Biomedical subjects

D Edelstein

Publications and source records attributed to D Edelstein.

At least 19 recordsLinked to original sources

PubMed2004

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells.

AIMS/HYPOTHESIS: Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. METHODS: Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. RESULTS: Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HG-cultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. CONCLUSIONS/INTERPRETATION: In diabetes, the expression and activity of eNOS is regulated through glucose-mediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1.

Animals↗

PubMed2001

Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A.

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. The plasma concentrations are correlated with body mass index, and are reported to be high in patients with insulin resistance, which is one of the major risk factors for cardiovascular disease. However, the direct effect of leptin on vascular wall cells is not fully understood. In this study, we investigated the effects of leptin on reactive oxygen species (ROS) generation and expression of monocyte chemoattractant protein-1 (MCP-1) in bovine aortic endothelial cells (BAEC). We found that leptin increases ROS generation in BAEC in a dose-dependent manner and that its effects are additive with those of glucose. Rotenone, thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), uncoupling protein-1 (UCP1) HVJ-liposomes, or manganese superoxide dismutase (MnSOD) HVJ-liposomes completely prevented the effect of leptin, suggesting that ROS arise from mitochondrial electron transport. Leptin increased fatty acid oxidation by stimulating the activity of carnitine palmitoyltransferase-1 (CPT-1) and inhibiting that of acetyl-CoA carboxylase (ACC), pace-setting enzymes for fatty acid oxidation and synthesis, respectively. Leptin-induced ROS generation, CPT-1 activation, ACC inhibition, and MCP-1 overproduction were found to be completely prevented by either genistein, a tyrosine kinase inhibitor, H-89, a protein kinase A (PKA) inhibitor, or tetradecylglycidate, a CPT-1 inhibitor. Leptin activated PKA, and the effects of leptin were inhibited by the cAMP antagonist Rp-cAMPS. These results suggest that leptin induces ROS generation by increasing fatty acid oxidation via PKA activation, which may play an important role in the progression of atherosclerosis in insulin-resistant obese diabetic patients.

Acetyl-CoA Carboxylase↗

PubMed2001

Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.

Endothelial nitric oxide synthase (eNOS) is activated by phosphorylation of serine 1177 by the protein kinase Akt/PKB. Since hyperglycemia-induced mitochondrial superoxide overproduction increases O-linked N-acetylglucosamine modification and decreases O-linked phosphorylation of the transcription factor Sp1, the effect of hyperglycemia and the hexosamine pathway on eNOS was evaluated. In bovine aortic endothelial cells, hyperglycemia inhibited eNOS activity 67%, and treatment with glucosamine had a similar effect. Hyperglycemia-associated inhibition of eNOS was accompanied by a twofold increase in O-linked N-acetylglucosamine modification of eNOS and a reciprocal decrease in O-linked serine phosphorylation at residue 1177. Both the inhibition of eNOS and the changes in its post-translational modifications were reversed by antisense inhibition of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme of the hexosamine pathway, or by blocking mitochondrial superoxide overproduction with uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD). Immunoblot analysis of cells expressing myc-tagged wild-type human eNOS confirmed the reciprocal increase in O-linked N-acetylglucosamine and decrease in O-linked serine 1177 phosphorylation in response to hyperglycemia. In contrast, when myc-tagged human eNOS carried a mutation at the Akt phosphorylation site (Ser1177), O-linked N-acetylglucosamine modification was unchanged by hyperglycemia and phospho-eNOS was undetectable. Similar changes in eNOS activity and covalent modification were found in aortae from diabetic animals. Chronic impairment of eNOS activity by this mechanism may partly explain the accelerated atherosclerosis of diabetes.

Acetylglucosamine↗

PubMed2001

Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction.

Alteration of platelet function contributes to microthrombus formation and may play an important role in the pathogenesis of diabetic micro- and macroangiopathies. However, the molecular mechanism for platelet dysfunction observed in patients with diabetes has not been fully elucidated. In this study, the direct effects of hyperglycemia on platelet function in vitro were investigated. Hyperglycemia increased reactive oxygen species generation in human platelets, and this effect was additive with that of collagen. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial electron transport chain complex II, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely prevented the effects of hyperglycemia, suggesting that reactive oxygen species arise from the mitochondrial electron transport chain. Hyperglycemia potentiated both platelet aggregation and the subsequent release of platelet-derived growth factor AB induced by a nonaggregating subthreshold concentration of collagen, which were also completely inhibited by TTFA or CCCP. Furthermore, hyperglycemia was found to inhibit protein tyrosine phosphatase (PTP) activity and increase phosphorylation of the tyrosine kinase Syk in platelets exposed to collagen. Hyperglycemia-induced PTP inhibition and Syk phosphorylation were found to be completely prevented by TTFA, CCCP, or Mn(III)tetrakis (4-benzoic acid) porphyrin, a stable cell-permeable superoxide dismutase mimetic. These results suggest that hyperglycemia-induced mitochondrial superoxide generation may play an important role in platelet dysfunction observed in patients with diabetes.

Blood Platelets↗

PubMed2000

Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation.

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Acetylglucosamine↗

PubMed2000

Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.

Diabetic hyperglycaemia causes a variety of pathological changes in small vessels, arteries and peripheral nerves. Vascular endothelial cells are an important target of hyperglycaemic damage, but the mechanisms underlying this damage are not fully understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C isoforms; increased formation of glucose-derived advanced glycation end-products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycaemia-induced abnormalities. Hyperglycaemia increases the production of reactive oxygen species inside cultured bovine aortic endothelial cells. Here we show that this increase in reactive oxygen species is prevented by an inhibitor of electron transport chain complex II, by an uncoupler of oxidative phosphorylation, by uncoupling protein-1 and by manganese superoxide dismutase. Normalizing levels of mitochondrial reactive oxygen species with each of these agents prevents glucose-induced activation of protein kinase C, formation of advanced glycation end-products, sorbitol accumulation and NFkappaB activation.

Animals↗

PubMed2000

The missing link: a single unifying mechanism for diabetic complications.

A causal relationship between chronic hyperglycemia and diabetic microvascular disease, long inferred from various animal and clinical studies, has now been definitely established by data from the Diabetes Control and Complications Trial (DCCT), a multicenter, randomized, prospective, controlled clinical study. A relationship between chronic hyperglycemia and diabetic macrovascular disease in patients with non-insulin-dependent diabetes mellitus (NIDDM) is also supported by the Kumamoto study. How does hyperglycemia induce the functional and morphologic changes that define diabetic complications? Vascular endothelial cells are a major target of hyperglycemic damage, but the mechanisms underlying this damage remain incompletely understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C (PKC) isoforms: increased formation of glucose-derived advanced glycation end products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these three pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycemia-induced abnormalities. Hyperglycemia increases reactive oxygen species (ROS) production inside cultured bovine aortic endothelial cells. In this paper, we show that ROS may activate aldose reductase, induce diacylglycerol, activate PKC, induce advanced glycation end product formation, and activate the pleiotropic transcription factor nuclear factor-kappa B (NF-kappaB). These data demonstrate that a single unifying mechanism of induction, increased production of ROS, serves as a causal link between elevated glucose and each of the three major pathways responsible for diabetic damage.

Animals↗

PubMed2000

Changes in diabetic retinal matrix protein mRNA levels in a common transgenic mouse strain.

Recently, all the structural features of non-proliferative diabetic retinopathy have been demonstrated in mice fed 30% galactose for 21-26 months. To determine whether changes in retinal matrix protein mRNA levels occur early in the course of murine diabetes we used a competitive RT-PCR method to quantitate retinal mRNA levels in an inbred mouse strain (FVB) commonly used for transgenic studies. Retinal mRNA was prepared from STZ-diabetic and non-diabetic FVB mice at 4, 8, 12 and 16 weeks and cDNA encoding basement membrane components was quantitated using MIMIC constructs that compete for the same primer pairs. alpha1 (IV) collagen, the beta1 and gamma1 chains of laminin, fibronectin, and vitronectin mRNAs were quantitated. For alpha1 (IV) collagen, statistically significant diabetes-induced increases were apparent by 8 weeks (3.11 +/- 0.20 vs. 1.29 +/- 0.19 x 10(6) molecules/mg total RNA, p < 0.005). Similarly, diabetes-induced increases were observed by 8 weeks for the beta1 chain of laminin (4.54 +/- 0.22 vs. 1.85 +/- 0.43 x 10(5) molecules/mg total RNA, p < 0.005), the gamma1 chain of laminin (7. 33 +/- 0.29 vs. 4.84 +/- 0.76 x 10(4)/microg total RNA, p < 0.05), and for fibronectin (2.22 +/- 0.21 vs. 1.35 +/- 0.15 x 10(6) molecules/mg total RNA, p < 0.05). The magnitude of change was greatest for alpha1 (IV) collagen (2.4-fold) and beta1 laminin (2. 5-fold) at 8 weeks, and least for fibronectin (1.6-fold). A smaller diabetes-induced increase in vitro nectin mRNA was also observed, but it failed to reach statistical significance at 12 and 16 weeks. These data provide the basis for assessing the effects of genetic manipulation on diabetic retinopathy in transgenic mouse models.

Animals↗

PubMed1999

Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck.

The purpose of this study was to determine whether cyclooxygenase-2 (COX-2) was overexpressed in squamous cell carcinoma of the head and neck (HNSCC). Quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry were used to assess the expression of COX-2 in head and neck tissue. Mean levels of COX-2 mRNA were increased by nearly 150-fold in HNSCC (n = 24) compared with normal oral mucosa from healthy volunteers (n = 17). Additionally, there was about a 50-fold increase in amounts of COX-2 mRNA in normal-appearing epithelium adjacent to HNSCC (n = 10) compared with normal oral mucosa from healthy volunteers. Immunoblotting demonstrated that COX-2 protein was present in six of six cases of HNSCC but was undetectable in normal oral mucosa from healthy subjects. Immunohistochemical analysis showed that COX-2 was expressed in both HNSCC and adjacent normal-appearing epithelium. Taken together, these results suggest that COX-2 may be a target for the prevention or treatment of HNSCC.

Carcinoma, Squamous Cell↗

PubMed1999

In vivo interleukin-2 gene therapy of established tumors with herpes simplex amplicon vectors.

In vivo cytokine gene transfer may greatly simplify autologous tumor vaccine production. Herpes simplex viral amplicon vectors (HSV) are efficient gene-transfer vehicles and may overcome many limitations of prior gene-transfer methods. The interleukin-2 (IL-2) and beta-galactosidase genes (lac) were inserted into an HSV amplicon vector and tested in a subcutaneous squamous cell carcinoma of lung origin to determine the efficiency of in vivo gene transfer and the utility of such a direct gene transfer approach in cancer therapy. Gene transfer and expression were assessed by histochemical staining and enzyme-linked immunosorbent assay (ELISA). Growth of injected tumors as well as non-injected tumors remote from the site of injection was assessed. Assessment of lymphocytic infiltrates into tumors was performed by immunohistochemistry. Survival was recorded. Direct in vivo injection of established tumors with a HSVi12 resulted in efficient gene transfer and production of IL-2 in the injected tumor but not at tumors remote from the sites of injection. There was a significant suppression of growth of the tumors injected with HSVi12 (P<0.01) when compared with tumors injected with HSV without i12. Of note, growth of tumors remote from sites of HSVi12 injection was also retarded and treatment was associated with a significant (P<0.05) improvement in survival. Direct intratumoral administration of HSV amplicon vectors can result in efficient transfer of cytokine genes and have antitumor efficacy. HSV vectors are therefore potentially useful agents in such in vivo gene-therapy strategies and simplify cytokine antitumor gene-therapy strategies.

Animals↗

PubMed1999

Inhibition of cyclooxygenase-2 expression. An approach to preventing head and neck cancer.

Cyclooxygenase (COX) catalyzes the formation of prostaglandins (PG) from arachidonic acid. A large body of evidence has accumulated to suggest that COX-2, the inducible form of COX, is important in carcinogenesis. In this study, we determined whether (1) COX-2 was overexpressed in squamous cell carcinoma of the head and neck (HNSCC) and whether (2) retinoids, a class of chemopreventive agents, blocked epidermal growth factor (EGF)-mediated activation of COX-2 expression. Levels of COX-2 mRNA were determined in 15 cases of HNSCC and 10 cases of normal oral mucosa. Nearly a 100-fold increase in amounts of COX-2 mRNA was detected in HNSCC. By immunoblot analysis, COX-2 protein was detected in 6 of 6 cases of HNSCC but was undetectable in normal mucosa. Because retinoids protect against oral cavity cancer, we investigated whether retinoids could suppress EGF-mediated induction of COX-2 in cultured oral squamous carcinoma cells. Treatment with EGF led to increased levels of COX-2 mRNA, COX-2 protein, and synthesis of PG. These effects were suppressed by a variety of retinoids. Based on the results of this study, it will be important to establish whether newly developed selective COX-2 inhibitors are useful in preventing or treating HNSCC. Moreover, the anticancer properties of retinoids may be due, in part, to inhibition of COX-2 expression. Combining a retinoid with a selective COX-2 inhibitor may be more effective than either agent alone in preventing cancer of the upper aerodigestive tract.

Carcinoma, Squamous Cell↗

PubMed1999

Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction.

Apoptosis has been identified as a mechanism of pancreatic islet beta-cell death in autoimmune diabetes. Proinflammatory cytokines are candidate mediators of beta-cell death in autoimmune diabetes, and these cytokines can induce beta-cell death by apoptosis. In the present study, we examined whether transfection of human islet beta-cells with an anti-apoptotic gene, bcl-2, can prevent cytokine-induced beta-cell destruction. Human islet beta-cells were transfected by a replication-defective herpes simplex virus (HSV) amplicon vector that expressed the bcl-2 gene (HSVbcl-2) and, as a control, the same HSV vector that expressed a beta-galactosidase reporter gene (HSVlac). Two-color immunohistochemical staining revealed that 95+/-3% of beta-cells transfected with HSVbcl-2 expressed Bcl-2 protein compared with 14+/-3% of beta-cells transfected with HSVlac and 19+/-4% of nontransfected beta-cells. The bcl-2-transfected beta-cells were fully protected from impaired insulin secretion and destruction resulting from incubation for 5 days with the cytokine combination of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. In addition, the bcl-2-transfected islet cells were significantly protected from cytokine-induced lipid peroxidation and DNA fragmentation. These results demonstrate that cytokine-induced beta-cell dysfunction and death involve mechanisms subject to regulation by an anti-apoptotic protein, Bcl-2. Therefore, bcl-2 gene therapy has the potential to protect human beta-cells in pancreatic islets, or islet grafts, from immune-mediated damage in type 1 diabetes.

Apoptosis↗

PubMed1998

Green tea regulates cell cycle progression in oral leukoplakia.

BACKGROUND: A complete in vitro multi-stage carcinogenesis model for oral cancer was developed to examine chemopreventive strategies. In the present study, the effects of EGCG [(-)-epigallocatechin-3-gallate], the major constituent of green tea, is being examined to understand mechanisms of action. METHODS: Effects of EGCG on the cell populations were examined with growth assays, cell cycle analysis, and western blots for retinoblastoma protein (pRB). RESULTS: In each cell type, EGCG inhibited growth, with a decrease in efficacy as cells progressed from normal to cancer. A G1 block was induced with an increase in the underphosphorylated form of pRB; EGCG-induced inhibition was not permanent, cells recovered, and no resistance developed. CONCLUSIONS: Our multistage carcinogenesis model for chemoprevention was effective in defining the chemopreventive value of EGCG. The observation that cancerous oral epithelium was less responsive than normal or dysplastic tissues has implication in the use of this agent, and the mechanisms responsible for this result remain to be defined.

Anticarcinogenic Agents↗

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

Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.

An in vitro model for oral cancer was used to examine the growth inhibitory effects of chemopreventive agents when used singly and in combination. The model consists of primary cultures of normal oral epithelial cells, newly established cell lines derived from dysplastic leukoplakia and squamous cell carcinoma. Two naturally occurring substances, (-)-epigallocatechin-3-gallate (EGCG) from green tea and curcumin from the spice turmeric were tested. Cells were treated singly and in combination and effects on growth determined in 5-day growth assays and by cell cycle analysis. Effective dose 50s and the combination index were calculated with the computerized Chou-Talalay method which is based on the median-effect principle. Agents were shown to differ in their inhibitory potency. EGCG was less effective with cell progression; the cancer cells were more resistant than normal or dysplastic cells. In contrast, curcumin was equally effective regardless of the cell type tested. Cell cycle analysis indicated that EGCG blocked cells in G1, whereas curcumin blocked cells in S/G2M. The combination of both agents showed synergistic interactions in growth inhibition and increased sigmoidicity (steepness) of the dose-effect curves, a response that was dose and cell type dependent. Combinations allowed for a dose reduction of 4.4-8.5-fold for EGCG and 2.2-2.8-fold for curcumin at ED50s as indicated by the dose reduction index (DRI). Even greater DRI values were observed above ED50 levels. Our results demonstrate that this model which includes normal, premalignant and malignant oral cells can be used to analyse the relative potential of various chemopreventive agents. Two such naturally-occurring agents, EGCG and curcumin, were noted to inhibit growth by different mechanisms, a factor which may account for their demonstrable interactive synergistic effect.

Anticarcinogenic Agents↗

PubMed1997

Phalangeal bone density and hip fracture risk.

OBJECTIVE: To assess the long-term predictive usefulness of radiographic absorptiometry measurements of phalangeal bone density for hip fracture risk. METHODS: Participants were members of the First National Health and Nutrition Examination Survey Epidemiologic Follow Up Study cohort. Subjects were followed up for a maximum of 16 years. The First National Health and Nutrition Examination Survey data were obtained from a nationally representative sample of non-institutionalized civilians. A cohort of 3481 white and black subjects (1559 white women) aged 45 through 74 years at baseline (1971-1975) were observed through 1987. Ninety-eight percent of the original cohort completed the study. Hospital records and death certificates were used to identify a total of 72 hip fracture cases. Phalangeal bone density at baseline was measured using photodensitometry (PD), and later reanalyzed by radiographic absorptiometry (RA), a newer, more sophisticated technique. RESULTS: Results were evaluated to determine the relative risk for hip fracture per 1-SD decrease in bone density, after controlling for age at baseline, race, gender, weight, and previous fractures. Both RA and PD measurements showed a significant inverse relationship to hip fracture risk, with RA density measurements showing a slightly higher adjusted relative risk per 1-SD density decrease than PD measurements. For RA bone density, the relative risk for all subjects was 1.81 (95% confidence interval, 1.34-2.44) compared with 1.57 (95% confidence interval, 1.19-2.07) for PD bone density after adjusting for age at baseline, race, gender, weight, and previous fractures. Results for white women were essentially the same as those for all subjects for RA bone density and PD bone density. CONCLUSIONS: Phalangeal bone density determined from standard hand x-ray films is a significant predictor of future hip fracture risk. Availability of a valid method to assess fracture risk using conventional radiographs will expand the ability to identify individuals with osteoporosis.

Absorptiometry, Photon↗

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↗