Cyclic AMP: A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells

Diabetologia, Jan 1982

The effect of dibutyryl cyclic AMP on DNA synthesis was studied in cultured human umbilical endothelial cells and rat aortic smooth muscle cells. Dibutyryl cyclic AMP (2×10-4mol/l) inhibited DNA synthesis in both arterial cell types when they were grown in medium supplemented with whole serum or with platelet poor serum, but had no effect in the absence of serum. An effect was seen one hour after the addition of the nucleotide, and the threshold concentration was between 2×10-6 and 2×10-5mol/l. These results may have relevance to the interaction of platelets and insulin with the arterial wall in the development of atherosclerosis in diabetes.

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Cyclic AMP: A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells

Diabetologia Cyclic A M P : A Potent Inhibitor of D N A Synthesis in Cultured Arterial Endothelial and Smooth Muscle Cells R.W. Stout 0 1 0 Department of Geriatric Medicine, The Queen's University of Belfast , Northern Ireland 1 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 Summary. The effect of dibutyryl cyclic A M P on D N A synthesis was studied in cultured human umbilical endothelial cells and rat aortic smooth muscle cells. Dibutyryl cyclic A M P (2 10-4tool/I) inhibited D N A synthesis in both arterial cell types when they were grown in medium supplemented with whole serum or with platelet poor serum, but had no effect in the absence o f serum. An effect was seen one hour after the addition of the nucleotide, and the threshold concentration was between 2 x 10-6 and 2 10-5mol/l. These results may have relevance to the interaction of platelets and insulin with the arterial wall in the development of atherosclerosis in diabetes. Cyclic AMP; dibutyryl cyclic AMP; endothelial cells; smooth muscle cells; artery; D N A synthesis; platelets; diabetes; atherosclerosis - 9 Springer-Verlag 1982 Atherosclerosis is a disease of the inner part of the arterial wall. In this part of the artery two cells predominate, endothelial cells, which line the inner surface of the vessel, and smooth muscle cells. M o d e m theories on the pathogenesis of atherosclerosis assign important roles to both these cells [ 1 ]. The endothelial cell is thought to act as a barrier preventing the constituents of circulating blood from entering the inner part of the artery. It is postulated that an early change in the development of atherosclerosis is an injury or alteration to the endothelial cell barrier allowing plasma constituents to enter the inner part of the arterial wall and act on the smooth muscle cells. Plasma constituents stimulate arterial smooth muscle cells to proliferate. If the stimulus is temporary, repair of the arterial damage occurs but if the stimulus continues, the cells become filled with lipid and eventually an atheromatous plaque develops. A number of factors have been proposed to have a role in the early development of the arterial lesion. These include platelet factors [ 2 ], lipoproteins [ 3 ] and insulin [ 4 ]. Both platelet and insulin action are related to the activity of cyclic adenosine 3' 5'-monophosphate (cyclic AMP) and situations associated with insulin overactivity [ 5 ] and with platelet adhesiveness [ 6 ] are associated with decreased intracellular cyclic AMP. Cyclic A M P may thus be a common factor linking the actions of these factors. This paper describes the effect of dibutyryl cyclic A M P (db-cAMP) on D N A synthesis in cultured arterial cells. Dibutyryl cyclic A M P was chosen as it is said to pass through cell membranes more readily than cyclic A M P itself [71. Materials and Methods Culture media, trypsin and fetal calf serum were obtained from Gibco Biocult, Paisley, Scotland, UK and Falcon plastic culture dishes and flasks from Becton Dickinson, Wembley, Middlesex, UK. Collagenase (Type I from Cl. histolyticum [Worthington]) was obtained from Millipore, London, UK. Human serum was obtained from the Northern Ireland Blood Transfusion Service, Belfast, UK. It was pooled from at least 20 donors and was filter sterilized before use. Platelet-poor serum was prepared according to the method of Rutherford and Ross [ 8 ] as described [ 9 ]. Isotopes were obtained from the Radiochemical Centre, Amersham, UK. dbcAMP was purchased from Sigma Chemicals, London and dissolved in a 4% bovine albumin solution. Cell Culture Techniques Human endothelial cells were cultured from umbilical vein by a modification [ 9 ] of the methods of Jaffe et al. [ 10 ] and Gimbrone et al. [ 11 ]. Briefly, umbilical cord was obtained within 12h of delivery. The umbilical vein was identified, canulated and washed with Dulbecco's phosphate buffered saline (PBS) to remove any residual Each value is the mean of six replicate plates. Results are expressed as counts min-~ mg cell protein-1 (mean + SEM). Figures in parentheses represent the values in the cells exposed to db-cAMP expressed as a percentage of the values in the control cells blood. Ten ml 0.025% collagenase in PBS was then infused and the vein was clamped. After 10min incubation at 37 ~C, the collagenase solution was flushed out together with the endothelial cells into medium 199. After centrifugation the cell pellet was resuspended in medium 199 supplemented with 20% human serum (v/v) and plated into a 25 cm culture flask. Cell growth occurred in about 6 h and the cells reached confluency in about 5 days. Endothelial cells were identified by the presence of Weibel-Palade bodies on electron microscopy [ 10 ] and by immunofluorescence against clotting factor VIII antibodies. When the cells had filled the surface of the flask, they were freed from each other and the flask by 10min exposure to trypsin (0.5g/l) in versene buffer (0.2g/l) and passed into two or more new flasks. The cells were used for experiments after two or three subcultivations. Each value is the mean of two replicate plates. Results are expressed as counts min ~mg cell protein- 1 Smooth muscle cells were cultured from rat aorta by methods previously described [ 13 ]. Briefly, the aorta was removed under sterile conditions from 150g male Wistar rats. The intima and media were carefully separated from the rest of the aorta, divided into small pieces and placed in plastic culture flasks with DulbeccoVogt medium supplemented with 10% fetal calf serum (v/v) at 37 ~ in a humidified atmosphere of 5% carbon dioxide in air. Cell growth was seen about 10 days after explantation and the cells grew in the hills and hollows typical of smooth muscle cells. The identity of the cells was confirmed as previously described [ 13 ]. The cells were treated by trypsinisation in a similar manner to the endothelial cells and in these studies were used after 4-12 subcultivations. Experimental Equal numbers of cells were plated into 12 30 mm plastic petri dishes and were grown to near confluency in their usual growth medium. For endothelial cells this was medium 199 supplemented with 20% pooled human serum (v/v) and for smooth muscle cells, Dulbecco-Vogt medium supplemented with fetal calf serum. When the platelet-poor serum was being tested, human serum was used for both cell types [ 9 ]. db-cAMP was then added in appropriate concentrations to the medium (see Tables) and the same volume of 4% albumen to the controls. After 24h exposure to db-cAMP, 2 ~tCi of (methyl-3H) thymidine was added to each dish. After a further 2 h the medium was removed, the cells were washed in PBS and removed from the dishes by trypsinisation. D N A was extracted and its activity measured as described [ 9 ]. Protein was measured by the Lowry method [ 14 ] and the results were expressed in counts per minute per milligram cell protein. The results were analysed by Student's 't' test. Results Smooth Muscle Cells In Table 1 the results o f duplicate experiments, each using six dishes in control and d b - c A M P medium, are shown, d b - c A M P ( 2 x 10-4mol/1) inhibited D N A synthesis b y more than 80% in cells grown in whole serum and by 75% in cells grown in platelet-poor serum, but had much less effect on cells grown in a serum free medium. In the latter medium, only one o f the duplicate experiments showed a significant difference between d b - c A M P and control. The counts in the control medium are much lower when it contained platelet-poor serum or no serum in keeping with the low level o f cellular activity in these media. Table 2 shows that the effect o f d b - c A M P on D N A synthesis is seen 1h after the cells are exposed to the nucleotide, the effect is more marked after 4h, and is (%) more marked again after 24h exposure (Table 1). Table 3 shows that the threshold concentration for the effect o f d b - c A M P on D N A synthesis in cultured arterial smooth muscle cells is between 2 x 10 .6 and 2 x 10-Smol/1. Endothelial Cells Tables 4, 5 and 6 show the effect o f d b - c A M P on D N A synthesis in cultured h u m a n endothelial cells. The inhibitory effect o f the nucleotide is more variable and perhaps not as potent on endothelial cells as on smooth muscle cells (Table 4) but the pattern o f effect on the different media is the same. The D N A c o u n t s / m i n in the cells in the control media are very variable between experiments, probably related to differing cell densities in the dishes. The time course o f the effect o f d b - c A M P in endothelial cells is similar to that in smooth muscle cells (Table 5), and the effect o f different concentrations o f the nucleotide is also similar, although a small effect o f 2 x 10-6tool/1 is shown. Discussion The results o f these experiments show that d b - c A M P potently inhibits D N A synthesis in cultured arterial smooth muscle and endothelial cells. Previous experiments have shown that d b - c A M P antagonized the stimulating effect o f insulin on the proliferation o f arterial smooth muscle cells cultured from primate aortas [ 4 ]. It also inhibited sterol synthesis from acetate in cultured rat arterial smooth muscle cells [ 15 ]. Cyclic A M P has been shown to be related to the growth o f a number of different cell lines in culture and it has been suggested that the growth response to serum constituents may be modulated by intracellular changes in cyclic AMP concentrations [ 16 ]. An inverse relationship between intracellular cyclic AMP levels and cell proliferation has been described [ 16 ]. Malignant and transformed cells have low intracellular cyclic AMP levels, and growth control and morphological appearance can be restored towards normal by addition of db-cAMP to the culture medium [ 7 ]. Most of the studies relating cyclic A M P to cell proliferation have used fibroblasts and it is not clear whether other cell types respond in the same way [ 17 ]. The present experiments suggest that growth of cultured arterial endothelial and smooth muscle cells is also influenced by cyclic AMP and that this effect is not dependent on the presence of added insulin in the medium [ 4 ]. Intracellular cyclic A M P levels do not appear to have been measured in arterial cells. The pattern of response of endothelial and smooth muscle cells to db-cAMP was strikingly similar in view of the different responses of these cells to insulin and to the platelet derived growth factor [ 9 ]. The fact that endothelial cells bind insulin to membrane receptors [ 18 ]and respond to db-cAMP, and yet are unresponsive to insulin with respect to D N A synthesis [ 9 ] suggests that some additional factor, absent in endothelial cells but present in smooth muscle cells, mediates the activity of insulin on D N A synthesis and cell proliferation. The identity of this factor remains unknown. Potent inhibitory effects of db-cAMP on D N A synthesis were only seen in the presence of serum. Serum was also necessary for db-cAMP to inhibit sterol synthesis in cultured smooth muscle cells [ 15 ]. This may be because the nucleotide only has effects on dividing cells. On the other hand, db-cAMP markedly inhibited D N A synthesis in smooth muscle cells exposed to platelet-poor serum despite the fact that these cells are quiescent in the absence of growth factors from platelets [ 2 ]. This suggests that some serum factor, other than one derived from platelets, is necessary for db-cAMP to have an effect on D N A synthesis. It is notable that db-cAMP had the same effect on D N A synthesis in endothelial cells and smooth muscle cells grown in platelet-poor serum despite the fact that the two cell types respond in opposite ways to this preparation of serum [91. This is further evidence for db-cAMP interacting with a serum factor unrelated to the platelet derived growth factor. In these experiments the endothelial and smooth muscle cells were derived from different species. Although there has been a report of successful smooth muscle cell culture from human umbilical vessels [ 19 ], the technique is difficult and attempts to reproduce it in this laboratory have been unsuccessful. H u m a n smooth muscle cells have been grown from small pieces of tissue obtained at operation or autopsy [ 20 ] but endothelial cell culture from such tissues has not been reported. On the other hand, the rat aorta is too small to allow sufficient quantities of endothelial cells to be harvested for culture purposes. A compromise could be made by studying bovine endothelial and smooth muscle cells as techniques have been reported for culturing both types of ceils from this source [ 21 ]. On the other hand, it would seem preferable to use human cells as far as possible. Smooth muscle cells have been cultured from a number of different species [ 3, 4, 20-23 ] and in general the effects of different stimuli have been the same irrespective of the origin of the cells. Furthermore, apart from whole serum, the stimuli being studied in these experiments are not species specific. An early event in the development of atherosclerosis is proliferation of smooth muscle cells in the arterial intima and adjacent media. Factors which control proliferation of arterial cells are of potential importance in the understanding of atherogenesis. The factors described include insulin [ 4 ] and a platelet derived growth factor [ 2 ]. Insulin has been shown to lower intracellular cyclic A M P levels in fat and hepatic cells [ 5 ] and to inhibit cyclic A M P elevations induced by other agents [ 5 ] while cyclic AMP inhibits the proliferative effect of insulin on arterial smooth muscle cells [ 4 ]. Platelet behaviour is influenced by two products of the arachidonic acid pathway, prostacyclin which causes vasodilatation and reduced platelet aggregation [ 24 ] and thromboxane A2 which has opposite effects [ 25 ]. Prostacyclin increases platelet cyclic AMP levels [ 24 ] while thromboxane A2 reduces cyclic A M P levels [ 25 ]. In diabetes, arterial prostacyclin levels have been reported to be low [ 26 ] and platelets from diabetics have increased sensitivity to aggregating agents [ 27 ]. The relation of cyclic A M P platelet and hormone action to the cellular changes in atherosclerosis and diabetes remains speculative and must be the subject of further studies. However, it has been reported that circulating cyclic A M P levels are low in diabetics [ 28 ], and that experimental atherosclerosis lesions in animals contain lower cyclic A M P levels than normal artery [ 29 ]. Acknowledgements.The skillful technical work of Mr. Jim Donnelly and Mrs. Nora Copeland and the careful preparation of the manuscript by Mrs. Melanie Hilary and Miss Andree Best are gratefully acknowledged. The work was supported by grants from the British Heart Foundation and the Department of Health and Social Services (Northern Ireland). 1. Ross R , Glomset JA ( 1976 ) The pathogenesis of atherosclerosis . N Engl J Med 295 : 369 - 377 , 420425 2. Ross R , Vogel A ( 1978 ) The platelet-derived growth factor . Cell 14 : 203 - 210 3. 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Pastan I ( 1975 ) Regulation of cellular growth . Adv Metab Disorders 8 : 7 - 16 17. Friedman DL ( 1976 ) Role of cyclic nucleotides in cell growth and differentiation . Physiol Rev 56 : 652 - 708 18. Bar RS , Peacock ML , Spanheimer RG , Veenstra R , Hoak JC ( 1980 ) Differential binding of insulin to human arterial and venous endothelial cells in primary culture . Diabetes 29 : 991 - 995 19. Gimbrone M A j r ( 1976 ) Culture of vascular endothelium . Prog Hemostasis Thromb 3 : 1 - 28 20. Albers JJ , Bierman EL ( 1976 ) The effect of hypoxia on uptake and degradation of low density lipoprotein by cultured human arterial smooth muscle cells . Biochem Biophys Acta 424 : 422429 21. Greenburg GB , Hunt TK ( 1978 ) The proliferative response in vitro of vascular endothelial and smooth muscle cells exposed to wound fluids and macrophages . J Cell Physio197 : 353 - 361 22. Bierman EL , Stein O , Stein Y ( 1974 ) Lipoprotein uptake and metabolism by rat aortic smooth muscle cells in tissue culture . Circ Res 35 : 136 - 150 23. Weinstein DB , Carew TE , Steinberg D ( 1976 ) Uptake and degradation of low density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis . Biochim Biophys Acta 424 : 404421 24. Tateson JE , Moncada S , Vane JR ( 1977 ) Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets . Prostaglandins 13 : 389 - 397 25. Miller OV , Johnson RA , Gorman RR ( 1977 ) Inhibition of PGErstimulated cAMP accummulation in human platelets by thromboxane A2 . Prostaglandins 13 : 599 - 609 26. Silberbauer K , Schernthaner G , Sinzinger H , Piza-Katzer H , Winter M ( 1979 ) Decreased vascular prostacyclin in juvenileonset diabetes . N Engl J Med 300 : 366 - 367 27. Halushka PV , Luric D , Colwell JA ( 1977 ) Increased synthesis of prostaglandin E-like material by platelets from patients with diabetes mellitus . N Engl J Med 297 : 1306 - 1310 28. Feinglos MN , Drezner MK , Lebovitz HE ( 1978 ) Measurement of plasma adenosine 3', 5'-monophospbate . J Clin Endocrinal Metab 46 : 824 - 829 29. Numano F , Maezawa H , Shimamoto T , Adachi K ( 1976 ) Changes of cyclic-AMP and cyclic AMP phosphodiesterase in the progression and regression of experimental atherosclerosis . Ann NY Acad Sci 275 : 311 320 Received: 16 April 1981 and in final form: 17 August 1981


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R. W. Stout. Cyclic AMP: A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells, Diabetologia, 1982, 51-55, DOI: 10.1007/BF00253870