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Biomedical subjects

A M Schmidt

Publications and source records attributed to A M Schmidt.

At least 55 records · Page 3Linked to original sources
PubMed1999

N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression.

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure, N(epsilon)-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-kappaB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.

Animals↗
PubMed1999

Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

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.

Animals↗
PubMed1999

RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

Amino Acid Sequence↗
PubMed1999

Activation of receptor for advanced glycation end products: a mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis.

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.

Animals↗
PubMed1999

Time-dependent cellular population of textured-surface left ventricular assist devices contributes to the development of a biphasic systemic procoagulant response.

OBJECTIVE: Textured-surface left ventricular assist devices (LVAD) have been shown to enhance ventricular function and survival in patients with end-stage heart failure. Furthermore, we have described a procoagulant physiology in our LVAD population with sustained thrombin generation (elevated thrombin-antithrombin III complex and prothrombin fragment 1+2) and fibrinolysis (D-dimers), even up to 335 days after LVAD placement. To explain such sustained activation of coagulation, we speculated that the LVAD surface selectively adsorbed and promoted activation of circulating blood cells. METHODS: In a prospective study of 20 patients with LVADs, we examined samples of peripheral blood as well as cells harvested from the surface of the LVADs at the time of their explantation for procoagulant proinflammatory markers. RESULTS: Analysis of the cells populating the LVAD surface revealed the presence of pluripotent hematopoietic CD34(+) cells, as well as cells bearing monocyte (CD14)/macrophage (CD68) markers, which also expressed procoagulant tissue factor. Reverse transcriptase-polymerase chain reaction confirmed cellular activation on the LVAD surface, revealing transcripts for interleukin 1alpha, interleukin 2, and tumor necrosis factor alpha, in addition to vascular cell adhesion molecule-1 consistent with their capacity to continually recruit and activate circulating cells, thereby propagating their response. In the periphery, elevated levels of tissue factor were found in the plasma of patients with LVADs, along with enhanced procoagulant activity. CONCLUSION: These observations suggest that the LVAD surface selectively absorbs and activates circulating hematopoietic precursor and monocytic cells, thereby creating a sustained prothrombotic and potentially proinflammatory systemic environment.

Adolescent↗
PubMed1999

Chronic aminoguanidine attenuates renal dysfunction and injury in aging rats.

We have previously shown that aging is associated with increased lipid peroxidation, reductions in renal function, and increased glomerular sclerosis. The mechanism(s) responsible for these age-related changes are not clear. The purpose of the present studies was to determine if there was an increase in inducible nitric oxide synthase (iNOS) with aging, and if so, whether inhibition of iNOS would prevent aging injury by preventing free radical-mediated lipid peroxidation. iNOS protein expression in the kidney increased by approximately 90% by 24 months. Inhibition of iNOS by aminoguanidine (0.1% in drinking water) for 9 months, beginning at 13 months of age, reduced blood pressure, improved glomerular filtration rate by 70%, and renal plasma flow by 40%, whereas glomerular sclerosis was considerably reduced. Renal F2-isoprostanes and malondialdehyde levels, markers of oxidative stress and lipid peroxidation, were not reduced by aminoguanidine. Aminoguanidine also did not attenuate immunostaining for advanced glycosylation end products (AGE) in the kidneys. These findings suggest that aminoguanidine attenuates aging renal dysfunction by inhibiting a pathophysiologic function of iNOS that is independent of free radical-mediated lipid peroxidation or significant effects on AGE deposition.

Aging↗
PubMed1999

A novel presenilin-2 splice variant in human Alzheimer's disease brain tissue.

Mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes account for the majority of cases of early-onset familial Alzheimer's disease (AD). Alternative splicing forms of the PS-1 and PS-2 gene products have previously been reported in fibroblast and brain tissue from both familial and sporadic AD patients, as well as from normal tissues and cell lines. We demonstrate here unusual alternative splicing of the PS-2 gene that leads to the generation of mRNA lacking exon 5 in human brain tissue. This product was more frequently detected in brain tissue from sporadic AD patients (70.0%; 21 of 30) than from normal age-matched controls (17.6%; three of 17). In cultured neuroblastoma cells, this splice variant was generated in hypoxia but not under other forms of cellular stress. Hypoxia-mediated induction of this splice variant was blocked by pretreatment of neuroblastoma cells with the protein synthesis inhibitor cycloheximide or antioxidants such as N-acetylcysteine and diphenyl iodonium, suggesting that hypoxia-mediated oxidant stress might, at least in part, underlie the alternative splicing of PS-2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N-terminal part of PS-2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.

Aged↗
PubMed1999

Soluble cellular adhesion molecules in proliferative vitreoretinopathy and proliferative diabetic retinopathy.

PURPOSE: To measure vitreous levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cellular adhesion molecule-1 (sVCAM-1) in the eyes of patients with retinal detachment (RD) due to proliferative diabetic retinopathy (PDR) or proliferative vitreoretinopathy (PVR) and to determine whether the levels of these mediators correlated with clinical parameters of disease. METHODS: Undiluted vitreous specimens were collected from 50 eyes of 48 patients undergoing vitrectomy for traction RD due to PDR (21 specimens) and recurrent RD due to PVR (19 specimens). Control vitreous specimens were obtained from patients undergoing macular hole repair (10 specimens). The levels of sICAM-1 and sVCAM-1 were measured in each sample by specific enzyme-linked immunoadsorbent assays. RESULTS: Vitreous levels of sICAM-1 were significantly increased in vitreous specimens from both PVR (median +/- SD; 12.0 +/- 76.3 ng/ml; P < 0.01) and PDR (8.4 +/- 24.0 ng/ml; P < 0.01) when compared to vitreous from eyes with macular holes (0. 3 +/- 4.2 ng/ml). Vitreous levels of sVCAM-1 were significantly increased in both PVR (36.5 +/- 255.2 ng/ml; P < 0.001) and PDR (26. 2 +/- 93.5 ng/ml; P < 0.01) when compared to control vitreous (17.7 +/- 7.8 ng/ml). The vitreous levels of sICAM-1 were higher in cases of PDR which developed recurrent proliferative disease (P < 0.01) and recurrent RD (P = 0.01), whereas the levels of sICAM-1 in PVR and sVCAM-1 in PDR and PVR did not significantly correlate with these clinical parameters. CONCLUSIONS: Soluble forms of ICAM-1 and VCAM-1 are increased in the vitreous cavity of patients with RD due to PDR or PVR, reflecting the inflammatory nature of these conditions and suggesting a possible role for these mediators in the pathogenesis of proliferative retinal disease. The vitreous levels of these sCAMs at the time of surgery may serve as a marker of inflammation, but their specific levels do not predict the likelihood of recurrent proliferation or surgical anatomic success in most cases of PVR and PDR.

Adult↗
PubMed1999

Acute modulation of albumin microvascular leakage by advanced glycation end products in microcirculation of diabetic rats in vivo.

Advanced glycation end products (AGEs) are nonenzymatic glycosylated adducts of proteins that accumulate in vascular tissue during diabetes and aging. The aim of this work was to study the role of AGEs and of the oxidative mechanisms in diabetes-induced changes in vascular permeability. Intravital videomicroscopy was used to study albumin microvascular leakage in cremaster muscle. The extravasation of a fluorescent macromolecular tracer (fluorescein isothiocyanate-albumin) was measured for 1 h and, after computer-aided image analysis, was expressed as variations of normalized gray levels (arbitrary units). Extravasation of the macromolecular tracer was much higher in diabetic rats than in control rats (slope of extravasation versus time increased by >100%, P < 10(-4)). This increase was significantly inhibited when we blocked AGEs binding to their endothelial receptor by intravenous bolus of soluble recombinant receptor to AGEs (rR-RAGE) (slope of extravasation versus time decreased by 19, 30, and 40%, for 0.5, 2.5, and 5.15 mg/kg rR-RAGE, respectively) or by a 6 mg/kg intravenous bolus of antibody against RAGE (slope decreased by 53%). Systemic injection of probucol (an antioxidant) also significantly inhibited the increase in the extravasation of the macromolecular tracer occurring in experimental diabetes (slope decreased by 51%, P < 10(-4)). These results strongly suggest that in experimental diabetes the interaction of circulating AGEs and endothelial RAGE mediates albumin micro-vascular leakage, possibly via AGE-RAGE-dependent enhanced oxidant stress.

Albumins↗
PubMed1999

Using telemedicine in the treatment of pressure ulcers.

Pressure ulcers are dynamic and therefore require frequent assessment and immediate treatment. For many patients who live long distances from rehabilitation hospitals, frequent assessment and immediate treatment are often unavailable. Recent advances during the last two decades have resulted in the development of telemedicine--long-distance delivery of medical education and services to patients. This pilot study reports on a patient enrolled in a telemedicine program during his fifth hospitalization for pressure ulcers in 16 months. Although this is only a single case study, the results suggest the potential efficacy of this new intervention.

Adult↗
PubMed1998

Sp1-binding elements in the promoter of RAGE are essential for amphoterin-mediated gene expression in cultured neuroblastoma cells.

Receptor for AGE (RAGE) and the polypeptide amphoterin are highly expressed and co-localized in neurons of the developing central nervous system of the rat. In vitro, the interaction of amphoterin with neuronal RAGE induces neurite outgrowth. We tested the hypothesis that interaction of amphoterin with neuronal cells enhances RAGE expression, thereby providing a mechanism by which amphoterin-mediated regulation of RAGE might contribute to promotion of neurite growth and spreading. Incubation of cultured neuroblastoma cells with amphoterin resulted in increased transcription and translation of RAGE, a process largely inhibited in the presence of anti-RAGE IgG but not by nonimmune IgG. To begin to delineate molecular mechanisms underlying these findings, we identified multiple putative binding elements within the 5'-flanking region of the RAGE gene for Sp1, a transcription factor that has been critically linked to the process of normal development. DNase I footprinting and electrophoretic mobility shift assays demonstrated multiple functional Sp1-binding sites within the region -245 to -40 of the RAGE promoter. Transient transfection of cultured SK-N-SH neuroblastoma cells with chimeric 5'-deletion constructs linked to luciferase reporter revealed that the region containing Sp1-binding elements did not contribute uniquely to basal expression of the RAGE gene. Simultaneous mutation of the multiple Sp1-binding elements in this region did not affect basal promoter function; however, promoter responsiveness to amphoterin was markedly attenuated. These results point to Sp1-dependent mechanisms underlying amphoterin-mediated increases in RAGE expression in neuroblastoma cells and further link amphoterin-RAGE interaction to development of the nervous system.

Animals↗
PubMed1998

Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

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.

Alzheimer Disease↗
PubMed1998

Vasculopathy and insulin resistance in the JCR:LA-cp rat.

The JCR:LA-cp rat is one of a number of strains incorporating the autosomal recessive cp gene that induces obesity. This strain is unique in the development of not only a profound insulin resistance, but an accompanying cardiovascular disease that correlates strongly with hyperinsulinemia. The hyperinsulinemia develops rapidly after 4 weeks of age, with an age at half-maximum of 5.5 weeks. This reflects postprandial plasma insulin levels that peak at 1000 mU/l in a standardized meal tolerance test. Defective acetylcholine-mediated vascular relaxation develops with a 1-week lag over the developing hyperinsulinemia. The frequency of staining for the vascular adhesion molecules, VCAM-1 and ICAM, does not show either age or genotype variation, although plasma levels do show an age variation. Treatment of the rats with the alpha-glucosidase inhibitor, miglitol (Bay m1099), obviates the exaggerated postprandial glucose and, especially, the insulin responses of the cp/cp rat. This causes an improvement in insulin sensitivity, prevention of the impaired vascular relaxation, and reduction in plasma levels of advanced glycated end-products. Arterial wall morphology, as visualized by both scanning and transmission electron microscopy, shows abnormal endothelium, adherent macrophages, and activated migrating smooth muscle cells in the intima. Oil-Red-O staining reveals lipid deposits in the intimal spaces, as confirmed by the presence of foam cells. The lesions resemble fatty streaks or modest atherosclerosis in man, rather than the extensive cholesterol-laden lesions seen in familial hypercholesterolemia or cholesterol-fed rabbit models. The lean rats of the strain show similar, but less marked, intimal abnormalities. The vasculopathy in this animal model appears to be precipitated by the developing hyperinsulinemia, but also requires an underlying abnormality of vascular smooth muscle and possibly also of the endothelium.

Animals↗
PubMed1998

Selective anticoagulation with active site-blocked factor IXA suggests separate roles for intrinsic and extrinsic coagulation pathways in cardiopulmonary bypass.

BACKGROUND: Multiple stimuli converge in cardiopulmonary bypass to create a tremendous prothrombotic stimulus. The ideal anticoagulant for cardiopulmonary bypass should selectively target only the intravascular stimuli, thereby eliminating pathologic clotting in the bypass circuit while preserving hemostasis in the thoracic cavity. We propose the inhibition of factor IX as such a targeted anticoagulant strategy. METHODS: We prepared an inhibitor of activated factor IX and applied it to a primate model of cardiopulmonary bypass to confirm the anticoagulant efficacy of activated factor IX in this setting and to assess more subtle markers of thrombin generation, macrophage procoagulant activity, and cellular tissue factor expression. Seven baboons that received activated factor IX (460 microg/kg) and 7 that received heparin (300 IU/kg) and protamine underwent cardiopulmonary bypass for 90 minutes and were followed after the operation for 3 hours. RESULTS: Analysis of plasma factor IX activity demonstrated adequate inhibition (<20%) of factor IX throughout cardiopulmonary bypass. Activated factor IX-treated baboons demonstrated similar circuit patency to heparin-treated baboons but had significantly diminished intraoperative blood loss. Preservation of extravascular hemostasis was further demonstrated in activated factor IX-treated animals by (1) significantly increased levels of thrombin-antithrombin III complex and prothrombin activation peptide (F1+2) without intravascular thrombosis, (2) significantly greater macrophage procoagulant activity in pericardial-derived monocytes, and (3) immunohistochemical evidence of tissue factor expression in pericardial mesothelial cells and macrophages. CONCLUSIONS: Anticoagulation with activated factor IX allows for intravascular anticoagulation with maintenance of extravascular hemostasis. These findings suggest activated factor IX as an agent that not only exemplifies a targeted approach to selective anticoagulation in cardiac surgery but also further characterizes the procoagulant milieu during cardiopulmonary bypass.

Animals↗
PubMed1998

Heparinless cardiopulmonary bypass with active-site blocked factor IXa: a preliminary study on the dog.

OBJECTIVE: Cardiopulmonary bypass is a potent stimulus for activation of procoagulant pathways. Heparin, the traditional antithrombotic agent, however, is often associated with increased perioperative blood loss because of its multiple sites of action in the coagulation cascade and its antiplatelet and profibrinolytic effects. Furthermore, heparin-mediated immunologic reactions (that is, heparin-induced thrombocytopenia) may contraindicate its use. Cardiopulmonary bypass with a selective factor IXa inhibitor was tested to see whether it could effectively limit bypass circuit/intravascular space thrombosis while decreasing extravascular bleeding, thereby providing an alternative anticoagulant strategy when heparin may not be safely administered. METHODS: Active site-blocked factor IXa, a competitive inhibitor of the assembly of factor IXa into the factor X activation complex, was prepared by modification of the enzyme's active site by the use of dansyl glutamic acid-glycine-arginine-chlormethylketone. Twenty mongrel dogs (five were given standard heparin/protamine; 15 were given activated site-blocked factor IXa doses ranging from 300 to 600 microg/kg) underwent 1 hour of hypothermic cardiopulmonary bypass, and blood loss was monitored for 3 hours after the procedure. RESULTS: Use of activated site-blocked factor IXa as an anticoagulant in cardiopulmonary bypass limited fibrin deposition within the extracorporeal circuit as assessed by scanning electron microscopy, comparable with the antithrombotic effect seen with heparin. In contrast to heparin, effective antithrombotic doses of activated site-blocked factor IXa significantly diminished blood loss in the thoracic cavity and in an abdominal incisional bleeding model. CONCLUSION: These initial studies on the dog suggest that administration of activated site-blocked factor IXa may be an effective alternative anticoagulant strategy in cardiopulmonary bypass when heparin is contraindicated, affording inhibition of intravascular/extracorporeal circuit thrombosis with enhanced hemostasis in the surgical wound.

Animals↗
PubMed1998

Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts.

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.

Animals↗