Electron microscopic analysis of glucose-induced endothelial damage in primary culture: possible mechanism and prevention.

We previously reported that high glucose treated cultured endothelial cells (ECs) showed intercellular gaps by transmission electron microscopy (TEM). These gaps were abrogated with insulin and/or heparin treatment. Our aims were to assess the severity of injury in ECs treated with high glucose for variable duration, and to further study the protective effects of insulin and/or heparin. Cells were also treated with L-buthionine sulfoximine (BSO), a glutathione inhibitor, to help understand the mechanism of high glucose injury. Primary porcine ECs were treated with high glucose (30 mM) for 2, 6 or 10 days; and glucose plus insulin (1 U/ml), glucose plus heparin (5 microg/ml), glucose plus insulin plus heparin for 6 days. ECs were treated with BSO (0.001-0.05 mM) for 2 days. Pellets from trypsinized cells were processed for TEM. High glucose treatment revealed apoptosis or necrosis showing variable cell size, abnormal nuclei, condensation of nuclear chromatin, few mitochondria, cell membrane disruption and needle-shaped structures. Changes increased with duration of exposure. In high glucose plus heparin or insulin treated cultures at least one-half of the cells appeared normal. Most ECs were intact when treated with high glucose plus insulin plus heparin. BSO treatment showed dose-dependent changes with low doses showing apoptosis whereas higher doses revealed necrosis similar to high glucose treatment for 6 or 10 days. High glucose-induced EC injury increased with duration of exposure. These data demonstrate that high glucose injury resembles that of BSO treatment, suggesting that glutathione depletion may be involved in EC injury. Insulin and/or heparin protect against high glucose-induced injury.

Marked variation in free radical injury between bovine and porcine endothelial cells cultured in different media.

Previous studies produced models of oxygen-derived free radical (OFR) injury, using H(2)O(2) or xanthine/xanthine oxidase (X/XO), in cultured porcine aortic endothelium (PAE) and rat coronary endothelium. H(2)O(2) at 0.1 mM resulted in 50% viability in both cell types. To determine if comparable H(2)O(2) or X/XO concentrations have the same injurious effect on endothelium from other sources, models of OFR injury were developed for bovine aortic endothelium (BAE) and bovine brain microvessel endothelium (BBME). Varying concentrations of H(2)O(2) (0.01 to 6 mM) or X/XO (10 microM/0.1 to 0.3 U/mL) were added to medium 24 h prior to evaluating cell damage. Injury was assessed using the Trypan blue exclusion test (% viability) and by measuring the release of lactate dehydrogenase into medium. H(2)O(2) concentrations required to produce 50% viability were >6 mM in BAE and BBME versus 1 mM in PAE when cells were grown in Dulbecco's modified Eagle's medium (DMEM). Similarly, BAE and BBME were less sensitive than PAE to damage by X/XO. Cells from both species were more sensitive to H(2)O(2) or X/XO injury when grown in Medium 199 (M199) versus DMEM. The most profound difference was observed with PAE where 50% viability was obtained with 0.12 versus 1.05 mM H(2)O(2) in M199 versus DMEM. These results indicate that bovine endothelial cells from aorta and brain are more resistant to free radical injury than PAE. The presence or absence of key media components (iron, pyruvate, cysteine, histidine) likely influences the extent of OFR injury.

Orally administered dextran sulfate is absorbed in HIV-positive individuals.

Preliminary in vivo studies suggested that oral dextran sulfate was poorly absorbed, but investigations were limited by inadequate methods for measuring the drug in the body. To determine absorption in HIV-positive subjects, hydrogenated dextran sulfate, average molecular weight 8000 (Usherdex 8), was orally administered in a short-term (single dose, 4 g/day for 5 days, 7 subjects) and in a long-term study (1 g, 4 times per day for 29 to 335 days, 8 subjects), which was a continuation of the short-term study with the inclusion of an additional subject. When an agarose gel electrophoresis technique with toluidine blue staining was used, the drug was recovered from plasma (67%, peak 2.2 microg/mL) and circulating peripheral blood lymphocyte (PBL) samples (50%, peak 333 microg/L blood) obtained at 5 and 15 minutes and 1, 3, 6, and 24 hours after the first day's dose and from plasma (56%) and PBL samples (38%) obtained 5 minutes after administration on 4 subsequent days in the short-term study. In the long-term study, the drug was found in plasma (67%, peak 2.4 microg/mL) and PBL samples (25%, peak 126 microg/L blood) obtained at monthly visits within 4 hours of the last dose. The drug was found in all urine samples from all subjects in both studies (short-term study, 24-hour samples up to 4 days after the final dose; long-term study, monthly samples within 4 hours of the last dose). In the long-term study, bone marrow preparations from 3 subjects showed metachromatic inclusions present in reticular cells when the cells were stained with toluidine blue, indicating the presence of sulfated polyanions. A significant rise in activated partial thromboplastin time and a drop in platelet count (P < .025) were demonstrated, with thrombocytopenia developing in 3 patients. Mild-to-moderate gastrointestinal disturbances were experienced by 6 subjects in the short-term study and by all subjects in the long-term study. One subject experienced mild central nervous system symptoms in the short-term study. These results indicate that dextran sulfate is absorbed after oral administration; therefore, further studies on its efficacy, particularly in the early stages of the disease, along with additional observations on its toxicity, are warranted.