Phyllis Y. Reaves

Losartan Versus Gene Therapy: Chronic Control of High Blood Pressure in Spontaneously Hypertensive Rats

Interruption of the renin-angiotensin system by pharmacological manipulations attenuates high blood pressure (BP) in the spontaneously hypertensive rat (SHR). However, these agents, such as losartan, need to be administered daily to maintain effective BP control. Therefore, we have hypothesized that a genetic intervention in the expression of angiotensin type 1 receptor (AT1R) should attenuate development of hypertension on a long-term basis in SHR. A retroviral-mediated AT1R antisense cDNA gene delivery system (LNSV-AT1R-AS) was used to test this hypothesis and to compare its BP-lowering effects with those of losartan. Introduction of LNSV-AT1R-AS into 5-day-old Wistar-Kyoto rats and SHR resulted in a robust expression of AT1R antisense (AS) within 3 days and persisted for at least 30 days. This expression was associated with a selective attenuation of high BP in SHR by 25 to 30 mm Hg. Although basal lowering of BP was exclusive to SHR, the angiotensin II (Ang II) pressor response was significantly reduced in all LNSV-AT1R-AS-treated rats. The decreased response to Ang II was associated with a similar attenuation of Ang II-induced dipsogenic responses in both strains of rats. The BP-lowering effects of LNSV-AT1R-AS treatment and losartan treatment were similar and primarily observed in SHR. However, the antihypertensive effect lasted less than 24 hours in losartan-treated SHR compared with 90 days in LNSV-AT1R-AS-treated SHR. In addition, losartan was unable to further lower BP in LNSV-AT1R-AS-treated SHR. Collectively, these results suggest that both losartan and LNSV-AT1R-AS treatment produces an antihypertensive response selectively in SHR that is mediated by interruption of AT1R function. However, a single, acute genetic treatment with LNSV-AT1R-AS can result in long-term control of high BP at a similar level of effectiveness as losartan, without altering plasma Ang II levels.

Reversal of hypertension by angiotensin II type 1 receptor antisense gene therapy in the adult SHR

Pharmacological blockade of the renin-angiotensin system in both hypertensive patients and animal models such as the spontaneously hypertensive rat (SHR) effectively reduces blood pressure (BP). Recent studies have established that virally mediated delivery (vector LNSV) of antisense to the angiotensin II type 1 receptor (LNSV-AT1R-AS) will attenuate or abolish the development of hypertension in the SHR. However, the effectiveness of this gene therapy approach to reduce high BP once it is established in the adult has not been ascertained. In this study, we investigated the hypothesis that viral delivery of AT1R-AS into the adult SHR will reduce BP and reverse the vascular reactivity associated with the hypertension. Intracardiac injection of virus particles containing LNSV-AT1R-AS into adult SHR resulted in a 30- to 60-mmHg reduction in BP that was maintained for up to 36 days compared with SHR treated with virus alone (LNSV without antisense). Measurement of renal resistance arteriolar reactivity demonstrated a leftward shift in the KCl and phenylephrine concentration-response relationships and an impaired endothelium-dependent relaxation to ACh in LNSV-treated SHR compared with control Wistar-Kyoto rats. These vascular alterations were reversed in the LNSV-AT1R-AS-treated SHR. Collectively, these data demonstrate that virally mediated gene delivery of AT1R-AS can effectively reduce BP and reverse renovascular pathophysiology associated with the hypertensive state when administered to the adult SHR.

Gene therapy attenuates the elevated blood pressure and glucose intolerance in an insulin-resistant model of hypertension.

Objective Fructose feeding in male Sprague–Dawley (SD) rats results in a mild hypertension and glucose intolerance. Although the mechanism of this glucose intolerance and hypertension is not completely understood, a role for the renin–angiotensin system (RAS) has been proposed. In the current study our aim was to test the hypothesis that intervention of the RAS with a gene therapy approach would be effective in preventing the development of hypertension and glucose intolerance in this animal model. Design and methods Five-day-old SD rats were administered either an empty retroviral vector (LNSV) or retroviral vector containing AT1receptor antisense DNA (AT1R-AS). The virus (25 μl, 8 × 109 CFU/ml) was injected into the heart and the animals were returned to their mothers. After weaning, half the animals from each group were placed on breeders chow or a 60% fructose diet. Indirect blood pressures (BP) were determined and an oral glucose tolerance test (OGTT) was performed when the animals had been on the respective diets for 2 months. Results Fructose-fed animals developed mild hypertension (145 ± 3 versus 132 ± 4 mmHg) by 6 weeks of dietary intervention. This increase in BP was prevented by AT1R-AS treatment (125 ± 3 mmHg). At 2 months of age, fasting blood glucose was comparable among the four groups; however, the glucose excursion during the OGTT was significantly greater and more prolonged in the LNSV-treated, fructose-fed group than the other three groups. AT1R-AS treatment significantly prevented glucose intolerance in the fructose rat to levels observed in the controls. Conclusions Early fructose dietary treatment results in moderate hypertension and glucose intolerance, which is prevented by a single neonatal treatment with AT1R-AS. These results suggest that the RAS is involved in the glucose intolerance associated with fructose feeding and that genetic intervention is effective in this rat model.

Targeting of the renin-angiotensin system by antisense gene therapy: a possible strategy for the long-term control of hypertension.

Traditional pharmacological agents have been successfully used for the treatment of hypertension for a number of decades. However, this therapeutic regimen has reached a conceptual plateau and a cure for the disease is far from appearing on the horizon. With this in mind, and recent advances in state of the art gene delivery system coupled with the anticipated completion of the human genome project, it is timely to think about the possibility of treating and/or curing hypertension using genetic means. In this review, we discuss the role of renin–angiotensin system (RAS) in hypertension; the current gene delivery/gene transfer systems and the RAS as a target for gene therapy to treat hypertension; the successful use of retroviral vectors to deliver antisense to the AT1 receptor (AT1-AS) to prevent the development of hypertension and cardiovascular pathophysiology; the potential use of the viral vectors for the reversal of hypertension; and the future of antisense gene therapy and potential advantages and limitations of this regimen in the treatment and/or control of hypertension.

Sustained Inhibition of Angiotensin I–Converting Enzyme (ACE) Expression and Long-Term Antihypertensive Action by Virally Mediated Delivery of ACE Antisense cDNA

Angiotensin I-converting enzyme (ACE) inhibitors have been proven to be highly effective and are for the most part the drugs of choice in the treatment and control of hypertension, congestive heart failure, and left ventricular dysfunction. Despite this, questions regarding side effects and compliance with this traditional pharmacological strategy remain. In view of these observations, coupled with recent advances in gene-transfer technology, our objective in this study was to determine whether the expression of ACE could be controlled on a permanent basis at a genetic level. We argued that the introduction of ACE antisense to inhibit the enzyme would be a prerequisite in considering the antisense gene therapy for the control of hypertension and other related pathological states. Retroviral vectors (LNSV) containing ACE sense (LNSV-ACE-S) and ACE antisense (LNSV-ACE-AS) sequences were constructed and were used in rat pulmonary artery endothelial cells (RPAECs) to determine the feasibility of this approach. Infection of rat RPAECs with LNSV-ACE-S and LNSV-ACE-AS resulted in a robust expression of transcripts corresponding to ACE-S and ACE-AS, respectively, for the duration of these experiments, ie, 8 consecutive passages. The expression of ACE-AS but not of ACE-S was associated with a permanent decrease of approximately 70% to 75% in ACE expression and a 50% increase in the B(max) for the AT(1)s. Although angiotensin II caused a concentration-dependent stimulation of intracellular Ca(2+) levels in both ACE-S- and ACE-AS-expressing cells, the stimulation was significantly higher in ACE-AS-expressing RPAECs. In vivo experiments demonstrated a prolonged expression of ACE-AS transcripts in cardiovascularly relevant tissues of rats. This was associated with a long-term reduction in blood pressure by approximately 15 mm Hg, exclusively in the spontaneously hypertensive rat. These observations demonstrate that delivery of ACE-AS by retroviral vector results in a permanent inhibition of ACE and a long-term reduction in high blood pressure in the spontaneously hypertensive rat.

Angiotensin I–Converting Enzyme Antisense Gene Therapy Causes Permanent Antihypertensive Effects in the SHR

The renin-angiotensin system plays a critical role in the control of blood pressure (BP), and its hyperactivity is associated with the development and maintenance of hypertension. Although traditional pharmacological therapies targeted toward the inhibition of the renin-angiotensin system are effective in the control of this disease, they pose significant limitations. We used an antisense gene delivery strategy to circumvent these limitations and established that a single intracardiac administration of angiotensin type 1 receptor antisense (AT(1)R-AS) causes permanent prevention of hypertension in the spontaneously hypertensive rat (SHR), an animal model of primary human hypertension. Our objectives in this study were 2-fold: to determine (1) whether the targeting of angiotensin I-converting enzyme (ACE) mRNA by a similar antisense strategy would prevent the SHR from developing hypertension and (2) whether the antihypertensive phenotype is transmitted to the offspring from the antisense-treated parents. Administration of a retroviral vector containing ACE antisense (LNSV-ACE-AS) caused a modest yet significant attenuation of high BP ( approximately 15+/-2 mm Hg) exclusively in the SHR. This was associated with a complete prevention of cardiac and renovascular pathophysiological alterations that are characteristic of hypertension. Like their parents, the F(1) generation offspring of the LNSV-ACE-AS-treated SHR expressed lower BP, decreased cardiac hypertrophy, and normalization of renal arterial excitation-coupling compared with offspring derived from the LNSV-ACE-tS (truncated sense)-treated SHR. In addition, the endothelial dysfunction commonly observed in the SHR renal arterioles was significantly prevented in both parents and offspring of the LNSV-ACE-AS-treated SHR. Polymerase chain reaction followed by Southern analysis revealed that the ACE-AS was integrated into the SHR genome and transmitted to the offspring. These observations suggest that transmission of ACE-AS by retroviral vector may be responsible for the transference of normotensive phenotypes in the SHR offspring.

Angiotensin II Type 1 Receptor Antisense Gene Therapy Prevents Altered Renal Vascular Calcium Homeostasis in Hypertension

Intracellular Ca2+ ([Ca2+]i) homeostasis regulates vascular smooth muscle tone, and alteration in [Ca2+]i handling is associated with the development and establishment of hypertension. We have previously established in the spontaneously hypertensive rat (SHR) that virally mediated delivery of angiotensin II type 1 receptor antisense (AT1R-AS) prevents the development of high blood pressure and some pathophysiology associated with hypertension for 120 days. In light of this, our objectives in this study were to determine whether AT1R-AS gene therapy (1) could have a longer duration in the prevention of hypertension and (2) would attenuate the alterations in renal vascular Ca2+ homeostasis and therefore vasoconstriction, characteristics of hypertension. Intracardiac delivery of AT1R-AS in neonates prevented the development of hypertension in SHR for at least 210 days. At this time, untreated SHR renal resistance arterioles showed a significantly enhanced contractile response to KCl and angiotensin II (Ang II) when compared with normotensive Wistar-Kyoto rats. In addition, L-type Ca2+ current density and Ang II-dependent increases in [Ca2+]i were significantly increased in cells dissociated from renal resistance arterioles of the untreated SHR. AT1R-AS treatment prevented all of the above vascular alterations associated with the hypertensive state in SHR. Finally, Western blot analysis of L-type Ca2+ channel (alpha1C) protein levels in renal resistance arterioles of untreated SHR showed no significant difference when compared with control. These results are novel and demonstrate that viral-mediated delivery of AT1R-AS not only attenuates the development of hypertension on a long-term basis but prevents changes in renal vascular Ca2+ homeostasis associated with the disease.

Endothelial‐Independent Prevention of High Blood Pressure in L‐Name‐Treated Rats by Angiotensin II type I Receptor Antisense Gene Therapy

It has previously been established that a single systemic administration of retroviral vector containing angiotensin II type I receptor antisense (AT1R‐AS) in the neonatal spontaneously hypertensive rat (SHR) prevents development of hypertension, and in addition cardiac hypertrophy and endothelial dysfunction. However, these studies could not determine whether the effects of AT1R‐AS on high blood pressure (BP) and endothelial function were independent. Angiotensin receptor blockers have been shown to reduce BP in the L‐NAME (N ω‐nitro‐L‐arginine methyl ester hydrochloride)‐induced rat model of hypertension. Our objective in the present study was to use the L‐NAME model of hypertension to determine whether AT1R‐AS treatment would lower high BP and attenuate cardiac hypertrophy under conditions of permanent endothelial damage. A single bolus of LNSV‐AT1R‐AS viral particles in neonatal Wistar‐Kyoto (WKY) rats was without affect on basal BP. Efficacy of the transgene incorporation was assessed by observing a significant reduction in angiotensin‐induced dipsogenic response in the AT1R‐AS‐treated animals. Introduction of L‐NAME in the drinking water for 10 weeks resulted in the establishment of hypertension only in the WKY rats treated with vector alone. These hypertensive (BP, 179 ± 4 mmHg) animals showed a 17% increase in heart weight/body weight ratio and a 60% reduction in ACh‐induced vasorelaxation in phenylephrine‐preconstricted arteries. The L‐NAME‐induced high BP and cardiac hypertrophy were attenuated in rats expressing AT1R‐AS. However, endothelial dysfunction could not be prevented with the antisense therapy. These observations demonstrate that attenuation of endothelial dysfunction is not a prerequisite for the antihypertensive effects of AT1R‐AS treatment.

Decrease in Hypothalamic Gamma Adducin in Rat Models of Hypertension

Abstract—We have previously shown that a decrease in hypothalamic gamma adducin (&ggr;-adducin) is associated with hypertension in the spontaneously hypertensive rat (SHR). In view of many inherent issues with SHR, our objective in the present study was to provide proof of this concept with the use of 2 nongenetic rat models of hypertension. Subcutaneous angiotensin II (Ang II) infusion for 2 weeks (55 ng/kg per day) resulted in an increase in blood pressure (BP) of 18 mm Hg. This was associated with a 70% decrease in hypothalamic &ggr;-adducin. Concomitant administration of losartan attenuated the development of hypertension and a decrease in &ggr;-adducin. Deoxycorticosterone acetate salt-induced hypertension also caused a 70% decrease in hypothalamic &ggr;-adducin. Finally, neuronal cultures from neonatal rat brains were incubated with 100 nmol/L Ang II for 4 hours to mimic the in vivo Ang II infusion rat model. This chronic incubation with Ang II resulted in a 60% decrease in the neuronal &ggr;-adducin. Taken together, these observations strengthen our hypothesis that a decrease in hypothalamic &ggr;-adducin is linked to hypertension.

[39] Retrovirally mediated delivery of angiotensin II type 1 receptor antisense in vitro and in vivo

Publisher Summary Despite the availability of excellent drugs for the control of hypertension, the pharmacological approach has major disadvantages such as compliance, side effects, and inability to cure the disease. A gene therapy concept based on the inhibition of the renin-angiotensin system (RAS) at a genetic level, with the use of an antisense to the ang II type 1 receptor (AT 1 R), is an exciting and viable approach for long-term control of hypertension without the disadvantages inherent in pharmaceutical therapy. In an experiment described in the chapter, a retrovirus-based vector was used to deliver AT 1 R-antisense (AT 1 R-AS) in ang II target tissues both in vitro and in vivo. The transduction efficiency was high and led to the attenuation of ang II action in vitro and prevention of hypertension in the spontaneously hypertensive (SH) rat, a model for primary human hypertension. These studies have unveiled a new avenue in which a similar approach could be attempted in the reversal of hypertension in adult animals.