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141 Lipoprotein metabolism and atherosclerosis in APOE∗3Leiden mice
Abstracts
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142
Use of scavenger receptor regulatory elemoots to target gene expression to otlwosclerotic pleques. C.K. Glass, A. Horvai, U! P&t&, A. Li ondJ. Collier University of California, San Diego, Department of Medicine, Division of Cellular and Molecular Medicine
Lack of aneurysm formation in atherosclerotic eorto from plosminogeo activator deficient mice doe to &ad metdloproteinow activity. P&r Carmelietl, Florea Lupu? Steve Shapiro~, Angela Drew! Peter lippi&, Roger Lijned, Lieve Moonsl, &Desire Collenl. (I) Center of Tramgene Technology and Gene transfer, Flanders Interuniversity of Biotechnology, Leuven, Belgium; (2) Thrombosis Research Institute, London, m (3) Tbe Jewish Hospital of St. Louis, Washington University, St-Louis; (4) Tbe Center for Inflammatory Diseases, Monasb Medical Center, Australia.
The development of macrophage foam cells witbin atherosclerotic lesions is thought to depend on the expression of one or more scavenger receptors that mediate the uptake of oxidized lipoproteins. We have cloned and characte&ed genomic regolatmy elements that control the expression of the scavenger receptor A gene. These elements confer macmphage-specific expression of lied genes in viva, and are capable of directing high levels of hansgene expression in foam cells of atherosclerotic lesions. Intriguingly, sequence elements that sre sufficient to direct expression in peritoneal macropbages and mediate M-CSF dependent expression in bone marrow-derived macrophages are not snfticient to direct expression in atherosclerotic lesions. Higb levels of expression in lesions appear to require the presence of additional regulatory elements that mediate transcriptional responses to GM-CSF, and which timctionally interact with M-CSF response elements. We and other laboratories are currently utilizing SR-A regolatoty elements to investigate the consequences of overexpression of various genes on the development of atherosclerosis. In addition, we have recently cloned and c!mracterized genomic regulatory elements for macrosialin, the mmine homologue of cd68. The macro&m promoter is approximately 200 times stronger than the SR-A promoter in macrophage cell lines, suggesting that it may offer some advantages over the SR-A promoter in experiments requiring bigb levels of tmnsgme expression.
Impaired plasmin proteolysis has been implicated in progression of atherosclerosis by promoting fibrin and matrix deposition, but recent evidence has suggested a role for increased plasmin pmteolysis in plaque ulceration, mptwe and mxwascolar&ion, and in aneurysm formation, possibly via interaction with metalloproteinases (MMP). In order to investigate the role of tbe plasminogen activators (PA), tissue-type PA (t-PA) and uroki“ax-type PA (u-PA) in atherosclerosis, mice with a deficiency of t-PA @PA-/-) or u-PA (uPA-/-) were intercrossed with apalipoprotein E deficient (apoE-/-) mice and fed a cbolesterol rich diet for prolonged periods. There were no genotypic differences in the plaque ma nor in their anatomical location across the aorta. However, the media in plaques from apoE-‘- or apoE-‘-:t-PAj- mice displayed different stagesof destruction, ranging from fmgmentation of the elastic lam&e to bansmedial perforation and microaneurysm formation. Ultmstructoml analysis revealed degradation of elastin and collagen. In contrast, the media in apoE-‘XI-PA-‘- arteries were protected against such destruction, and displayed only a minor degree of fragmentation of the elastic laminae. Plaque macropbages expressed abundant u-PA mRNA, immmweactivity and zymogmpbic activity. when cultored in the presence of plasminogen, wild type, t-PA-‘-, MMP-3-l-, MMP-7” and MMP9-j. but not u-PA-‘- or MMPlZ’- macrophages degraded 3H-elastin. Activation of MMP3, MM-9 and MMPIZ was increased by culturing wild type and t-PA-‘- but not u-PA-‘. macmphages in the presence of plasminogen. Immunostaining revealed the presence of MM?-3, MMP-9 and MMP-I2 in plaque macmphages. Taken together, these results suggest that u-PA, via generation of plasmin, activates elastolytic as well as collagenolytic proteinases, mediating thereby destruction of the media and aneurysm formation.
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143
Lipoprotein metabolism nod ntherosclerosis in APOE’JLeidea mice. Louis M. Havekesl, BwtJM van Ylijmenl, Miek C. Jongl, Ko Willems van Dgk2, Pie&r Groot3 andhiaren H. Hojker? IGaubios Laboratory ‘MO-Prevention and Health; 2Department of Homao Genetics, L&den, Tbe Netherlands, 3SmitbKlme Beecham, Welwyn, UK.
A role for interfemn~ but not iaterleukk1-4 ia graft arteriowlemsis Rich&N. Mitchell*, Hiroaki Nagano, Marta K. Taylor’, Satonr Hawgmva, andPeter Libby Departments of Medicine and Pathology (I), Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
APOE’3Leiden transgsnic mice develop bypexholesterolemia after feeding cholesterol containing diets. In contrast, tmnsgenic mice cat-tying the APOE2(argl58+cys) variant (apoE2) do not display hyperlipidemia. This confums the common assumption that apoE is a recessive variant, whereas with apoE3Leide.n the hyperlipidemia is dominantly inherited. Intrcducing these two apoE variants onto an endogenous apoE deficient background showed that apoE3lciden is able to rescue the extreme hyperlipidemia in apoE-/- mice, whereas apoEz is not. In vitm analyses showed that apoE3Leiden containii VLDL still displays LDLR bmding activity, whereas apoE VLDL is completely defective in this respect In both apoE x apoE-/- and apoE3Leiden x apoE-/- mice serum lipid levels strongly increased after Ad-RAP transfection, indicating that both apoE and apoE3Leiden bind to the LRP. In addition, Ad-VLDLR tmasfectioa in the liver of these two moose lines leads to a decrease in plasma lipid levels, indicating that these apoE variants also bind to the VLDLR. APOE’3Leiden tmnsgenic mice are highly susceptible to diet-induced atherosclerosis. Using quantitative image analysis we investigated the effect of diet composition and feeding pmiod on serum cbolesteml and atheroselerotis in tbe aortic sinus in these mice. The atherosclcmtic lesion ueas in cross sections were 5 to IO times greater in transgenic mice compared with non-tiansgenic littematcs. Highly significant positive correlations were found between the log-transformed data on lesion area and serum cholesteml exposure (r = 0.85), indicating that hypercholestemlemia is likely to be a major determ&nt in lesion formation. We conclude that the APOE*3Leiden moose represents a promising model for research on bypxlipidemia and atherosclerosis development.
Differential contii@ons of THI (e.g., interferon-y [TFN-y]) versus THZ (e.g., interleukin-t [IL-41 cytokines likely modulate the development of acute parenchymal rejectoin and g&l arteriosclerosis io solid tissue transplants. To evaluate the relative importance of these cytokines, we have wed geneticallydeficient mouse strains lacking IFN-y or IL-4 as wipicm in a vascularized hetemtopic mmine cardiac alk~gmtl model. Total MHC-mismatchedor MHC class II-disparate hea& were transplanted into wild-type, IFN-y deficient (GKO), or IL4deficient tecipients with or without anti-CD4 and anti-CDS monoclonal antibodies for immunosuppmssion. In the absence of prior immunowppression, total MHC-mismatched grafts in wild-type or GKO hosts all showed similar rates of g&l failure, manifested bistolog&lly ss severe acute rejection. However, g&s into GKO recipients chamct&sdcally ceasedbeating l-2 days earlier thao wild-type grafts; in addition to focal acute rejection, the g&s in GKO recipients exhibited profound coagulation necrosis with multiple arterial dxombi, suggestinga role for IFN-y in locally modulating thrombosis. In total MHC-mismatched grafts, long-term grail smvival (at least 12 weeks) could be acbicvcd by weekly anti-CD4 and anti-CD8 admi&mtion after an initial 4 day cotax of rejection. Similar long-term g& survival was obtained in MHC U-disparate combinations when the hose received anti-CD4 and antiCD8 before transplant only. In such longtezm Omsplants of eidwr straio combination, g&Is into wild-type ha& reliably developed vascolar lesions of g&l arteriosclerosis by S-12 weeks. In comparison, grafts into GKO recipients developed no grail arteriosclerosis, despite a severe ongoing parenchymal intilkate comparable to wild-type grafts. A similar lack of graft arterkwlemsis, even in the presence of parencbymal rejection, WBSseen in MHC IId&amte transplants intO wild-type recipients given weekly injections of anti-IFNy antibodies.Although g&s in GKO recipient? showed numbers of macmphages,CD4, and CD8 cells comparable to go&s in wild-type recipients, grafts iota GKO rwipients had diminished MHC Il. CD40, VCAM-I and ICIAM-1 expression relative to wild-type controls. In contrast, MHC II-disparate transplantsin IL4 deficient .mimds showed pwenchymal rejection and graft arteriosclerosis at rates and severities comparable to wild-type controls. Thus, deficiency of IL-4 does not affect the progress of acute rejection or gmtl arteriosclerosis in cardiac allogratk. However, IFN-y is @ortaut to the development of graft arteriosclemsis, despite the fact that lack of IFN-y does not dim&b acute pamnchymai rejection and graft faihm, EN-y may mcdiatc gmtl arterkwlerosis by mechwisms involving secondary activation of macmphages,or expression of MHC, adhesion, or accessorymolecoks.
140
142
Use of scavenger receptor regulatory elemoots to target gene expression to otlwosclerotic pleques. C.K. Glass, A. Horvai, U! P&t&, A. Li ondJ. Collier University of California, San Diego, Department of Medicine, Division of Cellular and Molecular Medicine
Lack of aneurysm formation in atherosclerotic eorto from plosminogeo activator deficient mice doe to &ad metdloproteinow activity. P&r Carmelietl, Florea Lupu? Steve Shapiro~, Angela Drew! Peter lippi&, Roger Lijned, Lieve Moonsl, &Desire Collenl. (I) Center of Tramgene Technology and Gene transfer, Flanders Interuniversity of Biotechnology, Leuven, Belgium; (2) Thrombosis Research Institute, London, m (3) Tbe Jewish Hospital of St. Louis, Washington University, St-Louis; (4) Tbe Center for Inflammatory Diseases, Monasb Medical Center, Australia.
The development of macrophage foam cells witbin atherosclerotic lesions is thought to depend on the expression of one or more scavenger receptors that mediate the uptake of oxidized lipoproteins. We have cloned and characte&ed genomic regolatmy elements that control the expression of the scavenger receptor A gene. These elements confer macmphage-specific expression of lied genes in viva, and are capable of directing high levels of hansgene expression in foam cells of atherosclerotic lesions. Intriguingly, sequence elements that sre sufficient to direct expression in peritoneal macropbages and mediate M-CSF dependent expression in bone marrow-derived macrophages are not snfticient to direct expression in atherosclerotic lesions. Higb levels of expression in lesions appear to require the presence of additional regulatory elements that mediate transcriptional responses to GM-CSF, and which timctionally interact with M-CSF response elements. We and other laboratories are currently utilizing SR-A regolatoty elements to investigate the consequences of overexpression of various genes on the development of atherosclerosis. In addition, we have recently cloned and c!mracterized genomic regulatory elements for macrosialin, the mmine homologue of cd68. The macro&m promoter is approximately 200 times stronger than the SR-A promoter in macrophage cell lines, suggesting that it may offer some advantages over the SR-A promoter in experiments requiring bigb levels of tmnsgme expression.
Impaired plasmin proteolysis has been implicated in progression of atherosclerosis by promoting fibrin and matrix deposition, but recent evidence has suggested a role for increased plasmin pmteolysis in plaque ulceration, mptwe and mxwascolar&ion, and in aneurysm formation, possibly via interaction with metalloproteinases (MMP). In order to investigate the role of tbe plasminogen activators (PA), tissue-type PA (t-PA) and uroki“ax-type PA (u-PA) in atherosclerosis, mice with a deficiency of t-PA @PA-/-) or u-PA (uPA-/-) were intercrossed with apalipoprotein E deficient (apoE-/-) mice and fed a cbolesterol rich diet for prolonged periods. There were no genotypic differences in the plaque ma nor in their anatomical location across the aorta. However, the media in plaques from apoE-‘- or apoE-‘-:t-PAj- mice displayed different stagesof destruction, ranging from fmgmentation of the elastic lam&e to bansmedial perforation and microaneurysm formation. Ultmstructoml analysis revealed degradation of elastin and collagen. In contrast, the media in apoE-‘XI-PA-‘- arteries were protected against such destruction, and displayed only a minor degree of fragmentation of the elastic laminae. Plaque macropbages expressed abundant u-PA mRNA, immmweactivity and zymogmpbic activity. when cultored in the presence of plasminogen, wild type, t-PA-‘-, MMP-3-l-, MMP-7” and MMP9-j. but not u-PA-‘- or MMPlZ’- macrophages degraded 3H-elastin. Activation of MMP3, MM-9 and MMPIZ was increased by culturing wild type and t-PA-‘- but not u-PA-‘. macmphages in the presence of plasminogen. Immunostaining revealed the presence of MM?-3, MMP-9 and MMP-I2 in plaque macmphages. Taken together, these results suggest that u-PA, via generation of plasmin, activates elastolytic as well as collagenolytic proteinases, mediating thereby destruction of the media and aneurysm formation.
141
143
Lipoprotein metabolism nod ntherosclerosis in APOE’JLeidea mice. Louis M. Havekesl, BwtJM van Ylijmenl, Miek C. Jongl, Ko Willems van Dgk2, Pie&r Groot3 andhiaren H. Hojker? IGaubios Laboratory ‘MO-Prevention and Health; 2Department of Homao Genetics, L&den, Tbe Netherlands, 3SmitbKlme Beecham, Welwyn, UK.
A role for interfemn~ but not iaterleukk1-4 ia graft arteriowlemsis Rich&N. Mitchell*, Hiroaki Nagano, Marta K. Taylor’, Satonr Hawgmva, andPeter Libby Departments of Medicine and Pathology (I), Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
APOE’3Leiden transgsnic mice develop bypexholesterolemia after feeding cholesterol containing diets. In contrast, tmnsgenic mice cat-tying the APOE2(argl58+cys) variant (apoE2) do not display hyperlipidemia. This confums the common assumption that apoE is a recessive variant, whereas with apoE3Leide.n the hyperlipidemia is dominantly inherited. Intrcducing these two apoE variants onto an endogenous apoE deficient background showed that apoE3lciden is able to rescue the extreme hyperlipidemia in apoE-/- mice, whereas apoEz is not. In vitm analyses showed that apoE3Leiden containii VLDL still displays LDLR bmding activity, whereas apoE VLDL is completely defective in this respect In both apoE x apoE-/- and apoE3Leiden x apoE-/- mice serum lipid levels strongly increased after Ad-RAP transfection, indicating that both apoE and apoE3Leiden bind to the LRP. In addition, Ad-VLDLR tmasfectioa in the liver of these two moose lines leads to a decrease in plasma lipid levels, indicating that these apoE variants also bind to the VLDLR. APOE’3Leiden tmnsgenic mice are highly susceptible to diet-induced atherosclerosis. Using quantitative image analysis we investigated the effect of diet composition and feeding pmiod on serum cbolesteml and atheroselerotis in tbe aortic sinus in these mice. The atherosclcmtic lesion ueas in cross sections were 5 to IO times greater in transgenic mice compared with non-tiansgenic littematcs. Highly significant positive correlations were found between the log-transformed data on lesion area and serum cholesteml exposure (r = 0.85), indicating that hypercholestemlemia is likely to be a major determ&nt in lesion formation. We conclude that the APOE*3Leiden moose represents a promising model for research on bypxlipidemia and atherosclerosis development.
Differential contii@ons of THI (e.g., interferon-y [TFN-y]) versus THZ (e.g., interleukin-t [IL-41 cytokines likely modulate the development of acute parenchymal rejectoin and g&l arteriosclerosis io solid tissue transplants. To evaluate the relative importance of these cytokines, we have wed geneticallydeficient mouse strains lacking IFN-y or IL-4 as wipicm in a vascularized hetemtopic mmine cardiac alk~gmtl model. Total MHC-mismatchedor MHC class II-disparate hea& were transplanted into wild-type, IFN-y deficient (GKO), or IL4deficient tecipients with or without anti-CD4 and anti-CDS monoclonal antibodies for immunosuppmssion. In the absence of prior immunowppression, total MHC-mismatched grafts in wild-type or GKO hosts all showed similar rates of g&l failure, manifested bistolog&lly ss severe acute rejection. However, g&s into GKO recipients chamct&sdcally ceasedbeating l-2 days earlier thao wild-type grafts; in addition to focal acute rejection, the g&s in GKO recipients exhibited profound coagulation necrosis with multiple arterial dxombi, suggestinga role for IFN-y in locally modulating thrombosis. In total MHC-mismatched grafts, long-term grail smvival (at least 12 weeks) could be acbicvcd by weekly anti-CD4 and anti-CD8 admi&mtion after an initial 4 day cotax of rejection. Similar long-term g& survival was obtained in MHC U-disparate combinations when the hose received anti-CD4 and antiCD8 before transplant only. In such longtezm Omsplants of eidwr straio combination, g&Is into wild-type ha& reliably developed vascolar lesions of g&l arteriosclerosis by S-12 weeks. In comparison, grafts into GKO recipients developed no grail arteriosclerosis, despite a severe ongoing parenchymal intilkate comparable to wild-type grafts. A similar lack of graft arterkwlemsis, even in the presence of parencbymal rejection, WBSseen in MHC IId&amte transplants intO wild-type recipients given weekly injections of anti-IFNy antibodies.Although g&s in GKO recipient? showed numbers of macmphages,CD4, and CD8 cells comparable to go&s in wild-type recipients, grafts iota GKO rwipients had diminished MHC Il. CD40, VCAM-I and ICIAM-1 expression relative to wild-type controls. In contrast, MHC II-disparate transplantsin IL4 deficient .mimds showed pwenchymal rejection and graft arteriosclerosis at rates and severities comparable to wild-type controls. Thus, deficiency of IL-4 does not affect the progress of acute rejection or gmtl arteriosclerosis in cardiac allogratk. However, IFN-y is @ortaut to the development of graft arteriosclemsis, despite the fact that lack of IFN-y does not dim&b acute pamnchymai rejection and graft faihm, EN-y may mcdiatc gmtl arterkwlerosis by mechwisms involving secondary activation of macmphages,or expression of MHC, adhesion, or accessorymolecoks.