Hypertension is a major health problem worldwide. In 1999-2000, 29% or 3.6 million Australians aged 25 yrs and over had high blood pressure (> 140 / 90 mmHg) or were on medication for the condition. It is estimated that about one billion of the world’s population has hypertension and that this will increase to 1.56 billion by 2025. Although antihypertensive drugs have been relatively successful in attenuating elevated blood pressure (BP) and in reducing adverse outcomes, control of BP depends on continuation of therapy. Drugs may have undesirable side effects which diminish compliance and BP may be resistant to treatment. Gene transfer approaches may potentially provide a tool to control BP. RNA interference (RNAi) is a new tool for the study of gene function, producing specific down regulation of protein expression. I tested the hypothesis that angiotensin II type 1 receptor (AT1R) inhibition using RNAi technology would result in sustained reduction of blood pressure in the spontaneously hypertensive rat (SHR). To enable in vivo gene delivery into animal models of hypertension, I have developed small interfering RNA (siRNA) inhibition of AT1R mRNA delivered in a DNA plasmid (pPlasRi-AT1R). Transfection of the recombinant plasmid into a mammanlian cell line resulted in strong expression of the transgenes and a significant reduction in the level of AT1R expression. pPlasRi-AT1R plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at 1.5mg/kg. Telemetric blood pressure transducers were implanted into eight month old male SHR for long-term recording of blood pressure. Twenty-four hour intra-arterial blood pressure was measured weekly. After a 2 week control period animals were injected via the tail vein with AT1R DNA plasmid (n=6), control plasmid containing green fluorescent protein (GFP, n=6) or saline (NaCl, n=6)) and followed for 8 weeks. Additional animals were treated with the DNA plasmid or saline and euthanized at 0, 1, 2, 4, 6 and 8 weeks for determination of tissue AT1R expression using RT-PCR. Aims: (i) To develop an accurate radio-telemetry BP recording method in the SHR, (ii) To design rational siRNA sequences and select of methods for effective silencing in vitro, (iii) To measure the expression of DNA delivered RNAi-AT1R plasmid in vitro and in vivo, and (iv) To determine the in vivo effect of systemic delivery of DNA AT1R plasmid on BP. Methods: Continuous 24 h arterial BP was recorded by radio-telemetry using Maclab hardware and a transducer fixed in the abdominal aorta connected to a transmitter in the abdominal cavity. Data was analyzed using software specifically written for the project. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect AT1R transcripts in various tissues following in vivo AT1R gene delivery. BP was monitored weekly for 8 weeks following 1.5 mg DNA delivered RNAi -AT1R plasmid delivery into 8-month-old SHR by tail vein injection. SHR injected with DNA enhanced green fluorescent protein (eGFP) plasmid or saline served as controls. Results: Weekly 24 h BP was successfully recorded for up to 10 weeks. Following transfection with DNA delivered RNAi -AT1R plasmid in vitro, expression of AT1R in transfected cells was determined by western blot, immunofluorescence and flow cytometry. Furthermore, RT-PCR was employed to confirm the AT1R mRNA levels. Following systemic delivery of RNAi-AT1R plasmid into middle-aged SHR, in animals injected with RNAi plasmid control blood pressure (150 +/- 1mmHg) was reduced 1week after injection (145 +/- 0.5 mm Hg, p<0.05) with maximal reduction 4 weeks after injection (127 +/- 1 mmHg, p<0.01). Blood pressure returned to control level by 8 weeks. There was no change in blood pressure in GFP plasmid or saline injected animals. Tissue expression of AT1R in heart, lung, kidney and liver was reduced following AT1R plasmid injection and was associated with reduction in pressure (r=0.99, p<0.05 for each tissue). There were no significant adverse clinical or biochemical effects. AT1R silencing resulted in significant blood pressure reduction in 8 month old male SHR for approximately 2 months. There was a significant decrease in endogenous AT1R gene expression in tissues as determined by RT-PCR. The results suggest that the systemic delivery of siRNA against AT1R mRNA by DNA-based plasmid vector may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant DNA vector for a long-lasting siRNA effect are warranted. RNAi technology with inhibition of AT1R offers a potential new paradigm for the management of high blood pressure. Conclusions: Transfection of cells with DNA delivered RNAi -AT1R plasmid resulted in detection of AT1R transcript in transfected cells confirming a silencing effect in vitro. Significant BP reduction was induced in a group of middle-aged SHR following systemic delivery of DNA plasmid incorporating the siRNA against the AT1R gene. This correlated with significant decrease of endogenous AT1R in various tissues which supported the role of the gene therapy approach in producing a reduction in BP. In summary, the thesis lays the foundation for DNA delivered RNAi mediated AT1R gene delivery as a therapeutic strategy for hypertension. Future work should consider the possible benefits of DNA vector driven AT1R shRNA plasmid containing a regulated tissue-selective promoter and explore approaches which might extend the time during which the hypotensive effect is present
| Identifer | oai:union.ndltd.org:ADTP/282229 |
| Creators | Jian Xu |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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