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Docosahexaenoic acid reduces vascular cell adhesion molecule 1 induction and monocyte adhesion to stimulated human endothelial cells
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Poster session abstracts / Atherosclerosis 115 (SuppL ) (1995) $45-$129 P4 Endothelial Cells
210
212
ENDOTHELIAL CELL CYTOTOX1C AUTOANTIBODIES AGAINST HEAT SHOCK PROTEIN 60 IN ATHEROSCLEROSIS G. Schett~2, A. Ambergert, H. Recheis:, Q. Xu ~, G.Wick~2 qnst for Biomedical Aging Research, Austrian Academy of Sciences, ~lnst for General and Experimental Pathology, University of Innsbruck, Austria
DISTRIBUTION OF CELL ADHESION MOLECULES 1N CULTURED ENDOTHELIAL CELLS FROM HUMAN AORTA T.V. Byzova, Y.A. Romanov, A.V. Mazurov Cardiology Research Cemre, 3rd Cherepkovskaya 15a, 121552 Moscow, Russia
Previous studies from our laboratory have proven that serum antibodies (Abs) against mycobacterial heat shock protein (hsp) 65 are increased in clinically healthy subjects with sonographically detectable carotid atherosclerosis (Xu et al., Lancet 341:255,1993). We hypothetized that these Abs thal bind to stressed human cells via a crossreaction to their hsp-homologue hsp6Q may therefore be of possible functional importance in the developement of the disease. We now investigated complement-mediated lysis and antibody dependent cellular cytotoxicity (ADCC) to cultured human umbilical vein endothelial cells (HUVEC) using these Abs, to determine whether they react with homologous human hsp60 and whether they play a potential pathogenetic role. Positive staining with these high titer antisera (hts) or with affinity purified antihsp65 antibodies (hspkAb) was observed on the surface of cultured HUVEC stressed at 42°C for 30 rain, while unstressed cells showed only weak or no fluorescence. When stressed endothelial cells labeled with ~Cr were incubated with hts or hsp-Ab in the presence of complement, significant lysis occurred. Similarly, ~Cr release by HUVEC was observed when his or hsp-Ab and peripheral blood mononuclear cells (effector) were added to the culture (ADCC). In summary, serumantibodies to mycobacterial hsp65 cross-react with human 60 kDa homologue present in high levels on the surface of stressed human endothelial cells, and are able to evoke their lysis via antibody-complement mediated cell lysis and ADCC. These data support the hypothesis that humoral-mediated immune reactions, in addition to cell mediated immunity, may play a role in athero genesis. (Supported by the Austrian Research- Council project 10677 and the Sandoz Foundation for Gerontological Research).
Distribution of cell adhesion molecules (CAM) in cultured human aortic endothelial cells (HAEC) was studied using immunohistochemistry and FACS analysis. Several features of CAM expression were specific for HAEC in comparison with the standard culture of human umbilical vein EC (HUVEC). In primary culture or at 1-2 passages of HAEC but not HUVEC: (1) about 20% of cells constitutively expressed VCAM (but not ELAM) on their surface, (2) 10-15% of cells were negative for PECAM, and (3) 10-20% of cells contained no P-selectin both on the surface and in Weibel-Palade bodies. No principal differences in TNF-a or IL-1 induced expression of VCAM, ELAM and ICAM were found between HAEC and HUVEC. In long-term cultures resting HAEC did not express VCAM and higher concentrations of TNF-a and IL-1 were necessary for VCAM induction. The number of cells negative for P-selectin increased up to 80-90% after 5-6 passages. Heterogeneous expression of CAM in HAEC could reflect their different ability for interaction with white blood cells in vascular inflamatory diseases and atherosclerosis.
211
213
EFFECT OF ATHERO- AND NONATHEROGENIC LIPOPROTEINS ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELL ARACHIDONICACID METABOLISM Ts. Gerassimova Y. Barannikova, N Perova National Research Centre for Preventive Medicine, Moscow, Russia
DOCOSAHEXAENOIC ACID REDUCES VASCULAR CELL ADHESION MOLECULE 1 INDUCTION AND MONOCYTE ADHESION TO STIMULATED HUMAN ENDOTHELIAL CELLS C. Weber. W. Erl, A. Pietsch. P.C. Weber Institut fftr Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-Universit/it, Munich, FRG
To elucidate whether athero- and nonatherogenic lipoproteins (LP) have specific effect on cell cyclooxygenase cascade human umbilical vein endothelial cells (HUVEC) were prelabe[d with 3H-arachidonic acid (AA) and incubated with human serum LP separated at densities (g/ml): 1,006 - VLDL; 1,03-1,05 - LDL; 1,065-1,125 - HDI<; 1,125-1,21 - HDL3; 1,25 - LPDS. The medium was analyzed for total radioactivity and 3H-AA metabolite spectrum by thin layer chromatography. All LP and LPDS increased the total radioactivity. SP and LPDS did not change 3H-AA raetabolite spectrum at 1:10 and 1:100 dilution of the original preparations, respectively. All undiluted LP any LPDS (1:10) decreased the proportion of 3H-metabolites in the region of prostaglandin (Pg) F28, F ~ , Fz~, 6-keto-PgF~ recovery and exept VLDL increased the proportion of PgA. Some new peaks with Rf between PgA and free AA were seen under the action of LDL, HDLz and HDL3. Thus, LP activate HUVEC AA metabolism but without evidence for specific action of athero- and nonatherogenic lipoproteins.
Incorporation of the n-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) but not eicosapentaenoic acid (EPA) or n-6 arachidonic acid (AA) into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor-alpha (TNF) induced surface expression of vascular cell adhesion molecule 1 (VCAM-I). In parallel, DHA inhibited TNF stimulated monocytic U937 cell adhesion to HUVEC, but did not affect TNF or IFN induced expression of intercellular adhesion molecule 1 (ICAM-1) and endothelial leukocyte adhesion molecule l (ELAM-1) or VCAM-1 induction by interleukin-lB (IL-IB). DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-I mRNA by TNF. VCAM-1 induction is regulated by activation of nuclear factor 103 (NF-kB), which can be mediated by a TNF-responsive phosphatidylcholine-specific phospholipase C (PCPLC). Gelshifl analysis showed inhibition of TNF-induced NF-kB mobilisation by DHA. While the PC-PLC inhibitor D609 dosedependently prevented VCAM-I induction by TNF, 1,2-diacylglycerol (DAG) stimulated VCAM-I expression, suggesting that VCAM-1 induction by TNF is mediated by activation of PC-PLC Treatment with DHA resulted in 4 fold enrichment in PC. In addition, DHA or D609 but not EPA or AA suppressed activation of PC-PLC by TNF, estimated as [~4C]DAG synthesis in labeled HUVEC. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF and mnnocyte adhesion by inhibition of PC-PLC activation.
Poster session abstracts / Atherosclerosis 115 (SuppL ) (1995) $45-$129 P4 Endothelial Cells
210
212
ENDOTHELIAL CELL CYTOTOX1C AUTOANTIBODIES AGAINST HEAT SHOCK PROTEIN 60 IN ATHEROSCLEROSIS G. Schett~2, A. Ambergert, H. Recheis:, Q. Xu ~, G.Wick~2 qnst for Biomedical Aging Research, Austrian Academy of Sciences, ~lnst for General and Experimental Pathology, University of Innsbruck, Austria
DISTRIBUTION OF CELL ADHESION MOLECULES 1N CULTURED ENDOTHELIAL CELLS FROM HUMAN AORTA T.V. Byzova, Y.A. Romanov, A.V. Mazurov Cardiology Research Cemre, 3rd Cherepkovskaya 15a, 121552 Moscow, Russia
Previous studies from our laboratory have proven that serum antibodies (Abs) against mycobacterial heat shock protein (hsp) 65 are increased in clinically healthy subjects with sonographically detectable carotid atherosclerosis (Xu et al., Lancet 341:255,1993). We hypothetized that these Abs thal bind to stressed human cells via a crossreaction to their hsp-homologue hsp6Q may therefore be of possible functional importance in the developement of the disease. We now investigated complement-mediated lysis and antibody dependent cellular cytotoxicity (ADCC) to cultured human umbilical vein endothelial cells (HUVEC) using these Abs, to determine whether they react with homologous human hsp60 and whether they play a potential pathogenetic role. Positive staining with these high titer antisera (hts) or with affinity purified antihsp65 antibodies (hspkAb) was observed on the surface of cultured HUVEC stressed at 42°C for 30 rain, while unstressed cells showed only weak or no fluorescence. When stressed endothelial cells labeled with ~Cr were incubated with hts or hsp-Ab in the presence of complement, significant lysis occurred. Similarly, ~Cr release by HUVEC was observed when his or hsp-Ab and peripheral blood mononuclear cells (effector) were added to the culture (ADCC). In summary, serumantibodies to mycobacterial hsp65 cross-react with human 60 kDa homologue present in high levels on the surface of stressed human endothelial cells, and are able to evoke their lysis via antibody-complement mediated cell lysis and ADCC. These data support the hypothesis that humoral-mediated immune reactions, in addition to cell mediated immunity, may play a role in athero genesis. (Supported by the Austrian Research- Council project 10677 and the Sandoz Foundation for Gerontological Research).
Distribution of cell adhesion molecules (CAM) in cultured human aortic endothelial cells (HAEC) was studied using immunohistochemistry and FACS analysis. Several features of CAM expression were specific for HAEC in comparison with the standard culture of human umbilical vein EC (HUVEC). In primary culture or at 1-2 passages of HAEC but not HUVEC: (1) about 20% of cells constitutively expressed VCAM (but not ELAM) on their surface, (2) 10-15% of cells were negative for PECAM, and (3) 10-20% of cells contained no P-selectin both on the surface and in Weibel-Palade bodies. No principal differences in TNF-a or IL-1 induced expression of VCAM, ELAM and ICAM were found between HAEC and HUVEC. In long-term cultures resting HAEC did not express VCAM and higher concentrations of TNF-a and IL-1 were necessary for VCAM induction. The number of cells negative for P-selectin increased up to 80-90% after 5-6 passages. Heterogeneous expression of CAM in HAEC could reflect their different ability for interaction with white blood cells in vascular inflamatory diseases and atherosclerosis.
211
213
EFFECT OF ATHERO- AND NONATHEROGENIC LIPOPROTEINS ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELL ARACHIDONICACID METABOLISM Ts. Gerassimova Y. Barannikova, N Perova National Research Centre for Preventive Medicine, Moscow, Russia
DOCOSAHEXAENOIC ACID REDUCES VASCULAR CELL ADHESION MOLECULE 1 INDUCTION AND MONOCYTE ADHESION TO STIMULATED HUMAN ENDOTHELIAL CELLS C. Weber. W. Erl, A. Pietsch. P.C. Weber Institut fftr Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-Universit/it, Munich, FRG
To elucidate whether athero- and nonatherogenic lipoproteins (LP) have specific effect on cell cyclooxygenase cascade human umbilical vein endothelial cells (HUVEC) were prelabe[d with 3H-arachidonic acid (AA) and incubated with human serum LP separated at densities (g/ml): 1,006 - VLDL; 1,03-1,05 - LDL; 1,065-1,125 - HDI<; 1,125-1,21 - HDL3; 1,25 - LPDS. The medium was analyzed for total radioactivity and 3H-AA metabolite spectrum by thin layer chromatography. All LP and LPDS increased the total radioactivity. SP and LPDS did not change 3H-AA raetabolite spectrum at 1:10 and 1:100 dilution of the original preparations, respectively. All undiluted LP any LPDS (1:10) decreased the proportion of 3H-metabolites in the region of prostaglandin (Pg) F28, F ~ , Fz~, 6-keto-PgF~ recovery and exept VLDL increased the proportion of PgA. Some new peaks with Rf between PgA and free AA were seen under the action of LDL, HDLz and HDL3. Thus, LP activate HUVEC AA metabolism but without evidence for specific action of athero- and nonatherogenic lipoproteins.
Incorporation of the n-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) but not eicosapentaenoic acid (EPA) or n-6 arachidonic acid (AA) into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor-alpha (TNF) induced surface expression of vascular cell adhesion molecule 1 (VCAM-I). In parallel, DHA inhibited TNF stimulated monocytic U937 cell adhesion to HUVEC, but did not affect TNF or IFN induced expression of intercellular adhesion molecule 1 (ICAM-1) and endothelial leukocyte adhesion molecule l (ELAM-1) or VCAM-1 induction by interleukin-lB (IL-IB). DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-I mRNA by TNF. VCAM-1 induction is regulated by activation of nuclear factor 103 (NF-kB), which can be mediated by a TNF-responsive phosphatidylcholine-specific phospholipase C (PCPLC). Gelshifl analysis showed inhibition of TNF-induced NF-kB mobilisation by DHA. While the PC-PLC inhibitor D609 dosedependently prevented VCAM-I induction by TNF, 1,2-diacylglycerol (DAG) stimulated VCAM-I expression, suggesting that VCAM-1 induction by TNF is mediated by activation of PC-PLC Treatment with DHA resulted in 4 fold enrichment in PC. In addition, DHA or D609 but not EPA or AA suppressed activation of PC-PLC by TNF, estimated as [~4C]DAG synthesis in labeled HUVEC. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF and mnnocyte adhesion by inhibition of PC-PLC activation.