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P-35 Effect of angiotensin-converting enzyme inhibition on gene expression in vascular smooth muscle cell
5. Poster p-33
NEURONAL NOS CONTRIBUTES TO B~ADYKININ” INDUCED CORONARY FLOW IN ENDQTHELIAL NOS KNOCKOUT (eNOS-KO) MICE Hassan M.A. Talukder, Hiroaki Shimokawa, Takako Fujiki, Keiko orikawa, Hiroshi K~bota, Tetsuya and Akira Takeshita. repayment of Matoba, Cardiovascular Medicine, Kyushu University School of Medical Sciences, Fukuoka 812-8582, Japan
p-34
Inhibitory effects of ursodeoxycholic acid (UDCA) on : the induction of nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) Ji Ma, Toshiaki Nakajima, Haruko Iida, Kuniko Terasawa, Kuniaki Iwasawa. Yukichi Okuda, Fumihiko Eto, Nobuhiko Yamada, Ryozo Nagai, Masao Omata. Dept of Internal Medicine, University of Tokyo & Tsukuba, Japan Expression of iNOS and the resultant increased NO production are associated with endotoxemia and atheroscierotic lesions observed in transplant hearts or balloon injured artery. UDCA has been shown to inhibit the development of transplant arteriosclerosis, but its mechanism remains unclear. We investigated the effects of UDCA on NO production and the expression of iNOS mRNA and protein inVSMCs isolated from adult rat aorta and rabbit coronary artery by using the Griess reaction, Northern and Western blot analyses. UDCA inhibited lipopolysaccaride (LPS) plus interferon (INF)-y-enhanced NO production. But, UDCA showed only small inhibitory effects on NO production that had already been induced by LPS plus INF-y. UDCA also suppressed the expression of iNOS mRNA and protein. UDCA had the most potent inhibitory effect among various kinds of bile acids examined. These results suggest that UDCA inhibits the induction of iNOS and then NO production in VSMCs, a possible mechanism for the cardiovascular protective effect of UDCA under various pathophysiological conditions such as endotoxemia and atherosclerosis.
Recent reports of an enhanced or compensator vascular relaxation to acetylcholine in eNOSK0 mica indicate a co-regulatory interactions between different isoforms of NOS. Bradykinin (BK) is a potent endotheiium-dependent vasodilator and endogenous IBK is involved in the regulation of basal and flow-mediated coronary vasomotor tone in humans. The present study was designed to examine BK-induced coronary flow (CF) in Langendorff-perfused mouse hearts from eNOS-1~0 and wild-type (WT) mice. In WT hearts, BK-induced increases in CF were unaffected with i~domethacin but were markedly inhibited by ~~-nitro-L-argin~ne (NOS inhibitor). In contrast, BK produced comparable increases in CF in eNOS-KQ hearts and an inhibitor of nNOS, S-methyl-L-thiocitrulline, significantly attenuated it, ODQ, an inhibitor of cGMP, also partially attenuated EIKinduced CF while it abolished SNP-induced CF in eNOSKO hearts. BK-induced CF was abolished by BK receptor
antagonist, HOE-140. These data suggest that both NO and EDHF contribute to BK-induced CF in isolated mouse hearts, plays a compensator p-35
and
nNOS-cGMP-mediated role in eNOS-KO mice.
pathway
EFFECT OF ANGIOTENSIN-CGNVERTING ENZYME INHIBITION ON GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE CELL Yuhei Nishimura, Patrice Ribiere and Toshio Tanaka. Dept of Pharmacology, Mie Univ. School of Medicine, Japan. The renin-angiotensin system and angiotensinconverting enzyme (ACE) are increasingly being implicated in the pathogenesis of artery disease and its sequelae. ACE inhibitors prevent stimulation of smooth muscle cell angiotensin II receptors, thereby blocking both contractile and proliferative actions of angiotensin II. In addition, ACE inhibition of kininase inhibits the breal~down of bradykinin, a direct stimulant of nitric oxide release from the intact endothelial cells. Thus, at the cellular level ACE inhibition shifts the balance of ongoing mechanisms in favor of those promoting vasodilatory, antiaggregatory, antithrombot~c and a~tiproliferat~vla effects. The purpose of this study was to examine the effects of ACE inhibition on gene expression in vascular smooth muscle ceils. W microarray technology to identify expressed genes in smooth muscle cells exposed to captopril and enalapri~at. The gene expression profile elucidates new aspects of therapy by AGE inhibitors,.
A33
Presentations
p-36
GENE TRANSFECTION OF NF-KB DECOY NUCLEOTIDES IMPROVJ!X VASCULAR PE~EAB~L~Y IN SEPTIC MOUSE LUNG
OLIGO-
Naoyuki Matsuda, Satoshi Cando, Osamu Kemmotsu & Yuichi Hattori. Departments of Pharmacology and Anesthesiology & Critical Care Medicine, Hokkaido University School of Medicine, Sapporo, Japan .
Septic shock is a serious systemic disorder characterized by vasodilation, enhanced vascular permeability and endotheliai dysfunction, leading to acute respiratory distress syndrome and extremely high mortality. A variety of proinflammatory cytokines are produced by activation of nuclear factor kB (NFkB) in septic shock. We investigated whether gene transfection of NF-kB decoy oligonucleotides (DON) which can block NF-kB region of nuclear DNA improves vascular permeability in septic mouse lung. ICR mice were rendered endotoxemic with IO mglkg LPS injection. Some mice received 5 pg/g of NF-LB DON (10 mer) by the liposome method before LPS injection. Accumulation of NF-LB DON into the nuclei of plumonary vasucular endotheliai cells and prevention of NF-kB activation in the lung were confirmed by the mobility gel shift assay. Northern blot analysis revealed that inducible NO synthase (iNOS) mRNA was markedly increased in lungs from mice at 10 h after LPS but was drama.tical!y reduced to the control level by NF-LB DON treatment. In contrast, endotbelial NO synthase (eNOS) mRNA was decreased by 73 % in septic lung and was partially improved to 89 % by NF-LB DON. Histamine Hl receptor (HIR) mRNA was increased 2.7-fold in the septic group and l&fold in the NF-kB DON transfected septic group. The lung permeability index (the ratio of BALF to blood for ‘*’ I-albumin mdioactivity) increased IS-fold in sepsis and 3.X-fold after NF-kB DON tmnsfection. NOS inhibitor and HIR antagonist significantly but slightly improved the index in sepsis. We suggest that sepsis causes transcriptional upregulation of iNOS and N iR and downregulation of eNOS. These transcriptional regulations arc strictly associated with NF-LB activation. However, NF-kB DON transfection could improve lung permeability in sepsis by regulating gene expressions of vascular permeability factors other than iNOS and HIR via inhibition of NF-kB activation.
NEURONAL NOS CONTRIBUTES TO B~ADYKININ” INDUCED CORONARY FLOW IN ENDQTHELIAL NOS KNOCKOUT (eNOS-KO) MICE Hassan M.A. Talukder, Hiroaki Shimokawa, Takako Fujiki, Keiko orikawa, Hiroshi K~bota, Tetsuya and Akira Takeshita. repayment of Matoba, Cardiovascular Medicine, Kyushu University School of Medical Sciences, Fukuoka 812-8582, Japan
p-34
Inhibitory effects of ursodeoxycholic acid (UDCA) on : the induction of nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) Ji Ma, Toshiaki Nakajima, Haruko Iida, Kuniko Terasawa, Kuniaki Iwasawa. Yukichi Okuda, Fumihiko Eto, Nobuhiko Yamada, Ryozo Nagai, Masao Omata. Dept of Internal Medicine, University of Tokyo & Tsukuba, Japan Expression of iNOS and the resultant increased NO production are associated with endotoxemia and atheroscierotic lesions observed in transplant hearts or balloon injured artery. UDCA has been shown to inhibit the development of transplant arteriosclerosis, but its mechanism remains unclear. We investigated the effects of UDCA on NO production and the expression of iNOS mRNA and protein inVSMCs isolated from adult rat aorta and rabbit coronary artery by using the Griess reaction, Northern and Western blot analyses. UDCA inhibited lipopolysaccaride (LPS) plus interferon (INF)-y-enhanced NO production. But, UDCA showed only small inhibitory effects on NO production that had already been induced by LPS plus INF-y. UDCA also suppressed the expression of iNOS mRNA and protein. UDCA had the most potent inhibitory effect among various kinds of bile acids examined. These results suggest that UDCA inhibits the induction of iNOS and then NO production in VSMCs, a possible mechanism for the cardiovascular protective effect of UDCA under various pathophysiological conditions such as endotoxemia and atherosclerosis.
Recent reports of an enhanced or compensator vascular relaxation to acetylcholine in eNOSK0 mica indicate a co-regulatory interactions between different isoforms of NOS. Bradykinin (BK) is a potent endotheiium-dependent vasodilator and endogenous IBK is involved in the regulation of basal and flow-mediated coronary vasomotor tone in humans. The present study was designed to examine BK-induced coronary flow (CF) in Langendorff-perfused mouse hearts from eNOS-1~0 and wild-type (WT) mice. In WT hearts, BK-induced increases in CF were unaffected with i~domethacin but were markedly inhibited by ~~-nitro-L-argin~ne (NOS inhibitor). In contrast, BK produced comparable increases in CF in eNOS-KQ hearts and an inhibitor of nNOS, S-methyl-L-thiocitrulline, significantly attenuated it, ODQ, an inhibitor of cGMP, also partially attenuated EIKinduced CF while it abolished SNP-induced CF in eNOSKO hearts. BK-induced CF was abolished by BK receptor
antagonist, HOE-140. These data suggest that both NO and EDHF contribute to BK-induced CF in isolated mouse hearts, plays a compensator p-35
and
nNOS-cGMP-mediated role in eNOS-KO mice.
pathway
EFFECT OF ANGIOTENSIN-CGNVERTING ENZYME INHIBITION ON GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE CELL Yuhei Nishimura, Patrice Ribiere and Toshio Tanaka. Dept of Pharmacology, Mie Univ. School of Medicine, Japan. The renin-angiotensin system and angiotensinconverting enzyme (ACE) are increasingly being implicated in the pathogenesis of artery disease and its sequelae. ACE inhibitors prevent stimulation of smooth muscle cell angiotensin II receptors, thereby blocking both contractile and proliferative actions of angiotensin II. In addition, ACE inhibition of kininase inhibits the breal~down of bradykinin, a direct stimulant of nitric oxide release from the intact endothelial cells. Thus, at the cellular level ACE inhibition shifts the balance of ongoing mechanisms in favor of those promoting vasodilatory, antiaggregatory, antithrombot~c and a~tiproliferat~vla effects. The purpose of this study was to examine the effects of ACE inhibition on gene expression in vascular smooth muscle ceils. W microarray technology to identify expressed genes in smooth muscle cells exposed to captopril and enalapri~at. The gene expression profile elucidates new aspects of therapy by AGE inhibitors,.
A33
Presentations
p-36
GENE TRANSFECTION OF NF-KB DECOY NUCLEOTIDES IMPROVJ!X VASCULAR PE~EAB~L~Y IN SEPTIC MOUSE LUNG
OLIGO-
Naoyuki Matsuda, Satoshi Cando, Osamu Kemmotsu & Yuichi Hattori. Departments of Pharmacology and Anesthesiology & Critical Care Medicine, Hokkaido University School of Medicine, Sapporo, Japan .
Septic shock is a serious systemic disorder characterized by vasodilation, enhanced vascular permeability and endotheliai dysfunction, leading to acute respiratory distress syndrome and extremely high mortality. A variety of proinflammatory cytokines are produced by activation of nuclear factor kB (NFkB) in septic shock. We investigated whether gene transfection of NF-kB decoy oligonucleotides (DON) which can block NF-kB region of nuclear DNA improves vascular permeability in septic mouse lung. ICR mice were rendered endotoxemic with IO mglkg LPS injection. Some mice received 5 pg/g of NF-LB DON (10 mer) by the liposome method before LPS injection. Accumulation of NF-LB DON into the nuclei of plumonary vasucular endotheliai cells and prevention of NF-kB activation in the lung were confirmed by the mobility gel shift assay. Northern blot analysis revealed that inducible NO synthase (iNOS) mRNA was markedly increased in lungs from mice at 10 h after LPS but was drama.tical!y reduced to the control level by NF-LB DON treatment. In contrast, endotbelial NO synthase (eNOS) mRNA was decreased by 73 % in septic lung and was partially improved to 89 % by NF-LB DON. Histamine Hl receptor (HIR) mRNA was increased 2.7-fold in the septic group and l&fold in the NF-kB DON transfected septic group. The lung permeability index (the ratio of BALF to blood for ‘*’ I-albumin mdioactivity) increased IS-fold in sepsis and 3.X-fold after NF-kB DON tmnsfection. NOS inhibitor and HIR antagonist significantly but slightly improved the index in sepsis. We suggest that sepsis causes transcriptional upregulation of iNOS and N iR and downregulation of eNOS. These transcriptional regulations arc strictly associated with NF-LB activation. However, NF-kB DON transfection could improve lung permeability in sepsis by regulating gene expressions of vascular permeability factors other than iNOS and HIR via inhibition of NF-kB activation.