- Email: [email protected]
Adhesion molecules and heat shock protein 60 (HSP 60) are concomitantly expressed in stressed human vascular endothelial cells
S I0
Oral session abstracts / Atherosclerosis 115 (Suppl.) (1995) $3-$42 05 Molecular Biology of Receptors
029
03O
I D E N T I F I C A T I O N O F F O U R L Y S I N E - B I N D I N G SITES IN A P O L I P O P R O T E I N ( A ) A N D T H E I R R O L E F O R T H E ASSEMBLY OF RECOMBINANT LIPOPROTEIN(A) A. Ernst, M. Helmhold', C. Brunner, A. PethO-Schramm, V.-W. Armstrong', H.-J. MOiler Boehringer Mannheim G m b H , Molekularbiologie, 68305 Mannheim and "Georg-August-Universiti0 GOttingen, Abteilung Klinische Chemie, 37075 G6ttingen, Germany
MICE DEFICIENT IN BOTH APOLIPOPROTEIN E AND CI GENERATED BY CONSECUTIVE GENE TARGETING J.H. van Ree, W.J.J.A. van den Brock, R.R. Frants, L. Havekes, M. Hofker Dept of Human Genetics, Leiden University, TNO Prevention and Health, Gaubius Laboratory, Leiden, and Dept of Cell Biology and Histology, Nijmegen University, The Netherlands
In order to localize lysine-binding sites in apolipoprotein(a) [apo(a)] and to investigate a possible role of these sites for lipoprotein(a) [Lp(a)] assembly, we performed a mutational analysis of an 18 kringle recombinant apo(a) [r-apo(a)] isoform. Wild type r-apo(a) as well as single or multiple point mutants thereof, carrying amino acid substitutions in the putative lysine-binding pockets of kringels 32 -37, were transiently expressed in HepG2-cells. F r o m a comparison o f wild type and mutant r-apo(a) species by lysine-Sepharose chromatography we identified four lysine-binding sites [LBS] in the r-apo(a) glycoprotein, positioned in the carboxyterminal kringles 32, 33, 34/35 and 37. No lysine-binding activity was detected in r-apo(a) kringles 1-31 or 36. Analysis of cell culture supernatants of transfected HepG2-cells by non-reducing S D S - P A G E and subsequent immunoblotting revealed that a r-apo(a) mutated in the LBS of the four kringles 32-35 was unable to form r-Lp(a). This r-apo(a) species was found exclusively as free r-apo(a) glycoprotein. In contrast, wild type r-apo(a) or mutants with substitutions in a single LBS were seen predominantly as component of r-Lp(a) particles. Our data are consistent with a two-step model for Lp(a) particle formation, where multiple non-covalent interactions between LBS in apo(a) kringles 32-35 and lysine-residues in apoB precede the disulfide bridging of the two proteins.
The Apolipoprotein E (Apoe) and Apocl genes are part of a conserved gene cluster in man and mouse. ApoE is the major structural component of very low density lipoprotein (VLDL), and serves as a ligand for the receptor mediated clearance of VLDL remnants by the liver. ApoC1 also resides on the VLDL, but its role is unclear. In vitro studies have shown that apoCl can block apoE mediated uptake of B-VLDL by the hepatic receptors. The interaction between apoE and apoCl is being studied in vivo using transgenic mice. A model has been generated in which APOE and APOC1 are simultaneously expressed. To eliminate the expression of the endogenous genes in such a model, mice have been generated by consecutive targeting of the Apoe and Apocl genes. First, an embryonic stemcell (ES) line was obtained, in which the neomycin gene replaces a part of the Apoe gene (Gift of Dr. A. Plump, Rockefeller University, New York). Second, a hygromycin-based targeting vector was used to disrupt the Apoc[ gene in the chromosome containing the targeted Apoe allele. Subsequently, mice were generated lacking both apoE and apoCl. Basic characterization reveals that the lipoprotein levels of the doubly targeted mice resemble that of Apoe knock-out mice. However, the expression of the Apoc2 gene, which is a member of the Apoe gene-cluster as well, is down regulated in the homozygous knock-out mice. This effect may be caused (1) directly at the level of gene regulation, due to the close proximity of the neomycin and hygromycin genes, or (2) could occur as a consequence of the altered [ipoprotein metabolism. The application of two rounds of gene targeting in the same ES cell line is very useful when more mutations axe desired in closely linked genes like this particular geoe cluster.
0 6 ADHESION MOLECULES 031
032
PLASMA LEVEL OF ADHESION MOLECULES IN PATIENTS SUFFERING FROM STABLE AND UNSTABLE ANGINA A. Mazzone G. Fossati, I. Mazzucchelli, D. Gritti, M . G Randine, C. Canale, M. Momagna, C. Pistone, G Ricevuti Dipartimemo di Medicina lnterna e Terapia Medica, Istitoto di Terapia Medica, Universita di Pavia, IRCCS Policlinico San Matteo, Pavia.
ADHESION MOLECULES AND HEAT SHOCK PROTEIN 60 (HSP 60) ARE CONCOMITANTLY EXPRESSED IN STRESSED HUMAN VASCULAR ENDOTHELIAL CELLS A. AmbergeP, Ch. Maczck 2, K. Triel~, Tit. EbesP, G. Wick ~'2 qnst for Biomedical Aging Research, Austrian Academy of Sciences, 21nst f General and Experimental Pathology; 3Dept of Surgery, University of lrmsbruck, Medical School, Austria
Adhesion molecules play a pivotal role in leukocyte-endothelial interaction. The aim of this work is to study the role of adhesion molecules in the pathogenesis of stable angina (SA) and unstable angina (UA). We studied 60 patients who undergoing coronary arteriography: 38 patients with unstable angina (UA); 22 patients with stable angina (SA). We collected 5 ml of eparinized blood in coronary sinus (CS) and in aorta (AO) before coronary arteriography. We prepared plasma for evaluating slCAM-I, sLselectin, HLA class I, TNF-~, lactoferrin by immunocuzymatic ELISA method. We show in table I our results. UA SA AO CS AO CS slCAM-1 (ng/ml) 239+23 254_+35 222_+15 267_+30 sL-selectin (ng/ml) 625_+35 609_+27 623_+46 650_+47 HLA class 1 (v.g/ml) 1.1_+0.95 1.19_+1.0 0.363-+0.1 0.33_+0.1 TNF ~ (pg/ml) 27.6_+2.7 31.6-+3.3" 28.5-+2.0 28.6_+3.5 lactoferrin (ng/ml) 1 9 3 _ + 3 8 420+89"" 1 9 3 _ + 5 3 180-+45 "p <0.1; "'p < 0.01 Our results show significative difference between AO and CS in UA for HLA class I, TNF-<~ and lactoferrin. There was no signiftcative difference in the expression of adhesion molecules in AO respect CS in SA. Our results support the hypothesis that in patients with angina, endotbelium and leukocytes network are involved in an inflammatory reaction. Particularly in patients with UA an acute inflammatory process takes place in coronary tree.
Recent data from our group have provided evidence that a high proportion of T-cells isolated from atherosclerotic intima reacts against HSP 60. These and other data in experimental animals suggest that HSP 60 may act as an autoantigen in the earliest stages of atherosclerosis. Since it is also well established that the migration of leucocytes into tissues is regulated by adhesion molecules expressed on endothelial cells, we studied the expression of vascular cell adhesion molecule (VCAM-I), intercellular adhesion molecule (ICAM-I), E-Selectin (ELAM-I) and heat shock protein 60 (HSP 60) in stressed human venous and arterial endothelial cells. Human vein endothelial cells (HVEC) and human arterial endothelial cells (HAEC) were grown as monolayers and then exposed to different types of stress, including inflammatory mediators (IL-1, TNF-~, IFN-'y), high temperature (42°C for 30 min), E. coil LPS and native or oxidized low densitiy lipoproteins (LDL and oxLDL). Except heat shock and native LDL, all stressors initiated co-expression of HSP 60 and adhesion molecules with a similar time course. On Northern blots, appearance of all transcripts investigated starts 1 hr after stress application, reaches the maximum after 2-4 hrs and is expressed c.ominously as long as stress is applied. On the protein level ELAMI, ICAM-I and HSP 60 appear 2-4 hrs and VCAM-I from 4-6 hrs after stress. Our results suggest that co-expression of adhesion molecules and HSP 60 in human endothelial cells is a normal physiological reaction against various forms of stress, providing the prerequisites for possible recruitment of HSP 60 reactive T-cells, subsequent transmigration into the imima and initiation of the atherosclerotic process. Supported by the Austrian Reseal"ch Council (Pr. Nr,: 8925)
Oral session abstracts / Atherosclerosis 115 (Suppl.) (1995) $3-$42 05 Molecular Biology of Receptors
029
03O
I D E N T I F I C A T I O N O F F O U R L Y S I N E - B I N D I N G SITES IN A P O L I P O P R O T E I N ( A ) A N D T H E I R R O L E F O R T H E ASSEMBLY OF RECOMBINANT LIPOPROTEIN(A) A. Ernst, M. Helmhold', C. Brunner, A. PethO-Schramm, V.-W. Armstrong', H.-J. MOiler Boehringer Mannheim G m b H , Molekularbiologie, 68305 Mannheim and "Georg-August-Universiti0 GOttingen, Abteilung Klinische Chemie, 37075 G6ttingen, Germany
MICE DEFICIENT IN BOTH APOLIPOPROTEIN E AND CI GENERATED BY CONSECUTIVE GENE TARGETING J.H. van Ree, W.J.J.A. van den Brock, R.R. Frants, L. Havekes, M. Hofker Dept of Human Genetics, Leiden University, TNO Prevention and Health, Gaubius Laboratory, Leiden, and Dept of Cell Biology and Histology, Nijmegen University, The Netherlands
In order to localize lysine-binding sites in apolipoprotein(a) [apo(a)] and to investigate a possible role of these sites for lipoprotein(a) [Lp(a)] assembly, we performed a mutational analysis of an 18 kringle recombinant apo(a) [r-apo(a)] isoform. Wild type r-apo(a) as well as single or multiple point mutants thereof, carrying amino acid substitutions in the putative lysine-binding pockets of kringels 32 -37, were transiently expressed in HepG2-cells. F r o m a comparison o f wild type and mutant r-apo(a) species by lysine-Sepharose chromatography we identified four lysine-binding sites [LBS] in the r-apo(a) glycoprotein, positioned in the carboxyterminal kringles 32, 33, 34/35 and 37. No lysine-binding activity was detected in r-apo(a) kringles 1-31 or 36. Analysis of cell culture supernatants of transfected HepG2-cells by non-reducing S D S - P A G E and subsequent immunoblotting revealed that a r-apo(a) mutated in the LBS of the four kringles 32-35 was unable to form r-Lp(a). This r-apo(a) species was found exclusively as free r-apo(a) glycoprotein. In contrast, wild type r-apo(a) or mutants with substitutions in a single LBS were seen predominantly as component of r-Lp(a) particles. Our data are consistent with a two-step model for Lp(a) particle formation, where multiple non-covalent interactions between LBS in apo(a) kringles 32-35 and lysine-residues in apoB precede the disulfide bridging of the two proteins.
The Apolipoprotein E (Apoe) and Apocl genes are part of a conserved gene cluster in man and mouse. ApoE is the major structural component of very low density lipoprotein (VLDL), and serves as a ligand for the receptor mediated clearance of VLDL remnants by the liver. ApoC1 also resides on the VLDL, but its role is unclear. In vitro studies have shown that apoCl can block apoE mediated uptake of B-VLDL by the hepatic receptors. The interaction between apoE and apoCl is being studied in vivo using transgenic mice. A model has been generated in which APOE and APOC1 are simultaneously expressed. To eliminate the expression of the endogenous genes in such a model, mice have been generated by consecutive targeting of the Apoe and Apocl genes. First, an embryonic stemcell (ES) line was obtained, in which the neomycin gene replaces a part of the Apoe gene (Gift of Dr. A. Plump, Rockefeller University, New York). Second, a hygromycin-based targeting vector was used to disrupt the Apoc[ gene in the chromosome containing the targeted Apoe allele. Subsequently, mice were generated lacking both apoE and apoCl. Basic characterization reveals that the lipoprotein levels of the doubly targeted mice resemble that of Apoe knock-out mice. However, the expression of the Apoc2 gene, which is a member of the Apoe gene-cluster as well, is down regulated in the homozygous knock-out mice. This effect may be caused (1) directly at the level of gene regulation, due to the close proximity of the neomycin and hygromycin genes, or (2) could occur as a consequence of the altered [ipoprotein metabolism. The application of two rounds of gene targeting in the same ES cell line is very useful when more mutations axe desired in closely linked genes like this particular geoe cluster.
0 6 ADHESION MOLECULES 031
032
PLASMA LEVEL OF ADHESION MOLECULES IN PATIENTS SUFFERING FROM STABLE AND UNSTABLE ANGINA A. Mazzone G. Fossati, I. Mazzucchelli, D. Gritti, M . G Randine, C. Canale, M. Momagna, C. Pistone, G Ricevuti Dipartimemo di Medicina lnterna e Terapia Medica, Istitoto di Terapia Medica, Universita di Pavia, IRCCS Policlinico San Matteo, Pavia.
ADHESION MOLECULES AND HEAT SHOCK PROTEIN 60 (HSP 60) ARE CONCOMITANTLY EXPRESSED IN STRESSED HUMAN VASCULAR ENDOTHELIAL CELLS A. AmbergeP, Ch. Maczck 2, K. Triel~, Tit. EbesP, G. Wick ~'2 qnst for Biomedical Aging Research, Austrian Academy of Sciences, 21nst f General and Experimental Pathology; 3Dept of Surgery, University of lrmsbruck, Medical School, Austria
Adhesion molecules play a pivotal role in leukocyte-endothelial interaction. The aim of this work is to study the role of adhesion molecules in the pathogenesis of stable angina (SA) and unstable angina (UA). We studied 60 patients who undergoing coronary arteriography: 38 patients with unstable angina (UA); 22 patients with stable angina (SA). We collected 5 ml of eparinized blood in coronary sinus (CS) and in aorta (AO) before coronary arteriography. We prepared plasma for evaluating slCAM-I, sLselectin, HLA class I, TNF-~, lactoferrin by immunocuzymatic ELISA method. We show in table I our results. UA SA AO CS AO CS slCAM-1 (ng/ml) 239+23 254_+35 222_+15 267_+30 sL-selectin (ng/ml) 625_+35 609_+27 623_+46 650_+47 HLA class 1 (v.g/ml) 1.1_+0.95 1.19_+1.0 0.363-+0.1 0.33_+0.1 TNF ~ (pg/ml) 27.6_+2.7 31.6-+3.3" 28.5-+2.0 28.6_+3.5 lactoferrin (ng/ml) 1 9 3 _ + 3 8 420+89"" 1 9 3 _ + 5 3 180-+45 "p <0.1; "'p < 0.01 Our results show significative difference between AO and CS in UA for HLA class I, TNF-<~ and lactoferrin. There was no signiftcative difference in the expression of adhesion molecules in AO respect CS in SA. Our results support the hypothesis that in patients with angina, endotbelium and leukocytes network are involved in an inflammatory reaction. Particularly in patients with UA an acute inflammatory process takes place in coronary tree.
Recent data from our group have provided evidence that a high proportion of T-cells isolated from atherosclerotic intima reacts against HSP 60. These and other data in experimental animals suggest that HSP 60 may act as an autoantigen in the earliest stages of atherosclerosis. Since it is also well established that the migration of leucocytes into tissues is regulated by adhesion molecules expressed on endothelial cells, we studied the expression of vascular cell adhesion molecule (VCAM-I), intercellular adhesion molecule (ICAM-I), E-Selectin (ELAM-I) and heat shock protein 60 (HSP 60) in stressed human venous and arterial endothelial cells. Human vein endothelial cells (HVEC) and human arterial endothelial cells (HAEC) were grown as monolayers and then exposed to different types of stress, including inflammatory mediators (IL-1, TNF-~, IFN-'y), high temperature (42°C for 30 min), E. coil LPS and native or oxidized low densitiy lipoproteins (LDL and oxLDL). Except heat shock and native LDL, all stressors initiated co-expression of HSP 60 and adhesion molecules with a similar time course. On Northern blots, appearance of all transcripts investigated starts 1 hr after stress application, reaches the maximum after 2-4 hrs and is expressed c.ominously as long as stress is applied. On the protein level ELAMI, ICAM-I and HSP 60 appear 2-4 hrs and VCAM-I from 4-6 hrs after stress. Our results suggest that co-expression of adhesion molecules and HSP 60 in human endothelial cells is a normal physiological reaction against various forms of stress, providing the prerequisites for possible recruitment of HSP 60 reactive T-cells, subsequent transmigration into the imima and initiation of the atherosclerotic process. Supported by the Austrian Reseal"ch Council (Pr. Nr,: 8925)