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Insulin and Nitric Oxide Synthase (NOS) gene expression and no generation by macrophages
$62
Poster session abstraets / Atherosclerosis 115 (Suppl.) (1995) $45-$129 P5 Vascular Biology
226
228
INSULIN AND NITRIC OXIDE SYNTHASE (NOS) GENE EXPRESSION AND NO GENERATION BY MACROPHAGES A. Dembirlska-Kie~, J. Dulak, J. Hartwich, E. Baczyftska, M. Polus, D. Zmudzifiska, A. Siedlecki, I. Guewara Department of Clinical Biochemistry, Collegium Medicum of the Jagiellonian University, Krak6w, Poland.
DISTINCT EFFECTS OF VASTATINS ON PROLIFERATION OF DIFFERENT HUMAN CELLS IN CULTURE A.K. van Vliet. P. N~gre-Aminou, A.C.J. de Vries, G.C.F. van Thiel, L.H. Cohen. Gaubius Laboratory TNO-PG, Zemikedreef 9, P.O.Box 2215, 2301 CE Leiden, The Netherlands
Islet B-cells death during the development of Type I diabetes mellitus (IDDM) is suggested to be mediated by massive NO secretion, induced by cytokines in inter-islet macrophages and endothelium (Corbett et al. J.Clin.Invest. 90.1992; Kolbet et al Diabetologia, 35, 1992). Treatment of the streptozotocin-induced diabetic animals with nitric oxide synthase (NOS) inhibitors attenuated hyperglycemia (Lukie et al BBRC. 178.1991). On the other hand, the impaired EDRF/NO generation by endothelium of patients with IDDM (Calver et al J.Clin.lnvest.90.1992) and in diabetic animals (Abiru et al Life sci 53, 1993) was found. We estimated the influence of insulin on NO generation and iNOS gene expression by rat macrophages and vascular smooth muscle cells in vitro. Rat peritoneal macrophages (108cellsfml) stabilized for 24 hours in Dubelcco medium, or smooth muscle cells, were incubated with insulin (10-100o.U/ml) without or in the presence of LPS (100ng/ml) for 30 min or 16 hours at 37°C. The generation of NO was measured according to Bredt and Snyder, (Proc.Natl. Acad Sci.87.1990) by the conversion of H3-L-arginine to citrulline. Induction of iNOS was analyzed by mRNA level for iNOS using RT-PCR and anti-sense primer. Insulin did not increase the NO generation after 30 min of preincubation. The about three time increase (from 5.91pmol/mg prot. to 16.3pmol/mg prot) of H 3 L-citrulline formation was observed after 16h coincubation of insulin with macrophages. LPS itself was the weak activator of NO generation after the long incubation, and completely prevented the induction of iNOS mediated by insulin. We conclude that the increased iNOS activity by hyperinsulinemia may contribute to the auto-immune destruction of islet fl-cells and microangiopaty.
Proliferation of smooth muscle cells plays an important role in the development of atherosclerotic plaques. Inhibition of this process might result in halt of progression and even regression of the lesion. Therefore we investigated the effects of three HMG-CoA reductase inhibitors (lovastatin (L), simvastatin (S) and pravastatin (P)) on proliferation of human smooth muscle cells isolated from internal mammary artery, but also on proliferation of human myoblasts from striated muscle biopsies, human cornea fibroblasts and human umbilical vein endothelial cells in culture. The latter three were cultured with fl,0.1 or 1 ~M of each drug for a period of three days. The slower growing smooth muscle cells were incubated for 6 days. Under these conditions viability of the cells was not influenced by the drugs. The effect on proliferation was measured by three different methods: cell counting, MTT-test and [3H]-thymidine incorporation into DNA. The proliferation of myoblasts and cornea fibroblasts was not influenced at 0.1 ,uM of L and P, but at 1 p.M of L or S a strong inhibition of proliferation of myoblast and to a lesser extent also of flbroblasts was observed. P did not inhibit the proliferation at either concentration. In endothelial cells even 0.1 /xM of L or S resulted in inhibition of DNA synthesis. Again P had no effect on the three parameters tested. Similar observations were made in smooth muscle ceils, with the exception that P in these cells inhibited the proliferation as well. It can be concluded that under the conditions used L and S were inhibitors of the proliferation of myoblasts, cornea flbroblasts, endothelial cells and smooth muscle cells. P had only an inhibitory effect on smooth muscle cells. The extent of inhibition of proliferation of human cells in culture by vastatins is dependent on the drug and on cell type.
227
229
NATIVE LOW DENSITY LIPOPROTEIN INDUCES TRANSITION OF MONOCYTIC CELL LINES INTO FOAM CELL-LIKE CELL LINES. M.L.C.M. Mevissen, J. Jansen, S.J.H. van Deventer Center for Hemostasis, Thrombosis, Atheroselerosis and Inflammation Research, Amsterdam, The Netherlands
C E L L U L A R LIPIDOSIS A N D P R O L I F E R A T I O N IN H U M A N AORTA A.N. Orekhov, E.R. Andreeva, V.V. Tertov, I.M. Khubulova Institute of Experimental Cardiology Moscow
Purpose: In order to study the role of foam cells in lesion development in an in vitro vessel wall model for atherosclerosis we tried to develop continuously growing foam cell-like cell lines/clones. Methods: Monocytic cell lines U937 and MonoMac 6 (MM6) were cultured for four weeks in the presence of 20 ,ug/ml native LDL (nLDL) followed by at least a one weeks culture in the absence of nLDL, in culture medium supplemented with Human Serum (HS). After this period cholesterolester-accumulation was visualized by Oil Red O staining. Cells (0.5 x 106 cells/ml) were subsequently incubated for 2 hours at 37°C and Interleukin-6 and -8 ( I L 6 and -8) and prostaglandin F-a (PGFa), Leukotrien B4 (LTB4) and Thromboxane 2 (TBX2) concentrations in the culture supernatants were measured by ELISA. MM6 cells were cloned by limiting dilution in the presence of 20 #g/ml nLDL. Using a ~Cr-assay, the effect of the presence of nLDL during culture on adhesion of monocytic cell lines and clones to endothelial cells was established. Results: Cholesterolester-accumulation was very heterogeneic within the cell lines and most pronounced in MM6. The presence of nLDL increased IL6, PGE 2, LTB 4 and TBX 2 (Increase: 0.25-2340 times control), in contrast to IL-8. Adhesion of monocytic cells, was increased by the presence of nLDL during pre-culture, especially for the clones. Conclusions: Establishing massive into monoeytic cells appears to be favoured by the presence of autologous serum. The results indicate that several features, common to foam cells, are found within these foamlike cell lines and clones. Furthermore, limiting dilution provides us with functionally different foam-like clones. These cells may therefore provide a powerfull tool in studying the role of foam ceils in the development of an atherosclerotic plaque.
Proliferating cells were revealed in human aorta by immunocytochemical technique with antibody against proliferating cell nuclear protein (PCNA). Proliferating cells were very seldom observed in grossly normal intima and in atherosclerotic lesions, namely: fatty infiltration, fatty streak, and plaque. However, the rate of cell proliferation in fatty infiltration was 6.5-fold and in fatty streaks was 9.7-fold higher, than in normal intima. In atherosclerotic plaque this rate was only 2.3-fold higher, than in normal intima. Thus, enhanced proliferative activity in human aorta is associated with fatty lesions which is characterised by intracellular lipid accumulation. To elucidate possible role of lipid accumulation in enhanced proliferative activity in fatty lesions of human aorta we stimulated intracellular lipid accumulation in primary culture of human aortic cells by incubation with naturally occurring modified low density lipoprotein. Lipid accumulation was accompanied with increased [~H]-thymidine incorporation (up to 2.5-fold). The study suggests, that the accumulation of intracellular lipids in fatty lesions of human aorta may stimulate proliferative activity of human aortic cells.
Poster session abstraets / Atherosclerosis 115 (Suppl.) (1995) $45-$129 P5 Vascular Biology
226
228
INSULIN AND NITRIC OXIDE SYNTHASE (NOS) GENE EXPRESSION AND NO GENERATION BY MACROPHAGES A. Dembirlska-Kie~, J. Dulak, J. Hartwich, E. Baczyftska, M. Polus, D. Zmudzifiska, A. Siedlecki, I. Guewara Department of Clinical Biochemistry, Collegium Medicum of the Jagiellonian University, Krak6w, Poland.
DISTINCT EFFECTS OF VASTATINS ON PROLIFERATION OF DIFFERENT HUMAN CELLS IN CULTURE A.K. van Vliet. P. N~gre-Aminou, A.C.J. de Vries, G.C.F. van Thiel, L.H. Cohen. Gaubius Laboratory TNO-PG, Zemikedreef 9, P.O.Box 2215, 2301 CE Leiden, The Netherlands
Islet B-cells death during the development of Type I diabetes mellitus (IDDM) is suggested to be mediated by massive NO secretion, induced by cytokines in inter-islet macrophages and endothelium (Corbett et al. J.Clin.Invest. 90.1992; Kolbet et al Diabetologia, 35, 1992). Treatment of the streptozotocin-induced diabetic animals with nitric oxide synthase (NOS) inhibitors attenuated hyperglycemia (Lukie et al BBRC. 178.1991). On the other hand, the impaired EDRF/NO generation by endothelium of patients with IDDM (Calver et al J.Clin.lnvest.90.1992) and in diabetic animals (Abiru et al Life sci 53, 1993) was found. We estimated the influence of insulin on NO generation and iNOS gene expression by rat macrophages and vascular smooth muscle cells in vitro. Rat peritoneal macrophages (108cellsfml) stabilized for 24 hours in Dubelcco medium, or smooth muscle cells, were incubated with insulin (10-100o.U/ml) without or in the presence of LPS (100ng/ml) for 30 min or 16 hours at 37°C. The generation of NO was measured according to Bredt and Snyder, (Proc.Natl. Acad Sci.87.1990) by the conversion of H3-L-arginine to citrulline. Induction of iNOS was analyzed by mRNA level for iNOS using RT-PCR and anti-sense primer. Insulin did not increase the NO generation after 30 min of preincubation. The about three time increase (from 5.91pmol/mg prot. to 16.3pmol/mg prot) of H 3 L-citrulline formation was observed after 16h coincubation of insulin with macrophages. LPS itself was the weak activator of NO generation after the long incubation, and completely prevented the induction of iNOS mediated by insulin. We conclude that the increased iNOS activity by hyperinsulinemia may contribute to the auto-immune destruction of islet fl-cells and microangiopaty.
Proliferation of smooth muscle cells plays an important role in the development of atherosclerotic plaques. Inhibition of this process might result in halt of progression and even regression of the lesion. Therefore we investigated the effects of three HMG-CoA reductase inhibitors (lovastatin (L), simvastatin (S) and pravastatin (P)) on proliferation of human smooth muscle cells isolated from internal mammary artery, but also on proliferation of human myoblasts from striated muscle biopsies, human cornea fibroblasts and human umbilical vein endothelial cells in culture. The latter three were cultured with fl,0.1 or 1 ~M of each drug for a period of three days. The slower growing smooth muscle cells were incubated for 6 days. Under these conditions viability of the cells was not influenced by the drugs. The effect on proliferation was measured by three different methods: cell counting, MTT-test and [3H]-thymidine incorporation into DNA. The proliferation of myoblasts and cornea fibroblasts was not influenced at 0.1 ,uM of L and P, but at 1 p.M of L or S a strong inhibition of proliferation of myoblast and to a lesser extent also of flbroblasts was observed. P did not inhibit the proliferation at either concentration. In endothelial cells even 0.1 /xM of L or S resulted in inhibition of DNA synthesis. Again P had no effect on the three parameters tested. Similar observations were made in smooth muscle ceils, with the exception that P in these cells inhibited the proliferation as well. It can be concluded that under the conditions used L and S were inhibitors of the proliferation of myoblasts, cornea flbroblasts, endothelial cells and smooth muscle cells. P had only an inhibitory effect on smooth muscle cells. The extent of inhibition of proliferation of human cells in culture by vastatins is dependent on the drug and on cell type.
227
229
NATIVE LOW DENSITY LIPOPROTEIN INDUCES TRANSITION OF MONOCYTIC CELL LINES INTO FOAM CELL-LIKE CELL LINES. M.L.C.M. Mevissen, J. Jansen, S.J.H. van Deventer Center for Hemostasis, Thrombosis, Atheroselerosis and Inflammation Research, Amsterdam, The Netherlands
C E L L U L A R LIPIDOSIS A N D P R O L I F E R A T I O N IN H U M A N AORTA A.N. Orekhov, E.R. Andreeva, V.V. Tertov, I.M. Khubulova Institute of Experimental Cardiology Moscow
Purpose: In order to study the role of foam cells in lesion development in an in vitro vessel wall model for atherosclerosis we tried to develop continuously growing foam cell-like cell lines/clones. Methods: Monocytic cell lines U937 and MonoMac 6 (MM6) were cultured for four weeks in the presence of 20 ,ug/ml native LDL (nLDL) followed by at least a one weeks culture in the absence of nLDL, in culture medium supplemented with Human Serum (HS). After this period cholesterolester-accumulation was visualized by Oil Red O staining. Cells (0.5 x 106 cells/ml) were subsequently incubated for 2 hours at 37°C and Interleukin-6 and -8 ( I L 6 and -8) and prostaglandin F-a (PGFa), Leukotrien B4 (LTB4) and Thromboxane 2 (TBX2) concentrations in the culture supernatants were measured by ELISA. MM6 cells were cloned by limiting dilution in the presence of 20 #g/ml nLDL. Using a ~Cr-assay, the effect of the presence of nLDL during culture on adhesion of monocytic cell lines and clones to endothelial cells was established. Results: Cholesterolester-accumulation was very heterogeneic within the cell lines and most pronounced in MM6. The presence of nLDL increased IL6, PGE 2, LTB 4 and TBX 2 (Increase: 0.25-2340 times control), in contrast to IL-8. Adhesion of monocytic cells, was increased by the presence of nLDL during pre-culture, especially for the clones. Conclusions: Establishing massive into monoeytic cells appears to be favoured by the presence of autologous serum. The results indicate that several features, common to foam cells, are found within these foamlike cell lines and clones. Furthermore, limiting dilution provides us with functionally different foam-like clones. These cells may therefore provide a powerfull tool in studying the role of foam ceils in the development of an atherosclerotic plaque.
Proliferating cells were revealed in human aorta by immunocytochemical technique with antibody against proliferating cell nuclear protein (PCNA). Proliferating cells were very seldom observed in grossly normal intima and in atherosclerotic lesions, namely: fatty infiltration, fatty streak, and plaque. However, the rate of cell proliferation in fatty infiltration was 6.5-fold and in fatty streaks was 9.7-fold higher, than in normal intima. In atherosclerotic plaque this rate was only 2.3-fold higher, than in normal intima. Thus, enhanced proliferative activity in human aorta is associated with fatty lesions which is characterised by intracellular lipid accumulation. To elucidate possible role of lipid accumulation in enhanced proliferative activity in fatty lesions of human aorta we stimulated intracellular lipid accumulation in primary culture of human aortic cells by incubation with naturally occurring modified low density lipoprotein. Lipid accumulation was accompanied with increased [~H]-thymidine incorporation (up to 2.5-fold). The study suggests, that the accumulation of intracellular lipids in fatty lesions of human aorta may stimulate proliferative activity of human aortic cells.