S. Murota

Increased arachidonic acid content in platelet phospholipids from diabetic patients

Platelet abnormalities have been suggested to be linked with vascular diseases. Diabetes mellitus has a high incidence of vascular complications. In the present study gas-liquid chromatography analyses were carried out to investigate the fatty acid composition in platelet phospholipids of Type-2 (non-insulin-dependent) diabetic patients. Fatty acid composition in platelet phospholipids was different between diabetics and age-matched control subjects. Arachidonic acid increased significantly (p less than 0.01), and both eicosatrienoic acid and eicosapentaenoic acid decreased in diabetes mellitus. A good correlation was found between fatty acid content of platelet phospholipids and that of plasma total lipids in the fatty acids examined, except for arachidonic acid. The levels of arachidonic acid in diabetic platelet phospholipids was disproportionately high to these in plasma total lipids. These results suggest that the system for arachidonic acid incorporation into platelet phospholipids is specifically accelerated in the diabetic metabolic state.

Effects of glucose on migration, proliferation and tube formation by vascular endothelial cells

SummaryIn order to elucidate the association between hyperglycemia and the vascular complications of diabetes, the effects of high glucose concentrations on the migration, proliferation and tube formation of bovine carotid artery endothelial cells were investigated. Cells treated with 16.7 and 33.3 mM glucose for 6 days showed 1.69- and 1.75-fold increase in serum-induced migration compared with cells treated with 5.6 mM glucose (p<0.05). The effect of glucose on cell proliferation was affected by serum concentration. When this was below 0.5%, a high glucose concentration stimulated cell growth to a maximum of 1.73 times that at a serum concentration of 0.05% (p<0.01) whereas at a serum concentration of 10%, growth was inhibited (p<0.05). Tube formation was studied by culturing the cells between two layers of collagen gel. Ultrastructurally, tubular structures were composed of one to several endothelial cells containing pinocytotic vesicles and cytoplasmic projections, and linked by junctional complexes. A basal lamina-like structure surrounded the abluminal surface. Treatment of the cells with 16.7 and 27.8 mM glucose for 4 days stimulated tubular elongation 1.85 and 1.71 times, respectively (p<0.01). Other osmogenic molecules such as mannitol and sucrose did not affect tube formation. These data imply that high glucose concentrations mimicking diabetic hyperglycemia may not inhibit the repair of endothelial injury and could act as a stimulator of neovascularization.

Platelet thromboxane synthesizing activity in non-insulin-dependent diabetes: Correlation with diabetic retinopathy and diabetic treatment

Since elevated thromboxane A2 synthesis could be expected to lead to microvascular injury in diabetic patients, it is important to determine the relationship between thromboxane A2 synthesis and diabetic microvascular complications. In the present study, the activity of thromboxane A2 synthesis in platelets was assessed in 57 Type 2 (non-insulin-dependent) diabetic patients by measuring the conversion of exogenously added 14C-arachidonic acid into 14C-thromboxane B2. The activity of thromboxane synthesis was significantly higher in diabetic patients than in 16 age-matched control subjects (2.61 +/- 0.07 vs 1.95 +/- 0.12 microgram . 10(9) platelets-1 . 3 min-1, respectively, p less than 0.001). When the diabetic patients were divided into three groups (nil, background, and proliferative retinopathy) on the basis of severity of diabetic retinopathy, there was a trend to increased thromboxane synthesizing activity as diabetic retinopathy develops. However, no statistically significant changes were demonstrated among these three groups. In the diabetic patients, a positive correlation was found between thromboxane synthesizing activities and glycosylated hemoglobin values (biochemical indicator of long-term glycemic control), but not between the activities and plasma glucose levels. To ascertain the effect of desirable diabetic treatment on thromboxane synthesizing activity in platelets, 9 newly diagnosed patients and 6 poorly controlled patients were examined. During treatment, all patients had declining plasma glucose and glycosylated hemoglobin levels. Thromboxane synthesizing activities in 6 patients treated by insulin decreased significantly during treatment (3.16 +/- 0.17 to 2.49 +/- 0.22 microgram . 10(9) platelets-1 .3 min-1, p less than 0.01), but 9 diabetic patients treated by diet or sulphonylureas still maintained their elevated thromboxane synthesizing activity levels (2.76 +/- 0.24 to 2.66 +/- 0.25 microgram . 10(9) platelets-1 .3 min-1,NS). These results suggest that the increased thromboxane synthesizing enzyme system is involved in the abnormal platelet function in diabetes independently of diabetic retinopathy, and that appropriate metabolic control using insulin might be successful in lowering the increased thromboxane synthesizing activity.

Regulation of platelet aggregation and arachidonate metabolism in streptozotocin-diabetic rats.

Thromboxane A2 generation during collagen-induced platelet aggregation was increased in platelets from streptozotocin-induced diabetic rats when washed platelets were resuspended in pooled-control rat plasma. 14C-Arachidonate incorporation into platelets and oxygenation into thromboxane B2 in these rats were significantly higher than in the control rats, whereas fatty acid analysis revealed markedly decreased arachidonate composition in platelet phospholipids. Meanwhile plasma isolated from diabetic rats inhibited collagen-induced platelet aggregation in a volume-dependent fashion. These results suggest that enhanced platelet thromboxane synthesis in streptozotocin-diabetic rats is accompanied by increased arachidonate metabolism in platelets, which may serve to aid efficient utilization of a decreased thromboxane A2 precursor pool. Plasma factor(s) in these rats may modify the platelet reactivity in vivo.

Hypoxia-reoxygenation inhibits gap junctional communication in cultured human umbilical vein endothelial cells.

We studied the change in gap junctional intercellular communication (GJIC) on human umbilical vein endothelial cells (HUVEC) under hypoxia-reoxygenation (H-R) conditions by the fluorescence redistribution after photobleaching (FRAP) method. Confluent HUVEC monolayers were exposed to hypoxia (pO2<0.1%) for 12 hours, and then were returned to normal atmospheric conditions for reoxygenation. Contrast microscopic observation showed no significant changes in the morphology of the HUVEC at any times after H-R. Reoxygenation following hypoxia caused time-dependent decrease in GJIC, that is, GJIC reduction was induced after 2 hours and reached maximum at 4-6 hours which recovered to normal levels after 18 hours. Oxidant sensitive fluorescence dye assay revealed that the generation of intracellular free radicals increased during the first 2 hours after reoxygenation. Hydroxyl radical scavengers (MCI-186, DMSO) and an iron chelator (deferoxamine) abolished the reduction of GJIC due to H-R. However, SOD, catalase and probucol were essentially inactive on this reduction. These data suggest that ischemia-reperfusion injury may be caused by a functional defect of GJIC induced by reactive oxygen radicals.

A proposed method for exploring anti-aggregatory effects of eicosapentaenoic acid in the rat

Eicosapentaenoic acid (EPA) has been reported to be anti-aggregatory in humans, but it is difficult to confirm that anti-aggregatory effect in rat. To induce an anti-aggregatory effect of EPA even in rats, an attempt was made to modify fatty acid composition of the diet, i.e., a diet low in linoleic acid, which is a precursor of arachidonic acid (AA) in vivo. A group of rats eating EPA with butter, which contained low linoleic acid, as a lipid source for 12 consecutive days revealed significant inhibition of platelet aggregation induced by collagen. The inhibition was complete at 3.0 micrograms of collagen, a dose which caused complete aggregation in rats which received EPA with corn oil. In these experiments, the ratio of EPA to AA in platelet phospholipids was 0.038 for corn oil, 0.306 for corn oil plus EPA, 0.050 for butter and 0.703 for butter plus EPA. These results indicated that if one blocks the in vivo supply of AA, it causes an increase in EPA to AA ratio in platelet phospholipids, and consequently leads to an inhibition of platelet aggregation even in the rat.

Augmentation of 12-lipoxygenase activity of lymph node and spleen T cells in autoimmune mice MRL/l

To investigate the relationship between development of autoimmune disease and arachidonic acid metabolism, we compared lipoxygenase and cyclooxygenase activities of lymphoid cells in MRL/1 mice during the development of the disease. Lipoxygenase activity per cell of lymph mo mice, although cyclooxygenase activity was low in both cases. These phenomena did not depend on aging since age-matched MRL/n mice lacking the lpr gene (congenic with MRL/1 mice), and BALB/c mice did not exhibit any change in the enzyme activity with aging. The lipoxygenase activity of 4 mo MRL/1 lymph node cells came mainly from nylon wool nonadherent cells. Lipoxygenase activity of nylon wool nonadherent spleen cells in MRL/1 mice was also higher in 4 mo mice than that in 1.5 mo mice, whereas we could not detect any difference in nylon wool adherent cells. The major lipoxygenase product was 12-hydroxyeicosatetraenoic acid (12-HETE), which was identified by a high performance liquid chromatography (HPLC). Our data suggest that change in arachidonic acid metabolism in T cells may be related to development of autoimmune disease in MRL/1.

Contribution of lipoxygenase metabolites to IL-2 production in the early phase of lymphocyte activation

Lipoxygenase inhibitors; eicosatetraynoic acid (ETYA), esculetin and caffeic acid, inhibited interleukin-2 (IL-2) production by Con A (concanavalin A)-stimulated murine spleen lymphocytes. They effectively inhibited IL-2 production without affecting cell viability when they were added to the cultures within around 6 hr after Con A stimulation. IL-1 production was also inhibited effectively when the inhibitors were added within 4 hr after stimulation. Therefore, immunosuppressive characteristics of these inhibitors can in part be explained by their inhibitory effects on production of lymphokines involved in the early phase of lymphocyte activation.

Dietary arachidonic acid supplementation increases thromboxane synthesizing activity in platelets from diabetics

Thromboxane A2 has been implicated in thrombotic vascular diseases. The diabetic population has a high incidence of vascular complications. In the present study, activity of thromboxane synthetase in platelets of type 2 (non-insulin dependent) diabetic patients was studied. Thromboxane synthesizing activity in diabetic platelets was significantly higher than that in controls (p less than 0.05). An effect of dietary arachidonic acid on thromboxane synthesizing activity in platelets was also studied. Dietary arachidonic acid supplementation containing 1.7g of arachidonic acid per day for 1 week caused a significant increase of arachidonic acid content in plasma total lipids (p less than 0.05), but the increase of arachidonic acid in plasma total lipids did not correlate with the level of arachidonic acid in platelet phospholipids. Thromboxane synthesizing activity in platelets from the diabetics and the controls was augmented by a diet enriched in arachidonic acid from 2.20 +/- 0.15 to 2.56 +/- 0.20 and from 1.71 +/- 0.23 to 2.01 +/- 0.34, respectively. The increase in diabetic subjects was statistically significant (p less than 0.05). These results suggest that fatty acids of dietary origin are important factors in development of thrombotic vascular diseases.

Effect of dietary protein level on platelet aggregation in rat

In order to investigate the effects of dietary protein level on the development of atherosclerosis and arterial thrombosis, studies were made on platelet function, plasma lipids and aortic prostaglandin I2 biosynthesis in rats. Under the same calorie intake, the group consuming a low level of protein (10% casein) increased plasma triglycerides and platelet aggregability in comparison with control (20% casein) and high protein groups (60% casein). Moreover, the low protein group produced more thromboxane A2 and less prostaglandin I2 compared with the high protein one. These data suggest that high protein diets may have beneficial effects on arterial thrombosis.