-stimulated Panc-1 cell proliferation (S100P, mean = 0.93 U, versus S100P + cromolyn, mean = 0.56 U, difference = 0.37 U; 95% confidence interval [CI] = 0.24 to 0.49 U; P = .001, n = 3), invasion (S100P, mean = 58.0%, versus S100P + cromolyn, mean = 9.4%, difference = 48.6%; 95% CI = 38.8% to 58.8%; P<.001, n = 3), and NFkappaB activity (S100P, mean = 14,460, versus S100P + cromolyn, mean = 7360 photons/s, difference = 7100 photons/s; 95% CI = 3689 to 10 510 photons/s; P = .005, n = 3). In vivo, cromolyn inhibited tumor growth in mice bearing tumor with endogenous S100P (BxPC-3: control, mean = 1.6 x 10(9) photons/s, versus cromolyn, mean = 4.4 x 10(8) photons/s, difference = 1.2 x 10(9) photons/s; 95% CI = 6.2 x 10(8) to 1.6 x 10(9) photons/s; P<.001, n = 5; MPanc-96: control, mean = 1.1 x 10(10) photons/s, versus cromolyn, mean = 4.8 x 10(9) photons/s, difference = 6.2 x 10(9) photons/s; 95% CI = 1.9 x 10(9) to 1.0 x 10(10) photons/s; P = .009, n = 5) and increased the effectiveness of gemcitabine (BxPC-3: gemcitabine, mean = 9.2 x 10(8) photons/s, versus combination, mean = 1.8 x 10(8) photons/s, difference = 7.4 x 10(8) photons/s; 95% CI = 4.5 x 10(8) to 1.0 x 10(9) photons/s; P<.001; MPanc-96: gemcitabine, mean = 4.1 x 10(9) photons/s, versus combination, mean = 2.0 x 10(9) photons/s, difference = 2.1 x 10(9) photons/s; 95% CI = 4.4 x 10(8) to 3.8 x 10(9) photons/s; P<.001). However, cromolyn had no effect on growth of tumors lacking S100P (Panc-1).

CONCLUSIONS

Cromolyn binds S100P, prevents activation of RAGE, inhibits tumor growth, and increases the effectiveness of gemcitabine in experimental models.

BACKGROUND

A non-enzymatic reaction between reducing sugars and amino groups of proteins, lipids and nucleic acids contributes to the aging of macromolecules, whose process has been known to progress at an accelerated rate under hyperglycemic and/or oxidative stress conditions. Over a course of days to weeks, early glycation products undergo further reactions such as rearrangements and dehydration to become irreversibly cross-linked, fluorescent protein derivatives termed advanced glycation end products (AGEs).

METHODS

In this paper, we review the role of AGE-oxidative stress axis and its therapeutic interventions in vascular complications in diabetes.

CONCLUSIONS

AGEs elicit oxidative stress generation and subsequently cause inflammatory and thrombogenic reactions in various types of cells via interaction with a receptor for AGEs (RAGE), thereby being involved in vascular complications in diabetes. In addition, mitochondrial superoxide generation has been shown to play an important role in the formation and accumulation of AGEs under diabetic conditions. Further, we have recently found that a pathophysiological crosstalk between AGE-RAGE axis and renin-angiotensin system (RAS) could contribute to the progression of vascular damage in diabetes.

CONCLUSIONS

These observations suggest that inhibition of AGE-RAGE-oxidative stress axis or blockade of its interaction with RAS is a novel therapeutic strategy for preventing vascular complications in diabetes.

The receptor for advanced glycation end products (RAGE) is an important cell surface receptor being pursued as a therapeutic target because it has been implicated in complications arising from diabetes and chronic inflammatory conditions. RAGE is a single membrane spanning receptor containing a very small approximately 40 residue cytosolic domain and a large extracellular region composed of 3 Ig-like domains. In this study, high level bacterial expression systems and purification protocols were generated for the extracellular region of RAGE (sRAGE) and the five permutations of single and tandem domain constructs to enable biophysical and structural characterization of its tertiary and quaternary structure. The structure and stability of each of these six protein constructs was assayed by biochemical methods including limited proteolysis, dynamic light scattering, CD, and NMR. A homology model of sRAGE was constructed to aid in the interpretation of the experimental data. Our results show that the V and C1 domains are not independent domains, but rather form an integrated structural unit. In contrast, C2 is attached to VC1 by a flexible linker and is fully independent. The interaction with a known RAGE ligand, Ca2+-S100B, was mapped to VC1, with the major contribution from the V domain but clearly defined secondary effects from the C1 domain. The implications of these results are discussed with respect to models for RAGE signaling.

BACKGROUND

The interaction of advanced glycation end products, including Nepsilon-(carboxymethyl)lysine-protein adducts (CML) and S100A12 protein, with their cellular receptor (RAGE) is implicated in the pathogenesis of diabetic vascular complications. RAGE has a circulating secretory receptor form, soluble RAGE (sRAGE), which, by neutralizing the action of advanced glycation end products, might exert a protective role against the development of cardiovascular disease.

OBJECTIVE

The objective of the study was to investigate whether plasma sRAGE levels are associated with glycemic control, proinflammatory factors, or circulating ligands of RAGE such as plasma CML and S100A12 protein.

METHODS

We studied 160 subjects, 84 subjects with type 2 diabetes (aged 60 +/- 7 yr) and 76 nondiabetic controls (aged 45 +/- 10 yr).

RESULTS

Plasma sRAGE was lower in diabetic patients than controls [141 (53-345) vs. 735 (519-1001) pg/ml, median (interquartile range), P < 0.0001], whereas CML levels were higher in diabetic patients than controls [67.9 (46.0-84.7) vs. 43.4 (28.0-65.0) microg/ml, P < 0.0001]. In stepwise regression analysis of the whole data set, hemoglobin A1c, insulin resistance (as homeostasis model assessment), and C-reactive protein were independently associated with plasma sRAGE, whereas age was not. In a subgroup of 26 diabetic and 24 nondiabetic subjects of similar age (54 +/- 3 yr), plasma S100A12 levels were higher in diabetic subjects [49 (39-126) vs. 28 (21-39) ng/ml]. Moreover, low sRAGE and high S100A12 were strongly associated with increased risk for cardiovascular disease (Framingham score). In this subgroup, the plasma S100A12 level was the only determinant of plasma sRAGE concentration.

CONCLUSIONS

Plasma level of sRAGE is down-regulated in chronic hyperglycemia; among its ligands, S100A12 protein, but not CML, appears to be associated with this effect.

Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is the receptor for advanced glycation products (RAGE). The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. Here, by using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface. Ca2+ binding creates two symmetric hydrophobic surfaces on Ca2+-calgranulin C that allow calgranulin C to bind to the C-type immunoglobulin domain of RAGE. Apo-calgranulin C also binds to sRAGE using a completely different surface and with substantially lower affinity, thus underscoring the role of Ca2+ binding to S100 proteins as a molecular switch. By using native gel electrophoresis, chromatography, and fluorescence spectroscopy, we established that sRAGE forms tetramers that bind to hexamers of Ca2+-calgranulin C. This arrangement creates a large platform for effectively transmitting RAGE-dependent signals from extracellular S100 proteins to the cytoplasmic signaling complexes.

BACKGROUND

The interaction of receptor for advanced glycation end products (RAGE) and its ligands often leads to inflammatory processes or tissue injury, although the effect of the blockade of RAGE signaling on lung injury remains to be investigated.

OBJECTIVE

Using a murine model of lung injury induced by intratracheal lipopolysaccharide (LPS), we evaluated RAGE expression in the airspace and the effect of recombinant soluble RAGE (sRAGE) on LPS-induced lung injury.

METHODS

First, the expression of sRAGE in bronchoalveolar lavage (BAL) fluid was determined at 24 hours after intratracheal instillation of LPS or phosphate-buffered saline. Next, to evaluate the effect of sRAGE, BAL fluid was collected for cell counting and measurements of lung permeability and cytokine concentrations 24 hours after intratracheal LPS in the mice with or without intraperitoneal administration of sRAGE 1 hour after the instillation. In another series, lungs were sampled for histopathology and detection of apoptotic cells. The activation of nuclear factor (NF)-kappaB was analyzed 4 hours after LPS instillation.

RESULTS

In response to LPS challenge, a RAGE isoform of 48 kD was detected in the BAL fluid. Treatment with sRAGE significantly attenuated the increases in neutrophil infiltration, lung permeability, production of inflammatory cytokines, NF-kappaB activation, and apoptotic cells in the lung as well as development of pathologic changes after LPS instillation.

CONCLUSIONS

RAGE plays an important role in the pathogenesis of LPS-induced lung injury in mice. It was suggested that sRAGE should be tested as a treatment modality in other models of acute lung injury.

BACKGROUND

Advanced glycation end products (AGEs) and receptor RAGE interaction play a role in diabetic vascular complications. Inhibition of dipeptidyl peptidase-4 (DPP-4) is a potential therapeutic target for type 2 diabetes. However, the role of DPP-4 in AGE-induced endothelial cell (EC) damage remains unclear.

METHODS

In this study, we investigated the effects of DPP-4 on reactive oxygen species (ROS) generation and RAGE gene expression in ECs. We further examined whether an inhibitor of DPP-4, linagliptin inhibited AGE-induced soluble DPP-4 production, ROS generation, RAGE, intercellular adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) gene expression in ECs.

RESULTS

DPP-4 dose-dependently increased ROS generation and RAGE gene expression in ECs, which were prevented by linagliptin. Mannose 6-phosphate (M6P) and antibodies (Ab) raised against M6P/insulin-like growth factor II receptor (M6P/IGF-IIR) completely blocked the ROS generation in DPP-4-exposed ECs, whereas surface plasmon resonance revealed that DPP-4 bound to M6P/IGF-IIR at the dissociation constant of 3.59 x 10⁻⁵ M. AGEs or hydrogen peroxide increased soluble DPP-4 production by ECs, which was prevented by N-acetylcysteine, RAGE-Ab or linagliptin. Linagliptin significantly inhibited the AGE-induced ROS generation, RAGE, ICAM-1 and PAI-1 gene expression in ECs.

CONCLUSIONS

The present study suggests that AGE-RAGE-induced ROS generation stimulates the release of DPP-4 from ECs, which could in turn act on ECs directly via the interaction with M6P/IGF-IIR, further potentiating the deleterious effects of AGEs. The blockade by linagliptin of positive feedback loop between AGE-RAGE axis and DPP-4 might be a novel therapeutic target for vascular injury in diabetes.

Oxidative stress has been well documented in the substantia nigra in Parkinson disease (PD), but little is known about oxidative damage, particularly lipoxidation, advanced glycation (AGE), and AGE receptors (RAGE) in other structures, including the cerebral cortex, in early stages of diseases with Lewy bodies. The present study was undertaken to analyze these parameters in the frontal cortex (area 8), amygdala, and substantia nigra in selected cases with no neurologic symptoms and with neuropathologically verified incidental Lewy body disease-related changes, comparing them with healthy age-matched individuals. Results of the present study have shown mass spectrometric and immunologic evidences of increased lipoxidative damage by the markers malondialdehyde-lysine (MDAL) and 4-hydroxynonenal-lysine (HNE), increased expression of AGE in the substantia nigra, amygdala, and frontal cortex, and increased and heterogeneous RAGE cellular expression in the substantia nigra and frontal cortex in cases with early stages of parkinsonian neuropathology. In addition, increased content of the highly peroxidizable docosahexaenoic acid in the amygdala and frontal cortex. These changes were not associated to alpha-synuclein aggregation in cortex, contrasting with aggregates found in SDS-soluble fractions of frontal cortex in dementia with Lewy bodies (DLB) cases. The pattern of lipidic abnormalities differed in DLB and incidental Lewy body disease. Furthermore, although AGE and RAGE expression were raised in DLB, no increase in the total amount of HNE and MDAL adducts was found in the cerebral cortex in DLB. Preliminary analyses have identified 2 proteins with lipoxidative damage, alpha-synuclein and manganese superoxide dismutase (SOD2), in incidentally Lewy body disease cortex. This study demonstrates abnormal fatty acid profiles, increased and selective lipoxidative damage, and increased AGE and RAGE expression in the frontal cortex in cases with early stages of parkinsonian neuropathology without treatment. These findings further support antioxidant therapy in the treatment of PD to reduce cortical damage associated with oxidative stress.