Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. EXPERIMENTAL APPROACH: A single s.c. injection of MCT (50 mg·kg(-1) ) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg(-1) C21, 3 mg·kg(-1) PD-123319 or 0.5 mg·kg(-1) A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. KEY RESULTS: Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

17beta-Estradiol modulates local cardiac renin-angiotensin system to prevent cardiac remodeling in the DOCA-salt model of hypertension in rats.

Ventricular remodeling can play a detrimental role in the progression of cardiovascular diseases, leading to heart failure. The current study was designed to investigate the effects of 17beta-estradiol (E2) on cardiac remodeling. Cardiac fibrosis and hypertrophy were examined in deoxycorticosterone acetate (DOCA)-salt treated rats with chronic, six-week administration of two different doses of E2. Bilaterally ovariectomized (Ovex) female Sprague-Dawley rats were randomly assigned to one of the following groups: Ovex-control; Ovex-DOCA; Ovex-DOCA+low-dose E2 (1.66 microg/day); or Ovex-DOCA+high-dose E2 (2.38 microg/day). All DOCA-treated rats were uninephrectomized and drinking water was replaced by 0.15M NaCl solution for the remainder of the study period. DOCA-salt treatment resulted in a significant increase in blood pressure, which was not altered by estrogen replacement. Histological examinations revealed marked cardiac remodeling (both ventricular hypertrophy and interstitial fibrosis) with DOCA treatment, which was attenuated in animals receiving estrogen therapy. Western blot analysis demonstrated increased cardiac levels of angiotensin converting enzyme (ACE) with DOCA treatment, which was attenuated by E2 replacement. Furthermore, increased levels of cardiac angiotensin converting enzyme 2 (ACE2) protein were observed in animals receiving high-dose E2 replacement. These findings suggest that physiologically relevant estrogen replacement therapy has blood pressure-independent cardioprotective effects, which are possibly mediated through modulation of the cardiac renin-angiotensin system.

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 AT1 receptor antisense DNA (AT1R-AS). The virus (25 microl, 8 x 10(9) 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 breeder's 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.

Gene therapy to control hypertension: current studies and future perspectives.

Hypertension is a complex pathophysiological state that leads to serious complications, including heart failure, coronary artery disease, and abnormal renal function. While traditional therapies can be effective in controlling the effects of hypertension, they offer no long-term cure and often lead to patient noncompliance, thereby diminishing their effectiveness. These reasons, coupled with the recent developments in gene transfer and somatic cell gene delivery, led researchers to explore alternative options that can produce long-term control of hypertension. Gene therapy offers the potential to yield lasting antihypertensive effects by influencing the genes associated with hypertension. In this review, we will discuss the merits of sense versus antisense strategies in controlling hypertension. We also discuss the advantages and disadvantages of both viral and nonviral vector types for the systemic delivery of genes for hypertension research. Results of our research group on the retrovirus-mediated delivery of the angiotensin type I receptor-antisense on the prevention of hypertension and related cardiovascular pathophysiology will be summarized. Finally, we discuss the future of this gene therapy approach in the reversal and long-term control of hypertension.

Perinatal dietary NaCl level: effect on angiotensin-induced thermal and dipsogenic responses in adult rats.

We have shown previously that administration of angiotensin II (Ang II) produces an apparent decrease in thermoregulatory set point. Exposure to high salt diets either perinatally or later in life has been shown to increase pressor responsiveness to administration of Ang II, so in the present studies we examine whether high dietary NaCl would also increase the thermal responsiveness to Ang II. In the first study, we show that exposure to a basal NaCl diet (0.12%) during gestation through 4 weeks postnatally produced very large elevations in plasma renin activity (PRA) and aldosterone concentrations in the offspring. Exposure to high salt diet (3%) did not decrease the levels of these parameters below those fed mid salt diet (1%). In the second study, we show that rats raised through 4 weeks of age on basal diet, but then fed standard chow until adulthood, showed greater changes in tail skin (T(sk)) and colonic (T(c)) temperatures following administration of Ang II (200 microg/kg sc) than either mid- or high-salt-raised groups. In the third study, we confirmed this finding and extended it to show that rats raised on a very high salt diet (6%) also did not differ from the mid-salt group. In both studies, acute water intake measured in a separate test following administration of Ang II did not differ as a function of perinatal salt diet. In a fourth study, the period of exposure to the diets was extended from the perinatal period through adulthood and, surprisingly, there was no longer an enhanced thermal response to Ang II in basal diet rats compared with rats fed the very high salt diet. In the final study, rats raised on a regular diet but exposed only as adults to the test diets showed a nonsignificant trend toward a decreased thermal response in the basal group. Thus, dietary salt level may have opposite effects on Ang II effects on adult thermoregulation, depending on the age at the exposure.

The future of hypertension therapy: sense, antisense, or nonsense?

Hypertension is a debilitating disease with significant socioeconomic and emotional impact. Despite recent success in the development of traditional pharmacotherapy for the management of hypertension, the incidence of this disease is on the rise and has reached epidemic proportions by all estimates. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau, and novel approaches for the treatment and control of hypertension must be explored. We have begun to investigate the possibility of treating and/or curing hypertension by using genetic means. In this review, we will provide evidence in favor of targeting of the renin-angiotensin system by antisense gene therapy as an effective strategy for the lifelong prevention of hypertension in the spontaneously hypertensive rat model. In addition, we will discuss the properties of an ideal vector for the systemic delivery of genes and the potential experimental hurdles that must be overcome to take this innovative approach to the next level of evaluation.

Gene therapy in cardiovascular disease. Current status.

Cardiovascular disease is the leading cause of mortality and morbidity in developed countries. Most conventional therapy is often inefficacious and tends to treat the symptoms rather than the underlying causes of the disorder. Gene therapy offers a novel approach for prevention and treatment of cardiovascular diseases. Technical advances in viral vector systems and the development of fusigenic liposome vectors have been crucial to the development of effective gene therapy strategies directed at the vasculature and myocardium in animal models. Gene transfer techniques are being evaluated as potential treatment alternatives for both genetic (familial hypercholesterolemia) and acquired occlusive vascular diseases (atherosclerosis, restenosis, arterial thrombosis) as well as for cardiac disorders including heart failure, myocardial ischemia, graft coronary arteriosclerosis and hypertension. Continued technologic advances in vector systems and promising results in human and animal gene transfer studies make the use of gene therapy a promising strategy for the treatment of cardiovascular disorders.

Gene therapy for cardiovascular disorders: is there a future?

Incidence of cardiovascular disease has reached epidemic proportions in spite of recent advances in improving the efficacy of pharmacotherapeutics. This has led many to conclude that drug therapy has reached a plateau in its effectiveness. As a result, our efforts have been diverted to explore the use of gene transfer approaches for long-term control of these pathophysiological conditions. The purpose of this review is to present various approaches that are being undertaken to provide "proof of principle" for gene therapy for cardiovascular diseases. Finally, we will discuss the future of gene therapy and other new technologies that may further advance this field of therapeutics.

Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb?

This article is based on an Experimental Biology symposium held in April 2001 and presents the current status of gene therapy for cardiovascular diseases in experimental studies and clinical trials. Evidence for the use of gene therapy to limit neointimal hyperplasia and confer myocardial protection was presented, and it was found that augmenting local nitric oxide (NO) production using gene transfer (GT) of NO synthase or interruption of cell cycle progression through a genetic transfer of cell cycle regulatory genes limited vascular smooth muscle hyperplasia in animal models and infra-inguinal bypass patients. The results of application of vascular endothelial growth factor (VEGF) GT strategies for therapeutic angiogenesis in critical limb and myocardial ischemia in pilot clinical trials was reviewed. In addition, experimental evidence was presented that genetic manipulation of peptide systems (i.e., the renin-angiotensin II system and the kallikrein-kinin system) was effective in the treatment of systemic cardiovascular diseases such as hypertension, heart failure, and renal failure. Although, as of yet, there are no well controlled human trials proving the clinical benefits of gene therapy for cardiovascular diseases, the data presented here in animal models and in human subjects show that genetic targeting is a promising and encouraging modality, not only for the treatment and long-term control of cardiovascular diseases, but for their prevention as well.

Systemic Delivery of aTransgene in Intact Animals by a Retroviral Vector.

Essential hypertension is a chronic cardiovascular disease that effects over 50 million people in the United States. It is a complex pathophysiological state that is primarily characterized by a sustained elevation in blood pressure (BP). If untreated, this chronically elevated BP can affect major target organs of the body including the heart, kidney, brain, and vascular system. As a consequence of the sustained high BP, there is an increased risk of mortality and morbidity that is characterized by myocardial infarction, congestive heart failure, stroke, end-stage renal failure, and peripheral vascular disease (1-3). Because hypertension is basically an asymptomatic disease, it confounds effective treatment and makes compliance a major issue in the treatment of this disease. Pharmacological agents currently utilized have to be administered daily in an attempt to control BP, and there are no agents available to cure essential hypertension. Thus, more effective therapeutic intervention is required in order to have a significant impact in alleviating this chronic disease and its lethal cardiovascular sequel.

Attenuation of hypertension by systemic delivery of retroviral vector containing type I angiotensin II receptor antisense cDNA.

Despite recent strides in the traditional pharmacological therapies in the control and management of hypertension, a successful prevention and cure for this disease by conventional drug strategy remain at a standstill. We have begun to investigate the conceptual possibility of the use of gene therapy in the control of hypertension. In this article we describe an experimental protocol that provides proof of the principle that antisense (AS) inhibition of Type I angiotensin II receptor (AT(1)-R) could prevent development of hypertension on a long-term basis. A retrovirus-based vector has been used to deliver AT(1)R-AS with high efficiency that attenuates development of high blood pressure and hypertension-associated cardiac and vascular pathophysiology in the spontaneously hypertensive rat.

Inability to induce hypertension in normotensive rat expressing AT(1) receptor antisense.

Our previous studies have shown that neonatal delivery of angiotensin type 1 receptor antisense (AT(1)R-AS) in a retroviral vector prevents spontaneously hypertensive rats from developing hypertension for life but has no effect on blood pressure (BP) in normotensive animals. Based on these results, we hypothesized that AT(1)R-AS transduction in normotensive rats would protect them from developing experimental hypertension. The present study was designed to evaluate this hypothesis. A single intracardiac administration of AT(1)R-AS by a retroviral-mediated delivery system (LNSV-AT(1)R-AS) in 5-day-old normotensive Sprague-Dawley rats resulted in long-term expression of the AT(1)R-AS without an effect on basal BP. However, angiotensin II (Ang II)-induced BP, dipsogenic responses, and renovascular contractility were significantly attenuated in the LNSV-AT(1)R-AS-treated rats. Chronic infusion of low-dose Ang II (55 ng. kg(-)(1). min(-)(1)) in LNSV-alone-treated rats caused a modest increase in BP, profound increase in cardiac hypertrophy, and increased vascular contractility. In contrast, the LNSV-AT(1)R-AS-treated rats were protected from developing these changes after Ang II infusion. These data establish that LNSV-AT(1)R-AS pretreatment protects healthy rats from developing Ang II-dependent hypertension.

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.

Current perspectives on the use of gene therapy for hypertension.

Systemic hypertension is a pathophysiological state that is manifested as high blood pressure and is a major risk factor for stroke, ischemic heart disease, peripheral vascular disease, and progressive renal damage. Pulmonary hypertension occurs in 3 distinct forms: primary pulmonary hypertension, pulmonary hypertension of the newborn, or secondary pulmonary hypertension attributable to a variety of lung and cardiovascular diseases. This review discusses the use of gene therapy in the control of systemic and pulmonary hypertension. Overexpression of vasodilator genes as well as antisense knockdown of vasoconstrictor genes has been successfully used in animal models of both forms of hypertension. Furthermore, the use of viral vectors to deliver these constructs has achieved long-term control of hypertension. The successful establishment of gene therapy techniques in the animal models of hypertension coupled with the anticipated advances in the genetic aspects of this disease would make it highly feasible to attempt gene delivery in the control of human hypertension.

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 I-converting enzyme antisense prevents altered renal vascular reactivity, but not high blood pressure, in spontaneously hypertensive rats.

The renin-angiotensin system plays a critical role in the control of blood pressure, and its hyperactivity is associated with the development of human primary hypertension. Because low-dose angiotensin I-converting enzyme (ACE) inhibitors cause small reductions in blood pressure that are associated with the complete reversal of altered vascular pathophysiology, our objective in this study was to determine whether ACE antisense (ACE-AS) gene delivery prevents alterations in renal vascular physiology in the parents and F(1) offspring of AS-treated spontaneously hypertensive rats (SHR). A single bolus intracardiac injection of ACE-AS (2x10(8) colony-forming units) in SHR neonates caused a modest (18+/-3 mm Hg, n=7 to 9) lowering of blood pressure, which was maintained in the F(1) generation offspring (n=7 to 9). Alterations in renal vascular reactivity, electrophysiology, and [Ca(2+)](i) homeostasis are underlying mechanisms associated with the development and establishment of hypertension. Renal resistance arterioles from truncated ACE sense-treated SHR showed a significantly enhanced contractile response to KCl and phenylephrine (n=24 rings from 6 animals, P<0.01) and significantly attenuated acetylcholine-induced relaxations (n=24 rings from 6 animals, P<0.01) compared with arterioles from ACE-AS-treated SHR. In addition, compared with cells dissociated from arterioles of ACE-AS-treated SHR, cells from truncated ACE sense-treated animal vessels had a resting membrane potential that was 22+/-4 mV more depolarized (n=38, P<0.01), an enhanced L-type Ca(2+) current density (2.2+/-0.3 versus 1.2+/-0.2 pA/pF, n=23, P<0.01), a decreased Kv current density (16.2+/-1.3 versus 5.4+/-2.2 pA/pF, n=34, P<0.01), and increased Ang II-dependent changes in [Ca(2+)](i) (n=142, P<0.01). Similar effects of ACE-AS treatment were observed in the F(1) offspring. These results demonstrate that ACE-AS permanently prevents alterations in renal vascular pathophysiology in spite of the modest effect that ACE-AS had on high blood pressure in SHR.

The effect of nitroglycerin on the gravid uterus in sheep and rabbits.

UNLABELLED: Clinical reports suggest that nitroglycerin (TNG) has an effect on the uterus that is detected by the obstetrician on palpation, but not obvious by measurement of uterine pressure. This study was designed to compare the effects of TNG on uterine compliance and tension. We used three sets of experiments to evaluate the effect of TNG on the uterus. In vivo experiments were conducted on laboring ewes and rabbits 2 h postpartum to measure the effect of TNG on active uterine tension. An in vitro experiment was also conducted by using isolated uterine strips from term pregnant rabbits to evaluate the effect of TNG on uterine compliance. TNG had no discernible effect on uterine intracavitary pressure or frequency of contraction in either laboring ewes or postpartum rabbits at doses that were sufficient to reduce the mean arterial pressure. TNG did, however, increase the compliance of uterine tissue from rabbits at term, whereas S-nitroso-N-acetylpenicillamine failed to produce a similar effect. Thus, the perceived relaxant effects of TNG may be caused by an increase in uterine compliance, which may facilitate efforts by the obstetrician to manipulate the uterus. While the mechanism of action remains unclear, it appears to be independent of nitric oxide. IMPLICATIONS: The administration of nitroglycerin may have a relaxant effect on the uterus. These experiments were conducted to elucidate the effect of nitroglycerin on the peripartum uterus by measuring active tension and compliance.

Retrovirally mediated delivery of angiotensin II type 1 receptor antisense in vitro and in vivo.

In spite of excellent drugs that are available for the control of hypertension, the pharmacological approach has major disadvantages including compliance, side effects, and inability to cure the disease. In the present chapter we provide evidence that a gene therapy concept based on the inhibition of the RAS at a genetic level, with the use of an antisense to the AT1R, is an exciting and viable approach for long-term control of hypertension without the disadvantages inherent in pharmaceutical therapy. A retrovirus-based vector has been used to deliver AT1R-AS in Ang II target tissues both in vitro and in vivo. The transduction efficiency is high and leads to the attenuation of Ang II action in vitro and prevention of hypertension in the 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.