- Email: [email protected]
Isolation and characterization of non-uniform shear stress-responsive genes, RECS
174
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Nieto POTENTIAL INVOLVEMENT OF SECRETORY PHOSPHOLIPASE A2 ACTIVITY IN ATHEROSCLEROSIS. CORRELATION W I T H THE DYAD C D 4 0 / C D 4 0 L
L. Fuentes 1, M. Sanchez-Crespo 1, M. Hernandez 1, J. Alonso 2, E Fernandez-Aviles 2, M.L. Nieto 1. JlBGM-Facultad de Medicina,
CSIC-Universidad de Valladolid," 2ICICOR-Hospital Clinico, Valladolid, Spain Acute coronary events commonly result from thrombosis triggered by disruption of an atherosclerotic plaque. The development of a new concept of assessing the pathogenesis of atherosclerosis in the context of immuneinflammation in the vascular wall open new vistas for the comprehension of this disease, but the mechanism underlying this network are widely unknown. Recent reports have pointed to the dyad CD40-CD40L as a stimulus for atheroma-associated cells. Since, it has been reported that vascular cells, endothelial cells and macrophages can express functional CD40/CD40L. To elucidate the biochemical pathways involved in the progression of the vascular lesion, we have studied the effect of secretory phospholipase A2 (sPLA2) on the expression and signaling of CD40 in the THP-1 cell line. This cell line shows functional similarities with the macrophages located in the vessel wall that exacerbate the local inflammatory response in the atherosclerotic lesion. We have also compared its effect with the cytokine TNF-c~. THP-1 ceils, express CD40 functional receptors and stimulation with sPLA2 or TNF-c~ led to its up-regulation. The cellular response to CD40 engagement includes cytosolic phospholipase A2 phosphorylation, arachidonic acid movilization and increase in the production of MCP-1 and in the expression of Fas/FasL. These effects were enhanced in the presence of sPLA2 and TNF-c~. Although, CD40 ligation modulates the expression of the system Fas/FasL does not affect survival and might represent a juxtacrine mechanism of signaling involving other cell types These data show that sPLA2 elicits CD40 expression on macrophages, linking the inflammatory and the immune responses. F b - ~ APOLIPOPROTEIN(a) ISOFORMS DISTRIBUTION IN MEN W I T H ISCHEMIC HEART DISEASE AND W I T H GALLSTONE DISEASE (EPIDEMIOLOGICAL STUDY) Y.P. Nikitin, A.V. Tikhonov, I.N. Grigorieva. Institute of Internal Medicine,
Novosibirsk, Russia To study apoprotein(a) - Apo(a) - isoforms in men with and without IHD or GSD. The representative sample of 830 men has been surveyed in 19941995 in the frame WHO MONICA Program. In GSD patients aged 35 54 (100% sonographic examination), IHD patients aged 25 64 - 'definite' IHD (at detection severe disturbances Q, Q-S, ST-T electrocardiograph intervals), and disease-free men isoforms Apo(a) were examined by immunoblotting. The frequency of isoforms B-S4 is conditionally adopted for 100%. In men with IHD Apo(a) isoforms B (M(m)%2(1,3), S1 (16,4(0,9), $2 (25,0(0,9), were found more frequently than without IHD (0,8(0,1)0(0,06), 17,4(0,08), respectively, p<0,05 in all cases). Isoforms 0 (30,1(0,8) vs. 58,5(0,2), $3 (11,2(0,8) vs. 31,5(0,09) were found more seldom in men with IHD than without IHD (p<0,05 in both cases). Isoform $4 frequency did not differ in men with and without IHD. In men with GSD isoforms $2 (50,0(1,3) and B (12,5(0,9) were found more frequently than without GSD (18,1(0,05) and,3(0,01), p<0,01 in both cases), and isoform $4 (12,5(0,9) were found more seldom than without GSD (47,6(0,07), p<0,01). Isoform, $3 frequency did not differ in men with and without GSD (p>0,05). This results allows to suppose certain connection of Apo(a) polymorphism in IHD and GSD pathogenesis, as possible risk factor in West Siberian men population. ~]
APO(a) ISOFORMS AND SERUM LIPID LEVELS IN MEN W I T H ISCHEMIC HEART DISEASE AND W I T H GALLSTONE DISEASE (EPIDEMIOLOGICAL STUDY)
Y.E Nikitin, A.V. Tikhonov, I.N. Grigorieva. Institute of Internal Medicine,
Novosibirsk, Russia To study serum lipid levels under different apolipoprotein(a) - Apo(a) i s o f o r m s in men with IHD and with GSD. The representative sample of 830 men has been surveyed in 1994 1995 in the frame WHO MONICA Program. In GSD patients aged 35 54 (100% sonographic examination) and in IHD patients aged 25 64 - 'definite' IHD (at detection severe disturbances Q, Q-S, ST-T electrocardiograph intervals) isoforms Apo(a) were examined by immunoblotting. The data (CHOL, HDL, TG) were ageadjusted. Was used ANOVA statistical method. For the men with IHD
were detected significant exceeding of CHOL, TG levels at all Apo(a) isoforms in relation to the nil Apo(a) isoform. Inter-group differences exceeded above innergroup dispersion of CHOL (pANOVA-0,0001), HDL (pANOVA-0,047), TG (pANOVA-0,016). Only tendency to such exceeding of CHOL was detected for the men with GSD (pANOVA-0,09). Minimum CHOL levels were in Apo(a) isoform nil - (M(m) 177(7) and 192(36) mg/dl in men with IHD and with GSD, maximum - in Apo(a) B, S1 in men with IHD (344(7) and 249(11) mg/dl) and in $2, $3 in men with GSD (253(63) and 223(15) mg/dl). In urban population of Western Siberia in men with 'definite' IHD elevated CHOL levels under all Apo(a) isoforms were found more frequently than in patients with nil Apo(a) isoform, ditto pertains and to TG. In men with GSD was discovered only trend to raising CHOL under Apo(a) isoforms B, $2.
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F O R LIPOPROTEIN RECEPTORS A T T H E CROSSROAD OF ENDOCYTOSIS AND SIGNALING
ADAPTERS
J. Nimpf. Institute of Medical Biochemistry, Department of Molecular
Genetics, University and BioCenter of Vienna, Austria Members of the LDL receptor gene family are type-I transmembrane proteins involved in endocytosis of macromolecules. Recent data however, demonstrated that via interaction with intracellular adapter proteins, some members of the family act as signal transducers. In particular, VLDL receptor and ApoER2 bind the extracellular matrix protein reelin which is secreted by specialized neurons in the developing CNS. This signal is translated into migrating neurons by the interaction of the intracellular domains of both receptors with Dabl which subsequently becomes phosphorylated and migrating target neurons detach from glial fibers and assume their final position in the developing brain. In addition, JNK-interacting proteins like JIP-1 and JIP-2 also interact with VLDL receptor and ApoER2 connecting the reelin pathway with the JNK-signaling cascade. On the other hand, binding of SNX17, a member of the family of sorting nexins, modulates the endocytic activity of these receptors. From these data a novel concept emerges in that the interplay of a variety of intracellular adapter proteins with LDL receptor family members may regulate their endocytic and signaling activity.
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]
ISOLATION AND CHARACTERIZATION OF NON-UNIFORM SHEAR STRESS-RESPONSIVE GENES, RECS
H. Noiima 1, H. Yoshisue 2, H. Zhao 3, T. Ohya3, S-Y. Kimura 1, S. Yamashita 3 , Y. Matsuzawa3 . JRIMD Osaka University, Suita, Osaka,"
2Kyowa Hakko Kogyo, Machida, Tokyo; 3Graduate School of Medicine, Osaka University, Suita, Osaka, Japan In order to investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have isolated non-uniform shear stressresponsive genes that are named RECS after responsive to centrifugal force and shear stress. We have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells, HUVEC, using a microcarrier culture system, extracted mRNA and prepared a cDNA library. We have isolated a number of genes upregulated by shear stress by a combination of subtraction and reverse-subtraction methods. The resultant subtracted library includes several known genes (eg, MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (eg, CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel RECS genes. Some RECS genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis. Detailed functional analysis on RECS1 will be presented.
73rd EAS Congress
•0-•
Nieto POTENTIAL INVOLVEMENT OF SECRETORY PHOSPHOLIPASE A2 ACTIVITY IN ATHEROSCLEROSIS. CORRELATION W I T H THE DYAD C D 4 0 / C D 4 0 L
L. Fuentes 1, M. Sanchez-Crespo 1, M. Hernandez 1, J. Alonso 2, E Fernandez-Aviles 2, M.L. Nieto 1. JlBGM-Facultad de Medicina,
CSIC-Universidad de Valladolid," 2ICICOR-Hospital Clinico, Valladolid, Spain Acute coronary events commonly result from thrombosis triggered by disruption of an atherosclerotic plaque. The development of a new concept of assessing the pathogenesis of atherosclerosis in the context of immuneinflammation in the vascular wall open new vistas for the comprehension of this disease, but the mechanism underlying this network are widely unknown. Recent reports have pointed to the dyad CD40-CD40L as a stimulus for atheroma-associated cells. Since, it has been reported that vascular cells, endothelial cells and macrophages can express functional CD40/CD40L. To elucidate the biochemical pathways involved in the progression of the vascular lesion, we have studied the effect of secretory phospholipase A2 (sPLA2) on the expression and signaling of CD40 in the THP-1 cell line. This cell line shows functional similarities with the macrophages located in the vessel wall that exacerbate the local inflammatory response in the atherosclerotic lesion. We have also compared its effect with the cytokine TNF-c~. THP-1 ceils, express CD40 functional receptors and stimulation with sPLA2 or TNF-c~ led to its up-regulation. The cellular response to CD40 engagement includes cytosolic phospholipase A2 phosphorylation, arachidonic acid movilization and increase in the production of MCP-1 and in the expression of Fas/FasL. These effects were enhanced in the presence of sPLA2 and TNF-c~. Although, CD40 ligation modulates the expression of the system Fas/FasL does not affect survival and might represent a juxtacrine mechanism of signaling involving other cell types These data show that sPLA2 elicits CD40 expression on macrophages, linking the inflammatory and the immune responses. F b - ~ APOLIPOPROTEIN(a) ISOFORMS DISTRIBUTION IN MEN W I T H ISCHEMIC HEART DISEASE AND W I T H GALLSTONE DISEASE (EPIDEMIOLOGICAL STUDY) Y.P. Nikitin, A.V. Tikhonov, I.N. Grigorieva. Institute of Internal Medicine,
Novosibirsk, Russia To study apoprotein(a) - Apo(a) - isoforms in men with and without IHD or GSD. The representative sample of 830 men has been surveyed in 19941995 in the frame WHO MONICA Program. In GSD patients aged 35 54 (100% sonographic examination), IHD patients aged 25 64 - 'definite' IHD (at detection severe disturbances Q, Q-S, ST-T electrocardiograph intervals), and disease-free men isoforms Apo(a) were examined by immunoblotting. The frequency of isoforms B-S4 is conditionally adopted for 100%. In men with IHD Apo(a) isoforms B (M(m)%2(1,3), S1 (16,4(0,9), $2 (25,0(0,9), were found more frequently than without IHD (0,8(0,1)0(0,06), 17,4(0,08), respectively, p<0,05 in all cases). Isoforms 0 (30,1(0,8) vs. 58,5(0,2), $3 (11,2(0,8) vs. 31,5(0,09) were found more seldom in men with IHD than without IHD (p<0,05 in both cases). Isoform $4 frequency did not differ in men with and without IHD. In men with GSD isoforms $2 (50,0(1,3) and B (12,5(0,9) were found more frequently than without GSD (18,1(0,05) and,3(0,01), p<0,01 in both cases), and isoform $4 (12,5(0,9) were found more seldom than without GSD (47,6(0,07), p<0,01). Isoform, $3 frequency did not differ in men with and without GSD (p>0,05). This results allows to suppose certain connection of Apo(a) polymorphism in IHD and GSD pathogenesis, as possible risk factor in West Siberian men population. ~]
APO(a) ISOFORMS AND SERUM LIPID LEVELS IN MEN W I T H ISCHEMIC HEART DISEASE AND W I T H GALLSTONE DISEASE (EPIDEMIOLOGICAL STUDY)
Y.E Nikitin, A.V. Tikhonov, I.N. Grigorieva. Institute of Internal Medicine,
Novosibirsk, Russia To study serum lipid levels under different apolipoprotein(a) - Apo(a) i s o f o r m s in men with IHD and with GSD. The representative sample of 830 men has been surveyed in 1994 1995 in the frame WHO MONICA Program. In GSD patients aged 35 54 (100% sonographic examination) and in IHD patients aged 25 64 - 'definite' IHD (at detection severe disturbances Q, Q-S, ST-T electrocardiograph intervals) isoforms Apo(a) were examined by immunoblotting. The data (CHOL, HDL, TG) were ageadjusted. Was used ANOVA statistical method. For the men with IHD
were detected significant exceeding of CHOL, TG levels at all Apo(a) isoforms in relation to the nil Apo(a) isoform. Inter-group differences exceeded above innergroup dispersion of CHOL (pANOVA-0,0001), HDL (pANOVA-0,047), TG (pANOVA-0,016). Only tendency to such exceeding of CHOL was detected for the men with GSD (pANOVA-0,09). Minimum CHOL levels were in Apo(a) isoform nil - (M(m) 177(7) and 192(36) mg/dl in men with IHD and with GSD, maximum - in Apo(a) B, S1 in men with IHD (344(7) and 249(11) mg/dl) and in $2, $3 in men with GSD (253(63) and 223(15) mg/dl). In urban population of Western Siberia in men with 'definite' IHD elevated CHOL levels under all Apo(a) isoforms were found more frequently than in patients with nil Apo(a) isoform, ditto pertains and to TG. In men with GSD was discovered only trend to raising CHOL under Apo(a) isoforms B, $2.
•0-•
F O R LIPOPROTEIN RECEPTORS A T T H E CROSSROAD OF ENDOCYTOSIS AND SIGNALING
ADAPTERS
J. Nimpf. Institute of Medical Biochemistry, Department of Molecular
Genetics, University and BioCenter of Vienna, Austria Members of the LDL receptor gene family are type-I transmembrane proteins involved in endocytosis of macromolecules. Recent data however, demonstrated that via interaction with intracellular adapter proteins, some members of the family act as signal transducers. In particular, VLDL receptor and ApoER2 bind the extracellular matrix protein reelin which is secreted by specialized neurons in the developing CNS. This signal is translated into migrating neurons by the interaction of the intracellular domains of both receptors with Dabl which subsequently becomes phosphorylated and migrating target neurons detach from glial fibers and assume their final position in the developing brain. In addition, JNK-interacting proteins like JIP-1 and JIP-2 also interact with VLDL receptor and ApoER2 connecting the reelin pathway with the JNK-signaling cascade. On the other hand, binding of SNX17, a member of the family of sorting nexins, modulates the endocytic activity of these receptors. From these data a novel concept emerges in that the interplay of a variety of intracellular adapter proteins with LDL receptor family members may regulate their endocytic and signaling activity.
•
]
ISOLATION AND CHARACTERIZATION OF NON-UNIFORM SHEAR STRESS-RESPONSIVE GENES, RECS
H. Noiima 1, H. Yoshisue 2, H. Zhao 3, T. Ohya3, S-Y. Kimura 1, S. Yamashita 3 , Y. Matsuzawa3 . JRIMD Osaka University, Suita, Osaka,"
2Kyowa Hakko Kogyo, Machida, Tokyo; 3Graduate School of Medicine, Osaka University, Suita, Osaka, Japan In order to investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have isolated non-uniform shear stressresponsive genes that are named RECS after responsive to centrifugal force and shear stress. We have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells, HUVEC, using a microcarrier culture system, extracted mRNA and prepared a cDNA library. We have isolated a number of genes upregulated by shear stress by a combination of subtraction and reverse-subtraction methods. The resultant subtracted library includes several known genes (eg, MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (eg, CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel RECS genes. Some RECS genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis. Detailed functional analysis on RECS1 will be presented.
73rd EAS Congress