PubMed Health⌕ Search

Biomedical subjects

M Brownlee

Publications and source records attributed to M Brownlee.

At least 19 recordsLinked to original sources
PubMed1998

Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis.

Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P < 0.001). In neo-transfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 MM (35.5+/-5.8 vs. 19.6+/-1.6, P < 0.02, n = 3, and 21.0+/-1.3 vs. 10.0+/-1.2 pmol/ 10(6) cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.

Acetone↗
PubMed1998

The relationship of glycaemic level to advanced glycation end-product (AGE) accumulation and retinal pathology in the spontaneous diabetic hamster.

To assess the relationship between glucose and advanced glycation end products (AGE) and the relationship between AGE and retinal changes in vivo, we studied the time course of retinopathy over 12 months in trypsin digest preparations and measured glycaemia and retinal AGE in spontaneous diabetic hamsters of mild (MD) and severe (SD) phenotypes. Blood glucose levels were elevated in MD (9.44+/-0.76 mmol/l) and in SD (3 months: 24.3+/-1.4 mmol/l; 12 months: 31.7+/-0.8 mmol/l) over non-diabetic controls (NC: 7.15+/-0.25 mmol/l; p < 0.05 or less vs MD; p < 0.001 vs SD). Similar relations were found for HbA1. Retinal AGE in mild diabetes was 405+/-11.3 arbitrary units (AU) (NC 245+/-7.7; p < 0.01) after 3 months and remained unchanged. A non-linear increase of AGE over time was found in severe hyperglycaemic hamsters (466+/-21 AU after 3 months and 758+/-21 AU after 12 months; p < 0.001 vs MD). Pericyte loss in mild diabetes progressed from -26% after 3 months to 41% after 12 months (p < 0.001 vs NC). Whereas the initial pericyte loss in severely diabetic hamsters was identical to the mildly diabetic group, a higher degree of pericyte loss occurred after 12 months (-57%; p < 0.05 vs MD). Endothelial cell numbers remained unaffected by mild hyperglycaemia, but significantly increased over time in severe diabetes reaching 31.7% above controls after 12 months (p < 0.001 vs NC and MD). Microaneurysms were absent in all retinae examined. Acellular capillary segments were increased in mild diabetes (3.83+/-0.31 per mm2 of retinal area) and severe diabetes (7.83+/-0.73) over controls (1.0+/-0.23). These data suggest that a threshold of glycaemia might exist above which AGE removal systems become saturated. Pericyte loss and acellular capillary formation are associated with mild increases in blood glucose and AGE levels while endothelial cell proliferation requires higher glucose and AGE levels.

Animals↗
PubMed1998

Transduction of non-dividing adult human pancreatic beta cells by an integrating lentiviral vector.

Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 +/- 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation.

Cell Division↗
PubMed1998

Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat.

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.

Animals↗
PubMed1998

Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis.

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the effects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H2O2, there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 micromol/l H2O2 (+AG 0 vs. 10 micromol/l, NS), and reduced the percentage of apoptotic cells in response to 50 micromol/l H2O2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dose-dependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H2O2-induced benzoate hydroxylation in a dose-dependent manner. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.

Animals↗
PubMed1997

Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways.

The switch from a quiescent tumor to an invasive tumor is accompanied by the acquisition of angiogenic properties. This phenotypic change likely requires a change in the balance of angiogenic stimulators and angiogenic inhibitors. The nature of the angiogenic switch is not known. Here, we show that introduction of activated H-ras into immortalized endothelial cells is capable of activating the angiogenic switch. Angiogenic switching is accompanied by up-regulation of vascular endothelial growth factor and matrix metalloproteinase (MMP) bioactivity and downregulation of tissue inhibitor of MMP. Furthermore, we show that inhibition of phosphatidylinositol-3-kinase leads to partial inhibition of tumor angiogenesis, thus demonstrating that activated H-ras activates tumor angiogenesis through two distinct pathways. Finally, we show evidence for two forms of tumor dormancy.

Androstadienes↗
PubMed1996

Rapid production of interleukin-2-secreting tumor cells by herpes simplex virus-mediated gene transfer: implications for autologous vaccine production.

Production of autologous tumor vaccines would be facilitated by the development of a rapid and efficient method for the transfer of genes into freshly isolated cells. To evaluate the potential of replication defective herpes simplex viral (HSV) amplicon vectors as gene transfer vehicles for tumor vaccine generation, a vector that expresses the human interleukin-2 (IL-2) gene (HSV-IL2) and one that expresses Escherichia coli beta-galactosidase (HSVlac) were tested in hepatoma cells of both murine and human origin. Gene transfer into murine hepatoma cells (HEPA 1-6) was both rapid and highly efficient: greater than 50% of cells expressed beta-Gal when infected at a multiplicity of infection (m.o.i.) of 1 with an exposure period of 20 min. Moreover, gene transfer was as efficient in tumor cells after irradiation with 10,000 rads as in nonirradiated tumor cells. Irradiated HEPA 1-6 cells infected with HSV-IL2 for 20 min secreted IL-2 at a rate of 1,200 +/- 160 ng/10(6) cells per day. C57B1/6J mice immunized with irradiated, HSV-IL-2-transduced tumor cells produced in this way demonstrated specific tumor immunity by in vitro splenocyte tumoricidal activity and by in vivo protection against tumor challenge. Human hepatobiliary tumor specimens harvested at the time of operation, irradiated, and infected with HSV-IL-2 also produced nanogram quantities of IL-2/10(6) cells per 24 hr. These results indicate that the HSV amplicon vector is a good candidate vehicle for gene transfer in the production of autologous tumor vaccines. By allowing rapid gene transfer to freshly harvested tumor specimens, these vectors bypass the requirement for cell culture and make feasible reinfusion of genetically modified and irradiated autologous cells within hours of tumor harvest.

Animals↗
PubMed1996

Expression of the bcl-2 gene from a defective HSV-1 amplicon vector protects pancreatic beta-cells from apoptosis.

It has been suggested that the mechanism of pancreatic beta-cell death in autoimmune diabetes mellitus and in immunoisolated transplantation devices involves cytokine-induced apoptosis. To explore the feasibility of a gene transfer strategy to protect beta-cells, we evaluated the use of replication defective HSV-1 amplicon vectors as gene transfer vehicles. Post-mitotic murine and human beta-cells were efficiently transduced by a herpes simplex virus (HSV) vector that expresses the reporting gene Escherichia coli lacZ under the transcriptional control of a HSV promoter (HSVlac) both as islets and as single cells. Insulin secretion, a marker of beta-cell function, was unaffected by HSVlac transduction of a beta-cell line. A HSV amplicon vector that expressed bcl-2 (HSVbcl2) in beta-cells was constructed, and its effects on cytokine-mediated apoptosis in both a beta-cell line and primary murine beta-cells assessed by measuring internucleosomal fragmentation. beta-Cell apoptosis was blocked by transduction with HSVbcl2 but not HSVlac. The prevention of cytokine-induced apoptosis in beta-cells by bcl-2 expression has the potential both to ameliorate primary autoimmune beta-cell destruction as type I diabetes develops, and to prevent the destruction of transplanted beta-cells inside immunoisolation devices.

Animals↗
PubMed1996

BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells.

Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.

Animals↗
PubMed1996

The cellular and molecular mechanisms of diabetic complications.

In this article, the cellular and molecular mechanisms of diabetic complications have been reviewed. Hyperglycemia-induced mechanisms that may induce vascular dysfunction in specific sites of diabetic microvascular damage include increased polyol pathway flux, altered cellular redox state, increased formation of diacylglycerol and the subsequent activation of specific PKC isoforms, and accelerated nonenzymatic formation of advanced glycation endproducts. Several of these mechanisms may be responsible for the potentially damaging overproduction of reactive oxygen species observed with hyperglycemia. Each of these mechanisms may contribute to the known pathophysiologic features of diabetic complications by a number of mechanisms, including the upregulation of cytokines and growth factors. Diabetic macrovascular disease may arise more from insulin resistance than from hyperglycemia, and the authors speculate that this may reflect a selective loss of insulin-dependent vascular homeostasis.

Blood Glucose↗
PubMed1996

Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization.

Retinal neovascularization is a major cause of blindness in such disorders as retinopathy of prematurity, proliferative diabetic retinopathy and senile macular degeneration. Because ligation of vitronectin receptor-type integrins appears to be required for the survival and maturation of newly formed but not quiescent blood vessels in several vascular beds including the retina, blockade of this downstream adhesion receptor system was investigated. In a mouse model of hypoxia-induced retinal neovascularization twice daily administration of 1 to 20 mg cyclic alpha v-integrin antagonist peptide per kilogram of body weight reduced capillary proliferation in a dose-dependent fashion--maximum 76%--without obvious side effects. A cyclic control peptide displayed no inhibitory effect on neovascularization. These findings indicate that systemic application of vitronectin receptor antagonists appears to be clinically feasible and is efficient in preventing retinal neovascularization and superior to cytokine-blocking strategies.

Amino Acid Sequence↗
PubMed1996

Modification of vitronectin by advanced glycation alters functional properties in vitro and in the diabetic retina.

Early diabetic retinopathy is characterized by areas of acellular capillaries and the accumulation of advanced glycation end products (AGE). Endothelial cell maintenance, growth, and migration is regulated by a complex system involving the partitioning of growth factors between extracellular matrix-bound proteoglycans and cells, as well as by the activation and control of pericellular proteolysis. Using specific antibodies, we compared the immunolocalization of the vascular heparan sulfate proteoglycan (HSPG)-binding protein vitronectin (VN), HSPG, basic fibroblast growth factor, and collagen type 1 in retinae prepared from 11-month-old diabetic and nondiabetic Wistar rats. In normal rats, VN immunostaining was prominent in vascularized parts of the inner retina and colocalized with HSPG, which itself showed costaining with basic fibroblast growth factor in the extracellular matrix, vascular walls, and the inner limiting membrane. In contrast, the recognition of VN, HSPG, and basic fibroblast growth factor in diabetic retinae was greatly reduced in the inner limiting membrane and the extracellular matrix, where increased immunoreactive AGE colocalizing with VN were detectable. Capillary labeling of retinal vessel walls for plasminogen activator inhibitor-1 was moderate in both normal and diabetic retinae. AGE-VN was demonstrated in tissue extracts from retina by immunoprecipitation and Western blotting and presented a similar increase of AGE-related immunoreactivity compared with AGE-VN generated in vitro. AGE-VN in vitro had lost its native conformation, yielded high Mr SDS-resistant products, and was resistant to proteolysis because of modification of about 30% of lysines. Because binding of glycosaminoglycans, as well as interaction of type I collagen and the morphoregulatory proteins osteonectin and tenascin with AGE-VN, were reduced to at least 50% of control, alteration of basic residues in the heparin-binding domain of VN is plausible. In comparison to nonmodified VN, AGE-VN exhibited reduced cell attachment-promoting activity. Together, these in vitro results suggest that AGE-VN found in vivo is related to morphologic and functional changes in the diabetic retina and may contribute to the genesis of acellular capillaries in early diabetic retinopathy.

Animals↗
PubMed1995

Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes.

The adenovirus (Ad) early region 3 (E3) genes code for at least four proteins that inhibit the host immune responses mediated by cytotoxic T lymphocytes and tumor necrosis factor alpha. To evaluate the potential use of these immunoregulatory viral functions in facilitating allogeneic cell transplantation, the Ad E3 genes were expressed in pancreatic beta cells in transgenic mice under control of the rat insulin II promoter. Transgenic H-2b/d (C57BL/6 x BALB/c) islets, expressing the Ad E3 genes, remained viable for at least 94 days after transplantation under the kidney capsule of BALB/c (H-2d) recipients. Nontransgenic H-2b/d control islets were rejected as anticipated between 14 and 28 days. Histological analysis of the transplanted transgenic islets revealed normal architecture. Immunohistochemical studies with antisera to islet hormones revealed the presence of both beta and non-beta islet cells, suggesting a propagation of the immunosuppressive effect of Ad proteins from beta cells to other islet cells. The use of viral genes, which have evolved to regulate virus-host interactions, to immunosupress the anti-genicity of donor transplant tissue suggests additional ways for prolonging allograft survival. In addition, these findings have implications for designing Ad vectors for gene therapy.

Adenovirus E3 Proteins↗
PubMed1995

Aminoguanidine does not inhibit the initial phase of experimental diabetic retinopathy in rats.

We have previously shown that long-term administration of aminoguanidine, an inhibitor of advanced glycosylation product formation, reduces the extent of experimental diabetic retinopathy in the rat by 85%. In order to determine whether the residual retinopathy that developed despite aminoguanidine was attributable to advanced glycation endproduct formation, a time-course study was performed in three different groups of male Wistar rats: non-diabetic controls (NC), streptozotocin-diabetic controls (DC) and streptozotocin-diabetic rats treated with aminoguanidine HCL, 50 mg/100 ml drinking water (D-AG). Eyes were obtained at 24, 32, 44 and 56 weeks of diabetes/treatment duration and morphologic evaluation was done on retinal digest preparations. At 56 weeks, retinal basement membrane thickness was additionally measured. After 24 weeks of diabetes, the number of acellular capillaries was significantly elevated in DC (44.6 +/- 5.7/mm2 of retinal area, NC 19.6 +/- 4.9; p < 0.001) and increased continuously over time (DC 56 weeks 87.4 +/- 15.1; p < 0.001 vs DC24 weeks). In contrast, acellular capillaries in D-AG increased over the first 24 weeks and then remained constant for the rest of the study (D-AG 24 weeks 35.7 +/- 5.18; p < 0.01 vs NC 24 weeks and NS vs DC 24 weeks; D-AG 56 weeks 42.0 +/- 6.20; p NS vs D-AG 24 weeks).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Secondary intervention with aminoguanidine retards the progression of diabetic retinopathy in the rat model.

Primary prevention with aminoguanidine-an inhibitor of advanced glycation end product (AGE) formation--has been successfully employed to prevent diabetic retinopathy in the rat. However, it is unknown whether inhibition of AGE formation is still effective in a secondary intervention strategy. The present study addresses this question by comparing secondary intervention with aminoguanidine with syngeneic islet transplantation in the rat model. After 6 months of diabetes, one group was treated with aminoguanidine (50 mg/100 ml drinking water; D-AG) while another group received syngeneic transplantation of collagenase-ficoll isolated islets by intraportal injection (Tx). After an additional 4 months, both groups were compared to a normal (NC 10) and diabetic (DC 10) control group. Retinal autofluorescence was increased 2.5-fold after 6 months and increased 3.7-fold after 10 months of diabetes (p < 0.001). Aminoguanidine and islet Tx retarded the further accumulation of autofluorescence equally (p < 0.001 vs DC 10), although the values were higher than those observed in DC at 6 months (p < 0.001). Diabetes was associated with a 2.7-fold increase in acellular capillaries after 6 months and a 4.1-fold increase after 10 months. Treatment with aminoguanidine or islet Tx reduced but did not completely attenuate the progression of vascular occlusion (p < 0.001 vs DC 10; D-AG vs DC 6, p < 0.05; Tx vs DC 6, p < 0.01). Both treatments reduced endothelial proliferation (22.4% after 10 months; p < 0.001) and completely arrested pericyte dropout (40% after 10 months; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1995

Advanced protein glycosylation in diabetes and aging.

Products of advanced protein glycosylation (advanced glycation end products, or AGEs) accumulate in tissues as a function of time and sugar concentration. AGEs induce permanent abnormalities in extracellular matrix component function, stimulate cytokine and reactive oxygen species production through AGE-specific receptors, and modify intracellular proteins. Pharmacologic inhibition of AGE formation in long-term diabetic animals prevents diabetic retinopathy, nephropathy, neuropathy, and arterial abnormalities in animal models. Clinical trials in humans are currently in progress.

Aged↗
PubMed1995

Evaluation of 2-(3-aminobenzamido)-2-deoxy-D-glucose as a radiosensitizer.

PURPOSE: 3-Aminobenzamide (3-AB), an inhibitor of poly(adenosine diphosphate [ADP]-ribose) synthetase, functions as a radiosensitizer in several human tumor cell lines. 2-(3-AB)-2-deoxy-D-glucose (3-AB-G) was designed to increase preferentially the intracellular concentration of the drug in tumor cells. Both the toxicity and effectiveness of 3-AB-G as a radiosensitizer were determined. MATERIALS AND METHODS: The toxicity of 3-AB-G was measured in HeLa and Chinese hamster ovary cells. The radiosensitizing effect of 3-AB-G was determined for both cell lines. RESULTS: 3-AB-G was not toxic to cells at concentrations of 10 mmol/L or less. 3-AB-G did not alter cell survival after irradiation. CONCLUSION: 3-AB-G was not an effective radiosensitizer for the cells tested. Coupling 2-deoxyglucose to 3-AB may block the uptake of the inhibitor into the cell by altering the ability of the receptor to recognize the molecule or may interfere with the specificity of the inhibitor for poly(ADP-ribose) synthetase.

Animals↗