Glucose inhibits replication of cultured human endothelial cells

Diabetologia, Nov 1982

Summary Diabetes is an important risk factor for atherosclerosis but the mechanism of the risk is unknown. As endothelial injury is considered to be an early event in the development of atherosclerosis, the effect of glucose on endothelial cell replication was studied. Concentrations of glucose of 11.2, 16.8 and 22.4 mmol/l inhibited DNA synthesis in cultured human umbilical venous endothelial cells by 8.1±10.8, 24.3±8.8 and 30.9±7.4%, respectively. Glucose also inhibited the proliferative response of endothelial cells to experimental wounds in the cell layer. Sorbitol (22.4 mmol/l) inhibited endothelial cell DNA synthesis by 50±13.6%, but mannitol (22.4 mmol/l) inhibited DNA synthesis by only 3±24.3%. It is suggested that in diabetic subjects, high blood glucose levels may cause endothelial injury, or inhibit its repair, and hence allow the exposure of the arterial media to plasma and its constituents.

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Glucose inhibits replication of cultured human endothelial cells

Glucose Inhibits Replication o f Cultured Human Endothelial Cells R. W. Stout 0 0 Department of Geriatric Medicine, The Queen's University of Belfast , Belfast, Northern Ireland , UK Summary. Diabetes is an important risk factor for atherosclerosis but the mechanism of the risk is unknown. As endothelial injury is considered to be an early event in the development of atherosclerosis, the effect of glucose on endothelial cell replication was studied. Concentrations of glucose of 11.2, 16.8 and 22.4 retool/1 inhibited D N A synthesis in cultured human umbilical venous endothelial cells by 8.1 ___10.8, 24.3 _+8.8 and 30.9 ___7.4%, respectively. Glucose also inhibited the proliferative response of endothelial cells to experimental wounds in the cell layer. Sorbitol (22.4 mmol/1) inhibited en- Glucose; endothelial cells; DNA; cell culture; diabetes mellitus; atherosclerosis - 9 Springer-Verlag 1982 A l t h o u g h atherosclerosis is m o r e c o m m o n in diabetic patients t h a n in n o r m a l subjects, the exact relationship b e t w e e n the m e t a b o l i c a b n o r m a l i t i e s o f diabetes a n d the pathogenesis o f the v a s c u l a r disease r e m a i n s unclear [ 1 ]. Risk factors for atherosclerosis in the general p o p u l a t i o n , including h y p e r t e n s i o n a n d a b n o r m a l s e r u m lipids, are related to atherosclerosis in diabetic patients in the s a m e w a y as in n o r m a l subjects, b u t do not a p p e a r to explain entirely the excess risk o f atherosclerosis in diabetes. The d e v e l o p m e n t o f large vessel disease in diabetic patients does not a p p e a r to b e p r e v e n t e d b y available m e t h o d s o f treating diabetes [ 1 ]. E p i d e m i o l o g i c a l studies suggest that there is a f a c t o r or factors u n i q u e to diabetes which is responsible for the i n c r e a s e d incidence o f large vessel disease [ 2 ]. T w o cells o f the arterial wall are closely associated with the d e v e l o p m e n t o f atherosclerosis [ 3 ]. Endothelial cells f o r m the lining o f the inner p a r t o f the artery a n d are c o n s i d e r e d to act as a barrier, p r e v e n t i n g the entry o f p l a s m a constituents into the inner p a r t o f the arterial wall. S m o o t h muscle cells c o m p r i s e the m e d i a o f the artery, a n d are also f o u n d in small n u m b e r s in the n o r m a l intima. A current t h e o r y suggests that the initial c h a n g e in atherosclerosis is the d e v e l o p m e n t o f an injury or alt e r a t i o n to the endothelial b a r r i e r [ 4 ]. This allows plasm a to enter the inner intima a n d m e d i a where s m o o t h m u s c l e cells are e x p o s e d to substances including a facdothelial cell DNA synthesis by 50+13.6%, but mannitol (22.4 mmol/1) inhibited D N A synthesis by only 3 + 24.3%. It is suggested that in diabetic subjects, high blood glucose levels may cause endothelial injury, or inhibit its repair, and hence allow the exposure of the arterial media to plasma and its constituents. tor f r o m platelets [ 5 ] a n d insulin [ 6, 7 ] which stimulate their proliferation. I f e x p o s u r e is p r o l o n g e d a n atherosclerotic lesion with lipid, connective tissue a n d t h r o m bosis develops. M u c h attention has b e e n p a i d to factors which stimulate arterial s m o o t h muscle cell proliferation [ 5-8 ]. O f equal i m p o r t a n c e are factors which inhibit endothelial cell proliferation a n d h e n c e retard the healing o f injury to the endothelium. The m a j o r m e t a b o l i c a b n o r m a l i t y in diabetes is a high p l a s m a glucose, a n d therefore the effect o f glucose o n h u m a n endothelial cell replication was studied. Methods Human endothelial cells were cultured from umbilical vein by a modification of the methods of Jaffe et al. [ 9 ] and Gimbrone et al. [ 10 ]as previously described [t 1]. Briefly,umbilical cord was obtained within 24 h of delivery. The umbilical vein was identified, cannulated and, after being flushed free of blood with phosphate-buffered saline (PBS) infused with a solution of collagenase (0.025% in PBS). Ten minutes later the collagenase solution was flushed from the vein together with the freed endothelial cells. The cells were concentrated by centrifugation and were then placed in plastic tissue culture flasks (Becton Dickinson, Wembley, Middlesex, UK) in medium 199 (Gibco-Biocult, Paisley, Scotland, UK) supplemented by 20% pooled human serum (obtained from the Northern Ireland Blood Transfusion Service). Cell growth was seen within 6 h. Endothelial cells could 11.2mmol/i 16.8mmol/I 22.4mmo1/I GLUCOSE z RI z L o,,~' -50 <{ Z z_ Ill Z t~ >t~ " 0 8 g z ~ o 0 identified by their immunological and ultrastructural characteristics as described previously [111. For the experiments, endothelial cells derived from three different umbilical cords were passed into twelve 30 mm Petri dishes (Falcon) and grown to sub-confluency. This was the first subcultivation of these cells. The medium was then changed, the six control dishes continuing in standard medium with 20% pooled human serum and glucose (5.6 mmol/1) and the other six dishes containing medium, serum and glucose concentrations (11.2, 16.8 or 22.4 mmol/1), which are representative of those found in diabetic patients under varying degrees of control. Each experiment tested one concentration of glucose against the control medium. After exposure to the test media for 24 h, 1 ktCi of 3H-thymidine (Radiochemical Centre, Amersham, UK) was added to each dish. Two hours later the medium was removed, the cells were freed from the dishes by trypsinization, D N A was isolated and radioactivity estimated as described previously [111. The results were expressed in terms of cell protein measured by the method of Lowry et al. [ 12 ]. For each experiment the mean of the six dishes exposed to the high glucose concentration was expressed as a percentage change of the mean of the control dishes. For each concentration of glucose the experiment was repeated on seven occasions. To test the effect of glucose on the proliferation of endothelial cells in response to experimental wounds, 30 mm Petri dishes containing almost confluent endothelial cells were scraped in two parallel lines with a 5 mm wide stainless steel spatula. The scraped cells were washed off and the medium changed to standard medium (six dishes) and medium containing glucose (22.4 mmol/1) (six dishes). After 24 h, 48 h or 7 days 1 gCi of 3H-thymidine was added for 2 h and the cells processed as before. In another series of experiments, sorbitol (22.4 mmol/l) or mannitol (22.4 mmol/l) instead of glucose was added to the medium. Statistical analysis was performed as follows. The significance of the seven replicate experiments was tested by paired t-test. As the results may not have been normally distributed, significance was also tested by the two-tailed binomial test and the Wilcoxon signed rank test. The significance levels were the same using all three tests and only the paired t-test results are reported here. A least-squares linear correlation between the change in the D N A synthesis and the increment in glucose concentration was calculated. Results The results illustrate a progressive depression o f D N A synthesis in h u m a n endothelial cells exposed to increasing concentrations o f glucose (Fig. 1). With glucose (1!..2 retool/l) the m e a n change was - 8 . 1 _+ 10.8% (mean _+ S E M ; p >0.20); with glucose (16.8 mmol/1) -24.3_+8.8% (p <0.025); and with glucose (22.4 retool/l) - 3 0 . 9 _+ 7.4% (p < 0.005). W h e n the m e a n percentage change in D N A synthesis was related to the increment concentration, there was a highly significant linear correlation (r --= - 0 . 9 9 ; p < 0.01). In cell layers that h a d been subjected to experimental w o u n d s , glucose (22.4 mmol/1) h a d a similar inhibitory effect o n D N A synthesis. This varied f r o m a change o f - 2 9 % (mean o f three experiments) 24 h after w o u n d i n g to - 54% (mean of two experiments) 7 days after wounding. When cells were exposed to sorbitol (22.4 mmol/1), the mean inhibition of D N A synthesis in four experiments was - 5 0 + 13.6% (p < 0.025; Fig. 2). Sorbitol also inhibited D N A synthesis (mean change - 68% at 24 h after wounding) in endothelial cell layers subjected to experimental wounds. However exposure o f the cells to mannitol (22.4 mmol/1) in five experiments resulted in a mean inhibition of D N A synthesis of - 3.0 _+24.3% (p > 0.45). Discussion These experiments show that glucose in increasing concentrations causes inhibition of incorporation of labelled thymidine into D N A in cultured h u m a n endothelial cells. This occurred both in subconfluent cell layers and in cell layers that had been subjected to experimental wounds. It has been suggested that the latter experimental model is more relevant to endothelial repair in vivo [ 13 ]. Although cell counts were not performed, other workers have found that the incorporation of thymidine into D N A reflects the number of cells when proliferation of endothelial cells has been studied [ 13 ]. These findings contrast with the effect of glucose on other cultured cells: glucose has no effect on smooth muscle cell proliferation but stimulates fibroblast proliferation [ 14 ]. The mechanism of the inhibition of endothelial cell proliferation by glucose may be related to its metabolic effects, to the osmotic effects of high glucose concentrations, or to cyclic AMP, a potent inhibitor of endothelial cell proliferation [ 15 ]. As reported with fibroblasts and smooth muscle cells [ 14 ] sorbitol, a nonutilizable corbohydrate, had similar effects to glucose but mannitol had no effect. However, endothelial cells do not have the capacity to convert glucose to sorbitol [ 16 ] so it is likely that extracellular effects of both molecules are involved in the inhibition of cell replication. However, the lack of effect of mannitol on endothelial cell proliferation argues against h3,perosmolarity being the explanation for the effect o f glucose. Detailed studies of the osmolarity of the culture medium are needed to confirm or deny hyperosmolarity as a mechanism for glucose's effect on endothelial cell proliferation. These results, if they apply to endothelial cells in vivo, suggest that high plasma glucose concentrations could inhibit endothelial cell replication. Glucose may, therefore, cause endothelial injury, perhaps by inhibiting the normal turnover of endothelial cells or, if an injury is caused by other means, the presence of a high glucose concentration may inhibit the repair of the endothelium. A recent study of alloxan-diabetic rabbits has shown aortic endothelial damage in hyperglycaemic animals [ 17 ]. A high glucose concentration could therefore contribute to the development of atherosclerosis by increasing the exposure of the subintimal tissues to plasma. Plasma constituents, including the platelet derived growth factor and insulin would then be able to stimulate the proliferation of medial smooth muscle cells and lipoproteins could be deposited in smooth muscle cells or macrophages. This hypothesis would be consistent with epidemiological evidence on the relationship of large vessel disease to diabetes [ 1 ]. Although diabetic patients are more prone to atherosclerosis than normal subjects, the relationship between blood glucose and atherosclerosis is not linear but more in the nature of a threshold effect [ 18 ]. Risk factors for atherosclerosis in the general population are related to atherosclerosis in diabetes but do not account for the excess incidence of this disorder in diabetic patients [ 2 ]. High plasma insulin levels are prospectively related to coronary artery disease in the general population [ 19-21 ]. If this hypothesis is correct, prevention of large vessel disease in diabetic patients would be achieved by a reduction o f blood glucose levels without the use of excess insulin concentrations. In non-insulin dependent diabetic patients this could be achieved by weight reduction and exercise. For insulin-requiting diabetic patients the development of new methods of insulin delivery resulting in insulin concentrations nearer the physiological should be encouraged. Acknowledgements.The skillful technical help of Mr. J. Donnelly and Mrs. N. Copeland, and the careful preparation of the manuscript by Miss A. Best is gratefully acknowledged. Supported in part by a grant from the Department of Health and Social Services (Northern Ireland). 1. Stout RW ( 1981 ) Blood glucose and atherosclerosis . Arteriosclerosis 1 : 227 - 234 2. Kannel WB , McGee DL ( 1979 ) Diabetes and cardiovascular risk factors:the Framingham study . Circulation 59 : 8 - 13 3. Ross R ( 1981 ) Atherosclerosis: A problem of the biology of arterial wall cells and their interactions with blood components . Arteriosclerosis 1 : 293 - 311 4. Ross R , Glomset JA ( 1976 ) The pathogenesis of atherosclerosis . N Engl J Med 295 : 369 - 377 , 420 - 425 5. Ross R , Vogel A ( 1978 ) The platelet-derived growth factor . Cell 14 : 203 - 210 6. Stout RW , Bierman EL , Ross R ( 1975 ) Effect of insulin on the proliferation of cultured primate arterial smooth muscle cells . Circ Res 36 : 319 - 327 7. Pfeifle B , Ditschuneit H ( 1981 ) Effect of insulin on growth of cultured human arterial smooth muscle cells . Diabetologia 20 : 155 - 158 8. Kosehinsky T , Bunting CE , Schwippert B , Gries FA ( 1981 ) Regulation of diabetic serum growth factors for human vascular cells by the metabolic control of diabetes mellitus . Atherosclerosis 39 : 313 - 319 9. Jaffe EA , Nachman RL , Becker CG , Minick GR ( 1973 ) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria . J Clin Invest 52 : 2745 - 2756 10. Gimbrone MA Jr, Cotran RS , Folkman J ( 1974 ) Human vascular endothelial cells in culture . J Cell Biol 60 : 673 - 684 11. Taggart H , Stout RW ( 1980 ) Control of DNA synthesis in cultured vascular endothelial and smooth muscle cells - response to serum, platelet-deficient serum, lipid-free serum, insulin and oestrogens . Atherosclerosis 37 : 549 - 557 12. Lowry OH , Rosebrough NJ , Farr AL , Randall ILl ( 1957 ) Protein measurement with the Folin phenol reagent . J Biol Chem 193 : 265 - 275 13. Schwartz SM , Gajdusek CM , Selden SC III ( 1981 ) Vascular wall growth control: the role of the endothelium . Arteriosclerosis 1 : 107 126 14. Turner JL , Bierman EL ( 1978 ) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth muscle ceils . Diabetes 27 : 583 - 588 15. Stout RW ( 1982 ) Cyclic AMP: a potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells . Diabetologia 22 : 51 - 55 16. Boot-Handford RP , Heath H ( 1978 ) The absence of sorbitol pathway activity in primary cultures of human umbilicalcord vein endothelial cells . IRCS Med Sci Biochem 9 : 451 (Abstract) 17. Dolgov VV , Zaildna OE , Bondarenko MF , Repin VS ( 1982 ) Aortic endothelium of alloxan-diabetic rabbits: a quantitative study using scanning electron microscopy . Diabetologia 22 : 338 - 343 18. Fuller JH , Shipley MJ , Rose G , Jarrett RJ , Keen H ( 1980 ) Coronary-heart-disease risk and impaired glucose tolerance . Lancet 1 : 1373 - 1376 19. Pyorala K ( 1979 ) Relationship of glucose tolerance and plasma insulin to the incidence of coronary heart disease: results from two population studies in Finland . Diabetes Care 2 : 131 - 141 20. Welborn TA , Weame K ( 1979 ) Coronary heart disease incidence and cardiovascular mortality in Busselton with reference to glucose and insulin concentrations . Diabetes Care 2 : 154 - 160 21. Ducimetiere P , Eschwege E , Papoz L , Richard JL , Claude JR , Rosselin G ( 1980 ) Relationship of plasma insulinlevels to the incidence of myocardial infarctionand coronary heart disease mortality in a middle-aged population . Diabetologia 19 : 205 -210 Received: 12 January 1982 and in revised form: 19 July 1982 Professor R. W. Stout Department of Geriatric Medicine The Queen's University of Belfast Whitla Medical Building 97 Lisburn Road Belfast BT9 7BL Northern Ireland , UK


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R. W. Stout. Glucose inhibits replication of cultured human endothelial cells, Diabetologia, 1982, 436-439, DOI: 10.1007/BF00260958