Role of vascular angiotensin generating system and mechanical stress in the vascular remodeling

WS3-J-5-02 ROLE OF VASCULAR ANGIOTENS1N GENERATING SYSTEM AND MECHANICAL STRESS 1N THE VASCULAR REMODELING H. Rakugi, T. Ogihara Dept. of Geriatric Medicine, Osaka University Medical School, Suite, Japan There are several recent reports that vascular All generating system is more important than circulating All for vascular remodeling, and that the All generating system is activated in the remodeling artery, however the mechanism of its augmentation is unclear. The present study was performed to clarify the role of mechanical stress and phenotypical change of vascular smooth muscle cells in an augmentation of vascular All generation. Two-kidney, one clip rats (2K1C) and one-kidney, one clip rats (1K1C) were examined to clarify the role of vascular All in aortic hypertrophy. 2KIC showed the activation of All generation both in circulating and vascular system. The suppression of All generation by ACE inhibitor, delapril, prevented the development of aortic hypertrophy, although hydralazine did not despite of the suppression of blood pressure. In contrast to 2KIC, IKIC is known to be an normoreninemic hypertensive model. In agreement with this, plasma All levels were not different between IK1C and uninephrectomized control rats (IK), but vascular ACE activity and vascular All concentration were elevated in 1K1C compared with 1K. In this model, the administration of delapril and All receptor antagonist (TCV-116) prevented vascular hypertrophy in 1KIC without the reduction of blood pressure. Taken together, these results suggest that vascular All is more important than circulating All. Mechanical stress caused by high blood pressure may be a factor to increase the vascular All leveles or affect vascular remodeling via other systems. To test this hypothesis, cultured rat aortic smooth muscle cells (SMC) and endothelial cells (EC) were deformed by a vacuum to 24% maximum elongation at 60 cycles/rain for 5 days. Cyclic stretch for EC themselves induced a tendency for a reduction in ACE activity compared with the static state (Stretch: 249 + 23 vs. Non-stretch: 362 5:51 pmol/min/mg, P<0.1). The conditioned medium from SMC augmented endothelial ACE activity compared with control MEM. Interestingly, cyclic stretch augmented this ACE activating effect by two times. Furthermore, the conditioned medium from EC increased thymidine incorporation of SMC maintained in a static condition compared with fresh control medium. Of interest, the conditioned medium from stretched-CPAE stimulated SMC growth greater than that from nonstretched-CPAE. These proliferative responses of SMC to cyclic stretch via interaction between SMC and EC may play a role in vascular remodeling to adapt to excessive hemodynamic forces. The increase of angiotensinogen gene expression and ACE activity were observed in the balloon injured blood vessel which is an hyperplastic model of smooth muscle cells. Furthermore, tissue ACE activity of injured artery showed the significant and the higher correlation with the neointimal formation compared with mean blood pressure or serum ACE activity in the rats treated with the different doses of quinapril, an ACE inhibitor. We observed that the cells expressed ACE in the neointimal lesion are the migrated SMC, and that cultured SMC from neonatal rats (pup cells), not from adult rats, expressed high level of ACE. These suggest that the phenotypical change in SMC contributes ACE expression in the injured blood vessels. in conclusion, interactions between EC and SMC under stretch stress and phenotipical change in SMC after balloon injury may be important factors for vascular remodeling via an augmentation of AII generation.

WS3-J-5-03

MOLECULAR CLONING OF HUMAN AND RAT PROSTACYCLIN RECEPTORS I.Tanaka, K. Nakao Department of Medicine and Clinical Science, Kyoto University Faculty of Medicine, Kyoto 606, Japan Prostacyclin is a labile prostanoid produced in endothelial cells of arterial wall. Chemically stable analogues of prostacyclin have been tried as inhibitors of platelet aggregation for the treatment of thrombotic diseases and as vasodilstors for the treatment of vascular occulusive diseases. In many tissues, prostacyclin receptors are the counterpart of thromboxane(TX) A2 receptors and maintain local homeostasis of the body. To clarify the structure, distribution and regulation of prostacyclin receptors, we tried to clone prostacyclin receptors from human and rat lung cDNA libraries. Human and rat clones isolated from the cDNA librades contained 1158 and 1248 bp open reading frames, respectively. The deduced amino acid residues of human and rat clones are 386 and 416 with relative molecular mass of 40,956 and 44,662. Hydropathiclty analysis revealed that both cDNA clones contain seven hydrophobic segments suggesting that they encode proteins which belong to G protein-coupled receptor superfamily. The amino acid sequence of the human clone contains two potential N-glycosylation sites and several serine and threonine residues in the cytoplasmic regions which are possible phosphorylaUon sites by protein kinase C. The homology in the amino acid sequences between the human and rat clones are 79%. However the homologies of these proteins to human and mouse TXA2 receptors and prostaglandin(PG) E receptor subtypes are less than 50%. Binding study using membrane fraction of COS-7 cells transfected with the human or rat clone revealed specific binding to [SH]lloprost, a proetecyclin receptor agonist. The dissociation constant was 7.5 nM and the maximal binding was 5 pmolhng protein in the human clone. This specific binding was inhibited by unlabeled PGs in the rank order of Iloprost = Cicaprost (a prostacyclin receptor agonist) • PGE1 > PGE2, STA2 (aTXA2 receptor agonist), PGD2 and PGF2c~. Iloprost also genrates cAMP in dose dependent manner from COS-7 cells translected with human or rat clone. From these results, we concluded that we have succeeded in the cloning of human and rat prostaoyclin receptors. Northern blot analysis of human tissues showed a mRNA band with a size of 2.5 kb. Prostacyclin receptor mRNA is most abundantly expressed in the aorta. Substantial amount of mRNA is also expressed in the lung, cardiac atrium and ventricle, kidney, liver, intestine, spleen, adrenal gland and placenta. No detectable band was observed in the cerebral cortex and cerebellum. In rat, a single mRNA band was detected in various tissues. The result showing abundant expression of prostacyclin mRNA in the aorta, heart, lung and kidney, not only is consistent with previous pharmacological studies but also provides a clue to investigate novel pathophysiologicai roles of the prostacyclin receptor in the cardiovascular system.

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