| Rennin angiotensin aldosterone system (RAS) plays important roles in the regulation of cardiovascular and renal hemostasis. The angiotensin II, a key element in RAS, constricts vessels, and stimulates the excretion of aldosterone, and the increasing absorption of Na+ and water, which induce hypertension. High salt as one important inducer of hypertension was controversial, and recently documented data revealed that high salt could stimulate RAS and induce AT1 receptor overexpression, which is responsible for elevation of blood pressure. In this study, we employed an approach of double strand recombinant adeno-associated virus-mediated somatic gene delivery to explore potential hypotensive effect and protective roles of AT1-Antisense gene in high salt diet-fed SD rats.Double stranded adeno-associated virus vector (rAAV-D(+)) is a new generation of rAAV vector and has been shown high efficiency of infecting dividing and nondividing cells and tissues. AAV-D(+) mediated gene infection leads to a stable, long-term expression without apparent immune response and other reverse effects. These properties and the broad host range of rAAV-D(+) vectors indicate that they constitute a powerful tool for gene therapy of chronic diseases. An additional potential benefit of rAAV-D(+) vectors is their ability to integrate site-specifically in the presence of Rep proteins that can be expressed transiently, thus limiting their suspected adverse effects. To explore the potential therapeutic effects of AT1-Antisense gene, and to gain the perspective of its long term and stable expression, we delivered rat AT1 -Antisense in rAAV-D(+). The rAAV-D(+) virons containing AT1-Antisense and GFP (as control), respectively, were packed by co-transfections of 293 cells and tittered by dot blots. A single tail vein injection of rAAV-D(+)-AT1-AS or rAAV-D(+)-GFP (about 2×1010 p.f.u/rat.) into the SD rats was performed, two weeks after gene delivery, the SD rats were given 8%NaCl diet. Animal blood pressure was measured and 24hs urine was collected biweekly for further analysis until the end of the experimental duration (12 weeks). Twelve weeks after gene delivery, all animals underwent cardiovascular dynamic assessments, including the heart rate (HR), the left ventricle systolic pressure and diastolic pressure (LVPSP and LVEDP) and the maximal/minimum rate of LVP (±LV dp/dtmax), were performed by inserting a catheter into the left ventricle before animals were sacrificed under anesthesia. Expression of AT1 in rat organs was assessed by reverse transcription–polymerase chain reaction (RT-PCR). Urinary excretion of micro albumin was assessed by using ELISA, urinary Na+,K+ was monitored in clinical laboratory of Tongji Hospital. Morphological analysis was done to evaluate the renal injury and the cardiovascular remodeling. Plasma AngII was monitored by Radio-immunity assay.Result:①One week after high salt dietary, the blood pressure of rAAV-D(+)-GFP group began to elevate, and reached the max(140±4.5mmHg) in the third week up to the end; During the first three weeks of high salt dietary , the blood pressure of rAAV-D(+)-AT1-AS group also elevate, but the BP were kept in normal level (117±3.8mmHg) until the end of the experimental duration (12 weeks).②Catheterization showed that the hemodynamic deterioration was induced by high salt diet but reversed by AT1-Antisense gene delivery compared with the rAAV-D(+)-GFP control: heart rates reduced (396.5±3.33 vs 413.2±4.59/minute); LVEDP, LVPSP, +dp/dtmax and -dp/dtmax were markedly improved in treatment animals.③The result of reverse transcription-polymerase chain reaction (RT-PCR) showed that the expressions of AT1 mRNA in heart, aorta, and kidney were decreased in rAAV-D(+)-AT1-AS group compared to rAAV-D(+)-GFP group.④AT1-AS delivery significantly decreased total urine micro albumin and the ratio of Na+/K+; morphological analysis of kidney demonstrated that gene treatment significantly improved lesions of tubules and glomerules, alleviated tubular dilation and protein casts in the cortex, and the glomerular sclerosis, as well as deposit of collagen.⑤Structural analysis of heart revealed AT1-AS gene treatment attenuated hypertrophy of cardiomyocytes and deposit of collagen in heart and the myocardial remodeling induced by high salt feeding.⑥radioimmuno-assay of AngII revealed that high salt induced low level of AngII, but AT1-AS gene treatment do not enhance plasma Ang II level,.Conclusions: Based on these results, we found that rAAV-D(+) mediated AT1-AS gene delivery resulted in a long term and stable expression of AT1-AS in animals and consequently a long-term reduction of systolic blood pressure, and protected against renal injury and cardiac remodeling in high salt dietary rat. No dominant reverse reactions induced by rAAV-D(+) delivery were found. All these lead us to make conclusion that rAAV-D(+) mediated AT1-AS gene can be an promising gene remedy for hypertension and complications in the future.
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