Search PubMed⌕ Search

Biomedical subjects

M Brownlee

Publications and source records attributed to M Brownlee.

At least 73 records · Page 4Linked to original sources
PubMed1994

Nerve growth factor administration protects against experimental diabetic sensory neuropathy.

Small fiber sensory neuropathy is one of the most common complications of diabetes mellitus. Currently there is no adequate therapy to prevent this often debilitating problem. Nerve growth factor (NGF) is a protein that promotes the survival and integrity of a large percentage of sensory neurons including the small fiber pain transmitting neurons which are often prominently affected in diabetic neuropathy. We report here that exogenously administered NGF is capable of preventing the behavioral and biochemical manifestations of diabetic sensory neuropathy in a streptozocin induced rat model. NGF administration prevented the elevation of tailflick threshold (a measure of the rat's response to a thermal noxious stimulus) which occurred in streptozocin-induced diabetic rats. Further, it prevented the induced reduction in levels of the neuropeptides substance P and calcitonin gene related peptide measured from cervical dorsal root ganglia. Finally, NGF did not ameliorate the prolonged latency of the compound action potentials measured from the caudal nerve of the tail. In view of these results, a clinical trial of NGF in diabetic neuropathy has now commenced.

Action Potentials↗
PubMed1994

Aminoguanidine inhibits the development of accelerated diabetic retinopathy in the spontaneous hypertensive rat.

Arterial hypertension has been identified as a major secondary risk factor for diabetic retinopathy. However, the mechanisms by which hypertension worsens retinopathy are unknown. Inhibition of advanced glycation product formation prevents the development of experimental diabetic retinopathy in normotensive diabetic rats. In this study the effect of hypertension on the rate of diabetic retinopathy development and the formation of arteriolar thrombosis was evaluated. We also evaluated the effect of aminoguanidine, an inhibitor of advanced glycation and product formation on retinal pathology of diabetic hypertensive rats. After 26 weeks of diabetes, hypertension accelerated the development of retinopathy despite a lower mean blood glucose level than in the non-hypertensive group (diabetic spontaneous hypertensive rats (SHR) 16.00 +/- 6.83 mmol/l; diabetic normotensive Wistar Kyoto rats (WKY) 34.9 +/- 3.64 mmol/l; p < 0.0001). Diabetic SHR had nearly twice as many acellular capillaries as diabetic WKY (SHR diabetic: 91.9 +/- 7.5 acellular capillaries per mm2 of retinal area vs WKY diabetic: 53.7 +/- 8.5 acellular capillaries per mm2 of retinal area), and a 3.8-fold increase in the number of arteriolar microthromboses (SHR diabetic 23,504 +/- 5523 microns2 vs SHR non-diabetic 6228 +/- 2707 microns2). Aminoguanidine treatment of SHR diabetic rats reduced the number of acellular capillaries by 50%, and completely prevented both arteriolar deposition of PAS-positive material and abnormal microthrombus formation. These data suggest that hypertension-induced deposition of glycated proteins in the retinal vasculature plays a central role in the acceleration of diabetic retinopathy by hypertension.

Aneurysm↗
PubMed1994

DNA damage by the glycation products of glyceraldehyde 3-phosphate and lysine.

In order to evaluate whether base modifications, apurinic/apyrimidinic site formation, strand breaks, or a combination of these lesions results from the interaction of glycation products with DNA, plasmid DNA was first reacted with these products, and then subjected to digestion with endonuclease III and endonuclease IV of Escherichia coli. Analysis of the differential effects of digestions with these enzymes by electrophoresis on agarose gels demonstrated that reactive glycation products produce both base modification and apurinic/apyrimidinic sites in DNA, in addition to the strand breaks observed after incubation with glycation products alone. These types of DNA damage may occur in specific diabetic cells where elevated levels of glycating sugars are associated with pathologic dysfunction.

DNA Damage↗
PubMed1994

Nonenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A model for intracellular glycosylation in diabetes.

Intracellular sugars are more reactive glycosylating agents than glucose. In vitro nonezymatic glycosylation of basic fibroblast growth factor (bFGF) by fructose, glucose-6-phosphate (G6P), or glyceraldehyde-3-phosphate (G3P) reduced high affinity heparin-binding activity of recombinant bFGF by 73, 77, and 89%, respectively. Mitogenic activity was reduced 40, 50, and 90%. To investigate the effects of bFGF glycosylation in GM7373 endothelial cells, we first demonstrated that GLUT-1 transporters were not downregulated by increased glucose concentration. In 30 mM glucose, the rate of glucose transport increased 11.6-fold, and the intracellular glucose concentration increased sixfold at 24 h and fivefold at 168 h. The level of total cytosolic protein modified by advanced glycosylation end-products (AGEs) was increased 13.8-fold at 168 h. Under these conditions, mitogenic activity of endothelial cell cytosol was reduced 70%. Anti-bFGF antibody completely neutralized the mitogenic activity at both 5 and 30 nM glucose, demonstrating that all the mitogenic activity was due to bFGF. Immunoblotting and ELISA showed that 30 mM glucose did not decrease detectable bFGF protein, suggesting that the marked decrease in bFGF mitogenic activity resulted from posttranslational modification of bFGF induced by elevated glucose concentration. Cytosolic AGE-bFGF was increased 6.1-fold at 168 h. These data are consistent with the hypothesis that nonenzymatic glycosylation of intracellular protein alters vascular cell function.

Animals↗
PubMed1993

Inhibition of matrix-induced bone differentiation by advanced glycation end-products in rats.

Glycation of long-lived proteins is an inevitable consequence of aging that is accelerated in patients with diabetes mellitus. Treatment of demineralized bone matrix particles from 35-week-old normal Long-Evans rats with glycoaldehyde, a precursor of advanced glycation end-products, was used to assess the effects of bone-matrix glycation on the process of bone differentiation. Matrix was incubated in phosphate buffered saline alone, phosphate buffered saline containing glycolaldehyde, glycolaldehyde plus the advanced glycation product-inhibitor aminoguanidine, or glycolaldehyde plus the advanced glycation product-inhibitor sodium cyanoborohydride. Glycolaldehyde increased the matrix advanced glycation product content as measured by specific fluorescence more than two-fold, while inhibiting bone differentiation more than 90% as measured by in vivo 45CaCl2 uptake, alkaline phosphatase levels, and histology. In contrast, simultaneous incubation with the advanced glycation product-inhibitor aminoguanidine or sodium cyanoborohydride not only reduced fluorescence to normal, but also restored bone differentiation. Furthermore, the inhibition of bone differentiation by glycolaldehyde was not reversed by subsequent application of recombinant bone morphogenetic protein-2. These observations suggest that formation of advanced glycation products on bone matrix alters its ability to induce bone formation, and probably involves alterations of binding sites for extractable proteins with direct bone inductive properties such as bone morphogenetic protein-2. Decreased bone formation associated with aging and diabetes may result, in part, from advanced glycation product formation on matrix proteins.

Acetaldehyde↗
PubMed1993

Localized gene transfer into organotypic hippocampal slice cultures and acute hippocampal slices.

Viral vectors derived from herpes simplex virus, type-1 (HSV), can transfer and express genes into fully differentiated, post-mitotic neurons. These vectors also transduce cells effectively in organotypic hippocampal slice cultures. Nanoliter quantities of a virus stock of HSVlac, an HSV vector that directs expression of E. coli beta-galactosidase (beta-gal), were microapplied into stratum pyramidale or stratum granulosum of slice cultures. Twenty-four hours later, a cluster of transduced cells expressing beta-gal was observed at the microapplication site. Gene transfer by microapplication was both effective and rapid. The titer of the HSVlac stocks was determined on NIH3T3 cells. Eighty-three percent of the beta-gal forming units successfully transduced beta-gal after microapplication to slice cultures. beta-Gal expression was detected as rapidly as 4 h after transduction into cultures of fibroblasts or hippocampal slices. The rapid expression of beta-gal by HSVlac allowed efficient transduction of acute hippocampal slices. Many genes have been transduced and expressed using HSV vectors; therefore, this microapplication method can be applied to many neurobiological questions.

Animals↗
PubMed1993

Nonenzymatic glycation of mesangial matrix and prolonged exposure of mesangial matrix to elevated glucose reduces collagen synthesis and proteoglycan charge.

Expansion of the mesangial matrix in diabetes occurs after prolonged exposure to the diabetic milieu. To mimic the long-term hyperglycemia of diabetes mellitus we developed tissue culture systems that might approximate the chronic state. This was accomplished in two ways: (1) by growing mesangial cells on extracellular matrix glycated and crosslinked in vitro and (2) by continuously growing cells on their own matrix on filters in elevated glucose medium (500 mg/dl) for up to eight weeks without passage. Synthesis of collagen and proteoglycans was evaluated in cells grown under these conditions. In both these situations, 3H-proline incorporation into collagenase sensitive protein and 35S incorporation into sulfated proteins were reduced compared to control cultures. Despite reduction in 35S incorporation into proteoglycans in the high glucose cultures, total glycosaminoglycan content was unchanged. However, proteoglycans generated by mesangial cells grown in elevated glucose media were of a lower negative charge than controls. In mesangial cells continuously grown on filters, the levels of messenger RNA for collagen types I and IV, biglycan and TGF-beta were not different in cells grown at elevated or standard glucose concentrations for two and four weeks. We conclude that crosslinking of mesangial matrix or continuous culture of cells for prolonged periods of time in high glucose medium, which may also crosslink matrix, suppresses collagen synthesis and reduces the negative charges on matrix proteoglycans without altering mRNA levels.

Animals↗
PubMed1993

Nonenzymatic glycosylation of laminin and the laminin peptide CIKVAVS inhibits neurite outgrowth.

Nerve regeneration in diabetic animals is delayed and qualitatively impaired, but the mechanisms responsible for these defects have not been elucidated. The extracellular matrix protein laminin promotes the extension of neuronal processes, and recent studies have localized neurite-promoting activity to a lysine-containing sequence (IKVAV) within the laminin molecule. Because long-lived molecules such as laminin are likely to accumulate excessive amounts of nonenzymatic glycosylation products in diabetic subjects, we have investigated whether such adduct formation on laminin or the IKVAV peptide affects their neurite-promoting properties. These studies used the murine neuroblastoma cell line NB2a, which extends neurites on laminin when differentiated by cAMP. Neurite outgrowth in NB2a cells plated on glycosylated laminin was significantly decreased from that occurring on unmodified laminin. Similarly, neurite outgrowth in NB2a cells plated on glycosylated IKVAV peptide was inhibited compared with that observed on native IKVAV. These data suggest that nonenzymatic glycosylation of a biologically active domain within laminin may contribute to impaired nerve regeneration in diabetes.

Amino Acid Sequence↗
PubMed1992

Altered cellular interactions between endothelial cells and nonenzymatically glucosylated laminin/type IV collagen.

Laminin and type IV collagen are two major basement membrane glycoproteins. In previous studies it has been shown that nonenzymatic glucosylation induces structural alterations of these macromolecules and also reduces their ability to self-associate. In the present study, endothelial cells were tested for their ability to adhere and spread on nonenzymatically glucosylated laminin and type IV collagen. Adhesion and spreading were reduced when glucosylated macromolecules were used as substrates. Glucosylation-induced changes in adhesion and spreading may be an important initial event signaling other phenotypic modifications of cells in the microvasculature and may be a crucial factor in order to understand the pathogenesis of diabetic microangiopathy at the molecular level.

Animals↗
PubMed1992

Enzymatic shot-gun 5'-phosphorylation and 3'-sister phosphate exchange: a two-dimensional thin-layer chromatographic technique to measure DNA deoxynucleotide modification.

DNA adducts occur through environmental, therapeutic, dietary, oxygen stress, and aging processes. A modified thin-layer chromatographic (TLC) technique can asses base composition and adduct formation. This requires labeling DNA by "shot-gun" 5'-phosphorylation of representative 32P-alpha-deoxyribonucleotide monophosphates. Subsequent 3'-monophosphate digest "sister exchanges" a radioactive 32PO4(2-) to the neighboring cold nucleotide. Separation in two-dimensional polyethyleneimine-cellulose TLC is carried out in acetic acid, (NH4)2SO4, and (NH4)HSO4. The technique was applied to control DNA, cold substitution of dUMP, methylation, depurination, and pBR322. This technique quantifies low-molecular-mass adducts and DNA integrity both in vivo and in vitro.

Animals↗
PubMed1992

Aminoguanidine ameliorates albuminuria in diabetic hypertensive rats.

We studied the effect of aminoguanidine, an inhibitor of advanced glycation product formation, on albuminuria in chronically diabetic spontaneously hypertensive rats. At the time of killing, there was no statistically significant difference in blood glucose concentration between the treated and untreated diabetic animals (18.2 +/- 0.69 mmol/l), nor was there any difference among the non-diabetic, diabetic untreated, and diabetic treated rats with respect to blood pressure (169 +/- 6.9 mm Hg). However, non-diabetic hypertensive animals had a mean quantitative 24-h urinary albumin excretion of 28 +/- 2 mg albumin/24-h, while untreated diabetic hypertensive animals averaged nearly four times that amount (106 +/- 3 mg albumin/24 h). Without affecting blood pressure, aminoguanidine treatment of diabetic hypertensive animals decreased the diabetic-associated elevation in urinary albumin excretion by 75% (48 +/- 2 mg/24 h). These data suggest than inhibition of advanced glycation product formation ameliorates the glomerular dysfunction caused by chronic hyperglycaemia.

Albuminuria↗
PubMed1992

Interactions of LDL and modified LDL with mesangial cells and matrix.

Hyperlipidemia may play a role in the progression of diabetic and other renal diseases. Low density lipoprotein (LDL) and other proteins including extracellular matrix components undergo nonenzymatic glycation in vivo. We examined the effects of glycation of LDL as occurs in diabetes (4 to 8%) on binding and uptake by mesangial cells and their proliferation. The glycation of LDL (g-LDL) significantly decreased its binding and uptake by mesangial cells by 15 to 20%, indicating that glycated LDL binds to the LDL receptor, but with lower affinity than LDL. Both LDL and g-LDL modestly stimulated [3H] thymidine incorporation into mesangial cells at 5 to 10 micrograms/ml. Native, oxidized (Ox-LDL) and glycated LDL all bound to the extracellular matrix generated by rat mesangial cells in culture. The binding of LDL, Ox-LDL and g-LDL to mesangial matrix was two to four times higher than to mesangial cells. Binding of LDL and g-LDL was significantly higher to glycolaldehyde modified matrix, which serves as an in vitro model for nonenzymatic glycation end-product cross-linking of matrix which occurs in long-standing diabetes. Based on these findings, we propose that glycation of LDL decreases its binding and uptake by the LDL receptor of mesangial cells and may slow its catabolism. Furthermore, LDL bound to extracellular mesangial matrix can undergo oxidation and generate cytotoxic LDL components. This process may be further enhanced by advanced glycation of the mesangial matrix in diabetes, contributing to glomerular pathology.

Animals↗
PubMed1992

Glucose and diabetic vascular disease.

The central therapeutic problem in diabetes mellitus is prevention and treatment of the chronic vascular disease associated with this disorder. Prolonged exposure to hyperglycemia is the primary factor associated with the development of diabetes-specific microvascular disease, and the relationship between deranged glucose metabolism and arterial disease is complicated by many other factors that influence atherogenesis in nondiabetics. Until relatively recently, knowledge about diabetic vascular disease was limited mainly to clinical description. New information about abnormal vascular physiology, ultrastructure, biochemistry, cell biology, and molecular biology now makes it possible to understand in an integrated fashion the major specific mechanisms by which hyperglycemia damages diabetic vessels. Continued progress in this area will further optimize the development of safe and effective drugs for the treatment of diabetic vascular disease.

Animals↗
PubMed1992

Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine.

Aminoguanidine-HCl inhibits the formation of advanced glycosylation end products (AGEs) in vitro and in vivo, but the mechanism by which this occurs has not been determined. Aminoguanidine inhibited glucose-derived AGE formation on RNase A by 67-85% at aminoguanidine-glucose molar ratios of 1:5 to 1:50 without affecting the concentration of Amadori products. Fast-atom-bombardment mass spectrometry of RNase peptides incubated with glucose alone or with glucose plus aminoguanidine showed that aminoguanidine inhibited the formation of AGEs without forming an adduct with glycosylated peptide. These data suggest that the primary mechanism of aminoguanidine action is reaction with Amadori-derived fragmentation products in solution. These findings are relevant to the potential clinical use of aminoguanidine in the prevention of diabetic complications.

Amino Acid Sequence↗
PubMed1992

Nonenzymatic glycosylation of macromolecules. Prospects of pharmacologic modulation.

Diabetes increases the risk of developing atherosclerotic arterial disease significantly. Although elevated glycohemoglobin was shown to be an independent risk factor in older women in the Framingham Heart Study, the relationship between hyperglycemia and macrovascular disease is complicated by the many other factors that influence atherogenesis in nondiabetic people. Studies in vitro suggest that chronic hyperglycemia may accelerate the atherogenic process through excessive glycation of various components of the arterial wall. These data are reviewed critically, and the biochemistry and pharmacological potential of the glycation-inhibitor aminoguanidine is discussed.

Animals↗
PubMed1992

Glycation products and the pathogenesis of diabetic complications.

Glucose irreversibly modifies long-lived macromolecules by forming AGEs as a function of glucose concentration and time. AGEs cause qualitative and quantitative changes in extracellular matrix components such as type IV collagen, laminin, and vitronectin. These AGE-induced changes can affect cell adhesion, growth, and matrix accumulation. AGE-modified proteins also alter cell function by interacting with specific receptors on macrophages and endothelial cells, inducing changes that promote matrix overproduction, focal thrombosis, and vasoconstriction. DNA and nuclear proteins also may be targets for AGE damage. The persistence of accumulated AGEs during periods of normal glucose homeostasis may explain the phenomenon of hyperglycemic memory. Pharmacological inhibition of in vivo AGE formation by aminoguanidine prevents or ameliorates diabetic retinopathy, nephropathy, and neuropathy in animal models. These data suggest that aminoguanidine and other AGE inhibitors have a potential therapeutic role in the treatment of diabetic patients.

Animals↗
PubMed1992

Glycosylation of proteins and microangiopathy.

Diabetic complications may result from chronic glycosylation of protein within cells and in the extracellular matrix. Prevention of glycosylation with aminoguanidine has forestalled complications in experimental diabetes.

Albuminuria↗