| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
The possibility of the brain-specific expression of a component of the renin-angiotensin system (RAS) was evaluated in the present study. We utilized the hemagglutinating virus of Japan (HVJ)-liposome complex to transfect human angiotensin converting enzyme (ACE) cDNA, driven by the cytomegalovirus enhancer and β-actin promoter, into the lateral cerebroventricle of male Sprague-Dawley rats. We evaluated the time course of hemodynamics, the tissue levels of angiotensin II and vasopressin, as well as ACE activity. Intracerebroventricular (ICV) transfection of the human ACE gene increased both blood pressure and heart rate. Transfected rats exhibited higher concentrations of brain Ang II and increased brain ACE activity. This activation of the brain angiotensin system was accompanied by increased vasopressin production. The increases in blood pressure and heart rate were abolished by ICV administration of an ACE inhibitor or Ang II type 1 receptor antagonist. The expression of the transge
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ne was widely distributed in the periventricular cell layer, the cortex, the hypothalamic nuclei, and the brainstem. Expression in the neuronal cells persisted for up o 14 days. Thus, this HVJ-liposome method is a highly efficient system for gene delivery and is extremely useful for functional gene transfection. This novel hypertensive model may enable characterization of the functions of the RAS in the brain, and determination of its role in the pathogenesis of hypertension.
Although the angiotensinogen gene is a possible candidate as a determinant of hypertension, the molecular mechanisms of tissue angiotensinogen gene regulation have yet to be clarified. We identified essential transcription regulators of angiotensinogen production in the central nervous system using synthetic double-stranded oligodeoxynucleotides (ODNs) as "decoy" cis-elements to block the binding of nuclear factors to promoter regions of the targeted gene. Using a gel mobility shift assay, angiotensinogen gene-activating element (AGE) 2 binding protein was detected in the brain nuclear extracts of both spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKYs). Importantly, the binding activity of AGE 2 and angiotensinogen mRNA level were significantly higher in the brain of SHRs than WKYs. Utilizing the decoy approach, we demonstrated a significant decrease in the blood pressure of SHRs by transfection of AGE 2 decoy, but not mismatched, ODNs into the lateral cerebroventricle, accompanied by a significant decrease in brain angiotensinogen concentration and mRNA, and angiotensin II level. We can ensure these effects demonstrated herein are due to central effects because no changes in circulating levels of angiotensinogen or angiotensin II concentrations were observed. Notably, AGE 2 decoy ODNs did not decrease the blood pressure of WKYs. We conclude that the abnormal expression of AGE 2 binding protein in the central nervous system plays a crucial role in high blood pressure of genetically hypertensive rat model. Less
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