Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

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

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18159号 / 医博第3879号 / 新制||医||1003(附属図書館) / 31017 / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 木村 剛, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.

Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.

Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.

Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.

The Angiotensin II Type I Receptor (AT1R) is the main receptor that produces most of the physiological actions of the Renin Angiotensin System (RAS). Alterations of AT1R expression in renal and cardiovascular diseases make this receptor an attractive target for developing an imaging agent to monitor its expression in disease states. [18F]FPyKYNE-Losartan has been developed as a derivative of the clinically used AT1R blocker Losartan. The aim of this work was to characterize this tracer and evaluate its potential as an imaging agent for AT1Rs, thereby, progressing towards human imaging of the AT1R. MicroPET imaging in rats and PET imaging in pigs displayed specific AT1R binding, high kidney-to-blood and image contrast, and slow clearance from kidneys. [18F]FPyKYNE-Losartan was shown to have 2 types of labeled metabolites in rat plasma and kidneys: hydrophilic and hydrophobic, whereas, only hydrophilic metabolite(s) in pig plasma. Plasma protein binding of [18F]FPyKYNE-Losartan was determined, by an in vitro ultrafiltration method, to be 97% which is very similar to that of Losartan (98%). FPyKYNE-Losartan displayed full antagonism of Ang II pressor effect in rats in vivo, with an ED50 of 25.5 mg/Kg and 4-times (25%) less potency than Losartan. In vitro binding studies confirmed the binding selectivity of [18F]FPyKYNE-Losartan. Bmax and Kd parameters were determined to be 348 ± 112 fmol/mm2 and 49.4 nM, respectively. Rat dosimetry studies exhibited that the sex averaged effective doses of [18F]FPyKYNE-Losartan according to ICRP 60 and 103 protocols are 2.97E-02 (mSv/MBq) and 3.06E-02 (mSv/MBq), respectively, which are within an acceptable range compared with other F-18 labeled tracers and within the safety limits of the FDA. In conclusion, [18F]FPyKYNE-Losartan has an excellent potential for translation towards human imaging to monitor AT1R expression and guide therapy.

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

Níveis elevados de hormônios tireoidianos (HTs) são comumente associados à ativação do sistema renina angiotensina local e ao desenvolvimento da hipertrofia cardíaca. O envolvimento do receptor de angiotensina II tipo 1 (AT1R) nos efeitos hipertróficos dos HTs fora descrito previamente. No entanto, os mecanismos subjacentes a essa interação ainda são desconhecidos. O AT1R pertence à família dos receptores acoplados à proteína G e, portanto, promove a transdução de sinal por mecanismos dependentes e independentes de proteína G. Recentemente, a sinalização dependente de beta-arrestinas (independente de proteína G) tem sido descrita por contribuir com a resposta hipertrófica em diferentes modelos experimentais. Assim, no presente estudo investigou-se o envolvimento da sinalização dependente de beta-arrestinas nos efeitos hipertróficos dos HTs, mediados pelo AT1R, bem como a participação de ERK&#189; nesse processo. Culturas primárias de cardiomiócitos foram estimuladas com T3 (triiodotironina; 15nM) para indução da hipertrofia. O tratamento dos cardiomiócitos com T3 por tempos rápidos (5-30 min) resultou na ativação transiente de ERK&#189;, a qual foi parcialmente atenuada quando da administração de Losartan (1&#181;M), antagonista do AT1R. A contribuição de ERK&#189; na hipertrofia dos cardiomiócitos foi verificada através do uso de PD98059 (20&#181;M), inibidor de MEK&#189;, o qual preveniu a transcrição de marcadores hipertróficos. Ensaios de imunoprecipitação revelaram o aumento da interação entre AT1R e beta-arrestina 2 sob estímulo do T3, sugerindo o recrutamento de beta-arrestina 2 e, possível, internalização do AT1R. Através de ensaios de imunofluorescência e fracionamento subcelular, foi demonstrado que o T3 estimula a translocação do AT1R, amentando sua expressão no núcleo dos cardiomiócitos. Além disso, tanto a ativação de ERK&#189; quanto a hipertrofia cardiomiocítica mostraram-se sensíveis à inibição da endocitose, a qual foi avaliada através de Concanavalina A (0,5&#181;g/ml). Ensaios de silenciamento gênico por RNA de interferência foram eficientes em demonstrar o envolvimento de beta-arrestina 2 na ativação de ERK&#189; e na hipertrofia cardiomiocítica induzida por T3. Desta forma, os resultados evidenciam o envolvimento da sinalização dependente de beta-arrestina 2 na ativação de ERK&#189;, através do AT1R, a qual contribui com a hipertrofia cardiomiocítica promovida pelo T3. / Elevated levels of thyroid hormones (THs) are commonly associated with activation of the local renin angiotensin system and the development of cardiac hypertrophy. The involvement of the angiotensin II receptor type 1 (AT1R) in the hypertrophic effects of the THs was previously described. However, the mechanisms underlying this interaction are still unknown. AT1R belongs to the G-protein coupled receptor family and promotes its signal transduction by G-protein dependent and independent mechanisms. Recently, beta-arrestin signaling (G-protein independent) has been described as contributing to the hypertrophic response in different experimental models. Thus, the present study investigated the involvement of beta-arrestin signaling in the hypertrophic effects of THs mediated by AT1R, as well as the participation of ERK&#189; in this process. Primary cardiomyocytes cultures were stimulated with T3 (triiodothyronine; 15nM) for the induction of hypertrophy. Cardiomyocytes acutely treated with T3 (5-30 min) resulted in transient activation of ERK&#189;, which was partially attenuated upon Losartan (1&#181;M) administration, an AT1R antagonist. The contribution of ERK&#189; to cardiomyocyte hypertrophy was verified by using PD98059 (20&#181;M), a MEK&#189; inhibitor, which prevented the transcription of hypertrophic markers. Immunoprecipitation assays revealed increased interaction between AT1R and beta-arrestin 2 under T3 stimulation, suggesting the recruitment of beta-arrestin 2 and, possibly, the internalization of AT1R. Through immunofluorescence and subcellular fractionation assays, T3 has been shown to stimulate AT1R translocation, enhancing its expression in the cardiomyocyte nucleus. In addition, both ERK&#189; activation and cardiomyocyte hypertrophy were sensitive to the inhibition of endocytosis, which was assessed by Concanavalin A (0.5&#181;g/ml). Interfering RNA assays were efficient in demonstrating the involvement of beta-arrestin 2 in ERK&#189; activation and in T3-induced cardiomyocyte hypertrophy. Therefore, the results evidenced the involvement of beta-arrestin-2-dependent signaling in the activation of ERK&#189;, through the AT1R, which contributes to the cardiomyocyte hypertrophy promoted by T3.