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Eicosapentaenoic acid (EPA) suppresses the proliferation of vascular smooth muscle cells (VSMC) by inhibiting the expression of c-fos mRNA
Tuesday I1 October 1994: Poster Abstracts Gene expression and targeting The result that TX synthase mRNA is highly expressed in rat thymus and spleen may suggest that TXA2 plays a role in the immune system.
CZII
The orphan receptor ABP-1 represses transcriptional activity of the human apolipoprotein B gene promoter by a mechanism independent of DNA binding
Achatz G, H&l B, Sandhofer F, 1st Dept. bat. Med., General Hosp. of Salzburg. Muellner-Hauptstr.48, 5020 Salzburg, Austria
mB,
Transcriptional activity of the human apolipoprotein B gene promoter is strongly repressed by the orphan receptor ARP-1. To identify the domains of ARP-1 required for its repressor activity a deletion analysis of the protein was performed. Residues 395-399 proved to be essential for repressor activity. A second domain required for repressor activity could be localized to the region between residues 131 and 192. Deletion of the DNA binding domain strongly reduced repressor activity of ARP-1. To ensure nuclear localization of the variant proteins a nuclear localization signal from the SV40 large T antigen was attached to the aminoterminus of various of the mutant proteins. Surprisingly, this modification completely abolished the ability of wild-type and mutant proteins to bind to the ARP-1 recognition site within the apo B promoter (-86 to -62), without affecting their ability to repress transcriptional activity of the apo B promoter. Deletion of the DNA binding domain of the modified ARP-1 protein only slightly reduced its repressor activity, indicating that the DNA binding domain is important for nuclear localization of the protein, but dispensable for its repressor activity. In transient transfection experiments overexpression of ARP-1 reduced transcriptional activity induced by HNF-3, HNF-4, and UEBP, but not by Spl or ATF, suggesting that ARP-I does not repress transcriptional activity by interaction with basal components of the transcriptional machinery. Transcriptional repression of the apo B promoter by ARP-1 was not relieved by overexpression of HNF3.HNF-4.CEBP. RXRa or TFBB. This result areues against heterodimerization of ARP-1 with one of these priteins being important for its repressor activity. Based upon our results it seems more likely that repression of transcriptional activity of the apo B gene promoter by ARP-I involves protein-protein interaction between ARP-1 and a limiting transcriptional coactivator protein.
1501
Eicosapentaenoic acid (EPA) suppresses the proliferation of vascular smooth muscle cells (VSMC) by inhibiting the expression of c-fos mBNA
Shiina T, Terano T, Tamura Y, Yoshida S, 2nd Dept. of Int. Med., Chiba Univ. Sch. of Med., I-R-1 Inohana, Chiba City, Chiba, 260 Japan Abnormal proliferation of VSMC plays an important role in the development of atherosclerosis. EPA has been reported to inhibit VSMC proliferation. PDGF binds to the cell surface receptor, and the signal is transduced to the nucleus via several signal transduction systems. c-fos mRNA, an immediate early growth gene, is induced by PDGF stimulation, and this expression is an important initial step in cell growth. In this study, the effect of EPA on PDGF- and TPA-stimulated intracellular signal messenger systems, and in particular the expression of c-fos mRNA, was investigated. Cultured VSMC were incubated with 80 or 16O~moUi EPA triglyceride for 24 h and stimulated by PDGF (10 @ml) or TPA (lo-’ mol/l). Total cellular RNA was extracted by the acid guanidinium method and puritied using oligo(dT)-cellulose spun columns, and expression of c-fos mRNA was measured by Northern blotting. The data were corrected for expression of betaactin.
10.5
Prior treatment of VSMC with EPA triglyceride increased the EPA content in the total phospholipid fraction. PDGF-induced [3H]thymidine uptake was dose-dependently inhibited by EPA treatment. The maximal expression of c-fos mRNA was observed 30 min after the PDGF or TPA stimulation. The induction of expression of c-fos mRNA by PDGF or TPA was dosedependently suppressed by EPA pre-treatment. We have previously reported that EPA suppressed the PDGF binding to its receptor. In this study we have shown that EPA suppressed the expression of an early growth gene. It suppressed not only PDGF- but also TPA-induced c-fos mRNA expression. These data might indicate that EPA inhibits VSMC proliferation by modulating the several steps of the signal transduction systems.
El
+ Use of recombinant adenovirus to facilitate gene delivery into human vascular cells in vitro
Hedrick, Brennan ML, Hama SY, Navab M, Meidell RS, Lusis AJ, Div. of Cardiol., Univ. of California, Los Angeles, CA 90024, USA We report the use of recombinant adenoviral vectors to successfully infect primary human aortic endotheiial cells (EC) and aortic smooth muscle cells (SMC) in vitro. Human aortic EC and SMC were infected at multiplicities of infection (MOI) of 1, 10, and 100 with recombinant adenovirus containing the p-galactosidase reporter gene. In this recombinant adenovirus, expression of the /3-galactosidase gene was driven by the cytomegalovirus (CMV) promoter. The number of cells which stained positive for Bgalactosidase increased as a linear function of viral dosage. Virtually 100% of SMC and EC were infected at a MO1 of 100; these cells were then passaged 96 h after infection to examine cell viability. These infected cells continued to divide and remain metabolically active for at least 10 days after infection. In addition, we successfully infected multilayer co-cultures of aortic SMC and EC using recombinant adenovirus containing the b-galactosidase gene. At an MO1 of 50, 100% of cells were infected. The human arterial co-culture system is a unique system for examining lipoprotein oxidation by cellular mechanisms. Examination of monocyte transmigration in the subendothelial space using fluorescein-labeled THP-1 monocytes revealed that infection of arterial co-cultures with recombinant adenovirus at an MO1 of 50 did not induce significant cellular inflammatory responses. Thus, we can successfully use recombinant adenoviruses to deliver genes into arterial wall co-cultures. This system will be useful in studies of the mechanism of oxidative modification of lipoproteins by cells.
Ex vivo gene therapy directed to liver in a patient with familial hypercholesterolemia QQWJ.U& Lupien P-J, Raper SE, Wilson JM, Inst. for Human
1
Gene Therapy, Univ. of Pennsylvania, 3601 Spruce St., Philadelphia, PA 19104, USA Familial hypercholesterolemia (FH) has emerged as an important model for the development of human gene therapies. The homozygous form of FH is an excellent candidate for early applications of gene therapy because it is a lethal disorder refractory to conventional therapies. The gene therapy treatment of a 29-year-old woman with homozygous FH by ex vivo gene therapy directed to liver was as follows. The patient was taken to the operating room where approximately 15% of the left lateral segment of her liver was removed via a left subcostal incision. A catheter was inserted into her inferior mesenteric vein with the distal end of the catheter brought through her incision providing access to the portal circulation for cell infusions. The resected liver (250 g) was perfused with collagenase to release 3.2 x lo9 hepatocytes. Medium containing the
Atherosclerosis X, Montreal, October I994
CZII
The orphan receptor ABP-1 represses transcriptional activity of the human apolipoprotein B gene promoter by a mechanism independent of DNA binding
Achatz G, H&l B, Sandhofer F, 1st Dept. bat. Med., General Hosp. of Salzburg. Muellner-Hauptstr.48, 5020 Salzburg, Austria
mB,
Transcriptional activity of the human apolipoprotein B gene promoter is strongly repressed by the orphan receptor ARP-1. To identify the domains of ARP-1 required for its repressor activity a deletion analysis of the protein was performed. Residues 395-399 proved to be essential for repressor activity. A second domain required for repressor activity could be localized to the region between residues 131 and 192. Deletion of the DNA binding domain strongly reduced repressor activity of ARP-1. To ensure nuclear localization of the variant proteins a nuclear localization signal from the SV40 large T antigen was attached to the aminoterminus of various of the mutant proteins. Surprisingly, this modification completely abolished the ability of wild-type and mutant proteins to bind to the ARP-1 recognition site within the apo B promoter (-86 to -62), without affecting their ability to repress transcriptional activity of the apo B promoter. Deletion of the DNA binding domain of the modified ARP-1 protein only slightly reduced its repressor activity, indicating that the DNA binding domain is important for nuclear localization of the protein, but dispensable for its repressor activity. In transient transfection experiments overexpression of ARP-1 reduced transcriptional activity induced by HNF-3, HNF-4, and UEBP, but not by Spl or ATF, suggesting that ARP-I does not repress transcriptional activity by interaction with basal components of the transcriptional machinery. Transcriptional repression of the apo B promoter by ARP-1 was not relieved by overexpression of HNF3.HNF-4.CEBP. RXRa or TFBB. This result areues against heterodimerization of ARP-1 with one of these priteins being important for its repressor activity. Based upon our results it seems more likely that repression of transcriptional activity of the apo B gene promoter by ARP-I involves protein-protein interaction between ARP-1 and a limiting transcriptional coactivator protein.
1501
Eicosapentaenoic acid (EPA) suppresses the proliferation of vascular smooth muscle cells (VSMC) by inhibiting the expression of c-fos mBNA
Shiina T, Terano T, Tamura Y, Yoshida S, 2nd Dept. of Int. Med., Chiba Univ. Sch. of Med., I-R-1 Inohana, Chiba City, Chiba, 260 Japan Abnormal proliferation of VSMC plays an important role in the development of atherosclerosis. EPA has been reported to inhibit VSMC proliferation. PDGF binds to the cell surface receptor, and the signal is transduced to the nucleus via several signal transduction systems. c-fos mRNA, an immediate early growth gene, is induced by PDGF stimulation, and this expression is an important initial step in cell growth. In this study, the effect of EPA on PDGF- and TPA-stimulated intracellular signal messenger systems, and in particular the expression of c-fos mRNA, was investigated. Cultured VSMC were incubated with 80 or 16O~moUi EPA triglyceride for 24 h and stimulated by PDGF (10 @ml) or TPA (lo-’ mol/l). Total cellular RNA was extracted by the acid guanidinium method and puritied using oligo(dT)-cellulose spun columns, and expression of c-fos mRNA was measured by Northern blotting. The data were corrected for expression of betaactin.
10.5
Prior treatment of VSMC with EPA triglyceride increased the EPA content in the total phospholipid fraction. PDGF-induced [3H]thymidine uptake was dose-dependently inhibited by EPA treatment. The maximal expression of c-fos mRNA was observed 30 min after the PDGF or TPA stimulation. The induction of expression of c-fos mRNA by PDGF or TPA was dosedependently suppressed by EPA pre-treatment. We have previously reported that EPA suppressed the PDGF binding to its receptor. In this study we have shown that EPA suppressed the expression of an early growth gene. It suppressed not only PDGF- but also TPA-induced c-fos mRNA expression. These data might indicate that EPA inhibits VSMC proliferation by modulating the several steps of the signal transduction systems.
El
+ Use of recombinant adenovirus to facilitate gene delivery into human vascular cells in vitro
Hedrick, Brennan ML, Hama SY, Navab M, Meidell RS, Lusis AJ, Div. of Cardiol., Univ. of California, Los Angeles, CA 90024, USA We report the use of recombinant adenoviral vectors to successfully infect primary human aortic endotheiial cells (EC) and aortic smooth muscle cells (SMC) in vitro. Human aortic EC and SMC were infected at multiplicities of infection (MOI) of 1, 10, and 100 with recombinant adenovirus containing the p-galactosidase reporter gene. In this recombinant adenovirus, expression of the /3-galactosidase gene was driven by the cytomegalovirus (CMV) promoter. The number of cells which stained positive for Bgalactosidase increased as a linear function of viral dosage. Virtually 100% of SMC and EC were infected at a MO1 of 100; these cells were then passaged 96 h after infection to examine cell viability. These infected cells continued to divide and remain metabolically active for at least 10 days after infection. In addition, we successfully infected multilayer co-cultures of aortic SMC and EC using recombinant adenovirus containing the b-galactosidase gene. At an MO1 of 50, 100% of cells were infected. The human arterial co-culture system is a unique system for examining lipoprotein oxidation by cellular mechanisms. Examination of monocyte transmigration in the subendothelial space using fluorescein-labeled THP-1 monocytes revealed that infection of arterial co-cultures with recombinant adenovirus at an MO1 of 50 did not induce significant cellular inflammatory responses. Thus, we can successfully use recombinant adenoviruses to deliver genes into arterial wall co-cultures. This system will be useful in studies of the mechanism of oxidative modification of lipoproteins by cells.
Ex vivo gene therapy directed to liver in a patient with familial hypercholesterolemia QQWJ.U& Lupien P-J, Raper SE, Wilson JM, Inst. for Human
1
Gene Therapy, Univ. of Pennsylvania, 3601 Spruce St., Philadelphia, PA 19104, USA Familial hypercholesterolemia (FH) has emerged as an important model for the development of human gene therapies. The homozygous form of FH is an excellent candidate for early applications of gene therapy because it is a lethal disorder refractory to conventional therapies. The gene therapy treatment of a 29-year-old woman with homozygous FH by ex vivo gene therapy directed to liver was as follows. The patient was taken to the operating room where approximately 15% of the left lateral segment of her liver was removed via a left subcostal incision. A catheter was inserted into her inferior mesenteric vein with the distal end of the catheter brought through her incision providing access to the portal circulation for cell infusions. The resected liver (250 g) was perfused with collagenase to release 3.2 x lo9 hepatocytes. Medium containing the
Atherosclerosis X, Montreal, October I994