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.

Quantification of Angiotensin-Converting Enzyme (ACE) Activity.

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase (EC 3.4.15.1) whose physiologic action is the conversion of AI to AII, and as such, it plays an important role in the regulation of blood pressure and fluid balance. ACE has been localized to endothelial cells throughout the body and epithelial cells in gut and kidney. It exists both as a membrane-bound enzyme and in a freely soluble form in plasma. The importance of the membrane-bound form is underscored by the recently described local renin-angiotensin system (RAS) in various organs, including heart, blood vessels, and kidney (1). In the heart and vascular smooth muscle, the local RAS is believed to play an important role in hypertrophy and remodeling. Accordingly, accurate and reliable methods for measurement of tissue (i.e., membrane bound) ACE activity are important, as well as soluble ACE activity.

Quantification of Angiotensin peptides.

There is an endogenous renin-angiotensin system in the kidney, and angiotensin (Ang) II generated within the kidney may play an important role in the regulation of renal hemodynamics and sodium excretion (1-6). Assessment of the functional significance of the intrarenal renin-angiotensin system requires accurate and sensitive measurement of the Ang peptides in renal tissue. These measurements have been problematic because of methodologic difficulties, and very variable Ang peptide levels have been reported in extracts of rat kidney (7-7). Radioimmunoassay (RIA) procedures carried out on crude tissue extracts have produced inaccurate results because of interference and/or crossreactivity with biologically inactive metabolites of the Ang peptides that are present in high concentrations in tissues. Additional problems include the instability of the Ang peptides during extraction from tissue, in part because of the cleavage of peptide bonds by tissue peptidases; incomplete extraction from tissue stores; variable recoveries from solid-phase extraction on octadecylsilica gel (C(18)) cartridges, which are commonly used for the preliminary clean-up of samples before high-performance liquid chromatography (HPLC), and the difficulties inherent in gradient elution techniques for HPLC (13). We have recently developed a simplified method for extracting Ang peptides from brain tissue (14).

Permanent cardiovascular protection from hypertension by the AT(1) receptor antisense gene therapy in hypertensive rat offspring.

Our previous studies have demonstrated that the introduction of angiotensin II type I receptor antisense (AT(1)R-AS) cDNA by a retrovirally mediated delivery system prevents the development of hypertension in the spontaneously hypertensive rat (SHR), an animal model for primary hypertension in humans. These results have led us to propose the hypothesis that an interruption of the renin-angiotensin system (RAS) activity at a genetic level would prevent hypertension on a permanent basis. F(1) and F(2) generations of offspring from a retroviral vector, LNSV- and LNSV-AT(1)R-AS-treated SHR, were generated, and various physiological parameters indicative of hypertension were studied and compared with those of their parents to investigate this hypothesis. Both F(1) and F(2) generations of LNSV-AT(1)R-AS-treated SHR expressed a persistently lower blood pressure, decreased cardiac hypertrophy and fibrosis, decreased medial thickness, and normalization of renal artery excitation-contraction coupling, Ca(2+) current, and [Ca(2+)](i) when compared with offspring derived from the LNSV-treated SHR. In fact, the magnitude of the prevention of these pathophysiological alterations was similar to that observed in the LNSV-AT(1)R-AS-treated SHR parent. The prevention of cardiovascular pathophysiology and expression of normotensive phenotypes are, at least in part, a result of integration and subsequent transmission of AT(1)R-AS from the SHR parents to offspring. These data demonstrate that a single intracardiac injection of LNSV-AT(1)R-AS causes a permanent cardiovascular protection against hypertension as a result of a genomic integration and germ line transmission of the AT(1)R-AS in the SHR offspring.

Lifetime treatment with captopril improves renal function in spontaneously hypertensive rats.

Lifetime treatment with captopril prevents the development of hypertension in spontaneously hypertensive rats (SHR). This study tests the hypothesis that compared to untreated hypertensive SHR, captopril-treated SHR display similar diuretic and natriuretic responses to an isotonic saline infusion despite significantly lower arterial pressure. Eight-week-old, male SHR were instrumented with femoral arterial, venous, and bladder catheters. Forty-eight hours later, each rat was infused intravenously with an isotonic saline load (5% of body weight; 0.5 ml/min). Lifetime captopril-treated SHR and untreated control SHR displayed nearly identical natriuretic and diuretic responses to the saline infusion. Thus, although lifetime captopril treatment significantly reduces mean arterial pressure in SHR, renal excretory responses appear to be unaltered. Moreover, histological examination of the kidneys of the lifetime captopril-treated SHR did not reveal significant structural damage in the kidneys at either 8 weeks of age or at 12 months of age. Together, the data suggest that lifetime captopril treatment does not adversely affect renal function and structure in SHR.

Interaction between lifetime captopril treatment and NaCI-sensitive hypertension in spontaneously hypertensive rats and Wistar-Kyoto rats.

DESIGN: Previous studies that were based on daytime arterial pressure recordings indicate that lifetime treatment with captopril exacerbates the hypertensive response to a high NaCl diet in spontaneously hypertensive rats (SHR) but has no such effect in normotensive Wistar-Kyoto (WKY) rats. The present study used 24-h recording methods to examine the hypothesis that during the normal waking hours of rats (night-time) the hypertensive response to a high NaCl diet is exacerbated in SHR and induced in WKY rats treated with lifetime captopril. METHODS: SHR and WKY rats were (1) untreated, (2), lifetime captopril treated or (3) lifetime captopril treated but removed from the treatment 2 weeks prior to exposure to a high (8%) NaCl diet RESULTS: Compared to untreated SHR, in SHR that were continuously treated with captopril, the high NaCl diet caused a more rapid and greater rise in arterial pressure. Discontinuation of the captopril treatment did not significantly diminish this NaCl-sensitivity. In untreated WKY rats, the high NaCl diet did not alter mean arterial pressure, but in the lifetime captopril-treated WKY rats the high NaCl diet induced a rapid rise in arterial pressure. In WKY rats, discontinuation of the lifetime captopril treatment did not diminish this NaCl-induced rise in arterial pressure, even though baseline mean arterial pressure in this group is similar to that in untreated WKY rats. CONCLUSIONS: Lifetime captopril treatment accelerates the hypertensive response to a high NaCl diet in SHR, and it induces a similar response in WKY rats. In both strains, the lifetime captopril treatment causes a change in the response that is not dependent on concurrent administration of the drug. This finding further suggests that lifetime captopril treatment causes a long-term resetting of cardiovascular response mechanisms.

Captopril treatment and its withdrawal prevents impairment of endothelium-dependent responses in the spontaneously hypertensive rat.

Angiotensin converting enzyme inhibitors (ACE-I) have been shown to prevent impairment of endothelial cell function in Spontaneous Hypertensive rats (SHR). The purpose of this study was to examine the effects of early, long-term captopril (ACE-I) treatment and its withdrawal on vascular reactivity in SHR. Three groups of male SHR were studied: 1) untreated SHR; 2) SHR treated with captopril in-utero and maintained on oral treatment post-weaning (SHRCAP); and 3) SHR treated with captopril in-utero followed by withdrawal of drug therapy at two months of age (OFFCAP). All rats were studied at six months of age. Isolated aortic ring segments were suspended in tissue chambers for measurement of isometric force. Ring segments were exposed to cumulative concentrations of serotonin or phenylephrine (3 x 10(-9)-3 x 10(-5) M). SHR demonstrated an enhanced sensitivity to serotonin induced contraction. EC50 value were: SHR 3.6+/-1.4 x 10(-7)M, SHRCAP 9.5+/-0.5 x 10(-7) and OFFCAP 8.1+/-0.9 x 10(-7). Endothelium-dependent relaxation to acetylcholine (ACh) was markedly impaired in the SHR. Maximum relaxation (Rmax) to ACh was 61.1+/-1.6% of serotonin induced contraction versus 91.4 +/- 1.2% and 90.7 + 1.8% relaxation in SHRCAP and OFFCAP, respectfully (p<0.05). These data suggest that early, long-term treatment with captopril can prevent alterations in endothelial function observed in SHR even after ACE-inhibitor therapy has been stopped.

Prevention of renovascular and cardiac pathophysiological changes in hypertension by angiotensin II type 1 receptor antisense gene therapy.

Hypertension produces pathophysiological changes that are often responsible for the mortality associated with the disease. However, it is unclear whether normalizing blood pressure (BP) with conventional therapy is effective in reversing the pathophysiological damage. The duration and initiation of treatment, site of administration, and agent used all appear to influence the reversal of the pathophysiological alterations associated with hypertension. We have previously established that retrovirally mediated delivery of angiotensin II type 1 receptor antisense (AT1R-AS) attenuates the development of high BP in the spontaneously hypertensive (SH) rat model of human essential hypertension. Our objective was to determine whether this attenuation of high BP is associated with prevention of other pathophysiological changes induced by the hypertensive state. Intracardiac delivery of AT1R-AS in neonates prevented the development of hypertension in SH rats for at least 120 days. Contractile experiments demonstrated an impaired endothelium-dependent vascular relaxation (acetylcholine) and an enhanced contractile response to vasoactive agents (phenylephrine and KCl) in the SH rat renal vasculature. In addition, the voltage-dependent K+ current density, which is believed to contribute to smooth muscle resting membrane potential and basal tone, was decreased in renal resistance artery cells of the SH rat. AT1R-AS treatment prevented each of these renal vascular alterations. Finally, AT1R-AS delivery prevented the pathological alterations observed in the SH rat myocardium, including left ventricular hypertrophy, multifocal fibrosis, and perivascular fibrosis. These observations demonstrate that viral-mediated delivery of AT1R-AS attenuates the development of hypertension on a long term basis, and this is associated with prevention of pathophysiological changes in SH rats.

Pressure-independent effects of AT1-receptor antagonism on cardiovascular remodeling in aortic-banded rats.

To determine the role of angiotensin II-receptor blockade on cardiovascular remodeling in a pressure-overload model of cardiac hypertrophy, a subdiaphragmatic aortic band was placed in adult male Sprague-Dawley rats. Aortic-banded (AB) rats were left untreated or were losartan (Los; 250 mg/l) treated (AB-Los). Sham-operated (S) controls were either left untreated or treated with Los (S-Los). After 4 wk, rats were catheterized for measurement of mean arterial pressures [carotid (CMAP) and femoral (FMAP), in mmHg]. Hearts were perfused on a modified Langendorff system, and minimal coronary resistance (MCR) was determined. Hearts were then perfusion fixed, total and regional heart weights were recorded, and sections were processed for morphology. Changes in coronary artery medial thickness and perivascular fibrosis were assessed by quantitative image analysis. CMAP was significantly higher in AB and AB-Los than in S or S-Los (P < 0.05). There was no difference in FMAP in AB vs. S, but AB-Los and S-Los had lower FMAPs than S. Total heart weight and left ventricular weight-to-body weight ratios were increased in AB and AB-Los compared with S and S-Los (P < 0.05). MCR of AB was greater than S and S-Los. MCR of AB-Los was significantly lower than AB and was not significantly different from S and S-Los. In coronary vessels, medial thickness was greatest in AB, whereas there was no difference among AB-Los, S, and S-Los. Similarly, the increase in perivascular fibrosis was greatest in AB, and there was no difference among AB-Los, S, and S-Los. These data suggest that angiotensin II, independent of increased arterial pressure, is critical for the development of the vascular and fibrotic changes that occur in this model of pressure-overload hypertrophy.

Early, short-term treatment with captopril permanently attenuates cardiovascular changes in spontaneously hypertensive rats.

The purpose of the current study was to determine if early, short-term treatment of spontaneously hypertensive rats (SHR) with captopril would cause a persistent attenuation of the structural alterations of the heart, aorta, and coronary arteries that are commonly seen in adult SHR. Therefore, mating pairs of SHR were treated with captopril and the pups were kept on captopril (SHRC) or were taken off captopril at two months (SHROC). Untreated SHR and Wistar-Kyoto (WKY) rats were mated and served as controls. At 8-10 months of age, heart weight and left ventricular weight/body weight ratios were increased in SHR compared to WKY, SHRC, and SHROC. Aortic medial areas of SHR and SHROC were similar and were larger than WKY and SHRC. Nuclear density in SHR and SHROC was less than WKY and SHRC suggesting hypertrophy of the medial wall. In coronary vessels, medial thickness was greatest in SHR, while there was no difference among WKY, SHRC, SHROC. These data suggest that early, short-term treatment of SHR with captopril permanently attenuated the structural alterations in the heart and coronary vessels that are commonly seen in adult SHR.

Captopril prevents vascular and fibrotic changes but not cardiac hypertrophy in aortic-banded rats.

To determine the role of the renin-angiotensin system (RAS) on cardiovascular remodeling in a pressure overload model of cardiac hypertrophy, a subdiaphragmatic aortic band was placed in adult male, Sprague-Dawley rats. Rats were left untreated (AB) or given captopril (Cap, 400 mg/l) (AB-Cap). Sham-operated controls were either left untreated (S) or given Cap (S-Cap). After 4 wk, rats were catheterized, and carotid and femoral mean arterial pressures (CMAP and FMAP in mmHg, respectively) were recorded. Hearts were isolated, and minimal coronary resistance (MCR) was determined. Hearts were then perfusion fixed, total and regional heart weights were recorded, and sections were processed for vessel morphology. Changes in coronary artery medical thickness and perivascular fibrosis were assessed by quantitative image analysis. CMAP was significantly higher in AB and AB-Cap than S or S-Cap rats (P < 0.05). There was no difference in FMAP in AB vs. S rats, but AB-Cap and S-Cap had lower FMAP values than S rats. Total heart weight and left ventricular weight-to-body weight ratios were increased in AB and AB-Cap rats compared with S and S-Cap rats (P < 0.05). MCR of AB was greater than S and S-Cap rats. MCR of AB-Cap rats was significantly greater than S and S-Cap rats but was significantly less than AB rats. In coronary vessels, medial thickness was greatest in AB, whereas there was no difference among AB-Cap, S, and S-Cap rats. Similarly, the increase in perivascular fibrosis was greatest in AB rats, and there was no difference among AB-Cap, S, and S-Cap rats. These data suggest that the RAS, independent of increased arterial pressure, is critical for the development of the vascular and fibrotic changes that occur in this model of pressure overload hypertrophy.

Effects of early captopril treatment and its removal on plasma angiotensin converting enzyme (ACE) activity and arginine vasopressin in hypertensive rats (SHR) and normotensive rats (WKY).

The purpose of this study was to evaluate the effects of early administration and removal of the ACE inhibitor, captopril (CAP) on the plasma ACE activity, AVP levels, and mean arterial pressure (MAP) in groups of rats, control and CAP treated SHR and WKY (SHR, WKY SHRCAP, WKYCAP, respectively and in SHR taken off CAP (OFFCAP) and their progeny (2nd generation, 2ndG). Plasma ACE activity in SHRCAP (54.8 +/- 2.1 mU/ml/min) was significantly greater than in SHR (25.96 +/- 0.34 mU/ml/min) and their offspring (OFFCAP, 26.32 +/- 2.71 and 2ndG, 17.62 +/- 2.47 mU/ml/min, P < 0.001, respectively). Plasma level of AVP in SHR (14.18 +/- 0.98 pg/ml) were greater than in SHRCAP (9.1 +/- 1.01 pg/ml, P < 0.01). A decrease in plasma AVP levels were also noted in OFFCAP (10.48 +/- 0.51 pg/ml) and their offspring 2ndG (10.34 +/- 0.46 pg/ml). Our results did not show a relationship between plasma ACE activity and blood pressure reduction. However, treatment of SHR with captopril produced a decrease in plasma AVP levels which may participate in its antihypertensive mechanism of action.

Pressor and bradycardic effects of centrally administered relaxin in conscious rats.

The current study tested the hypothesis that centrally administered relaxin elevates arterial pressure in conscious rats and that this hypertensive effect is mediated, at least in part, by central or peripheral vasopressin. Injection of human relaxin (0.068 or 0.34 microgram in 200 nL artificial cerebrospinal fluid) into the right lateral ventricle of conscious, unrestrained Sprague-Dawley rats caused significant dose-related increases in arterial pressure and decreases in heart rat. The pressor and bradycardic responses to intracerebroventricular injections of relaxin were significantly blunted by pretreatment with either intracerebroventricular or intravenous injection of a vasopressin receptor (V1) antagonist, suggesting that the cardiovascular effects of central relaxin are mediated, at least in part, by V1 receptors in the brain and perhaps also by vasopressin released into the peripheral circulation. Neither intracerebroventricular injection of the vehicle alone nor intravenous injection of relaxin (0.34 microgram) altered arterial pressure or heart rate. In contrast to the above, intravenous injections of relaxin (40 micrograms/kg) elicited pressor and tachycardic responses that were not blunted by pretreatment with either intracerebroventricular or intravenous injection of the V1 receptor antagonist. Together, these data suggest that in the central nervous system relaxin contributes to the regulation of cardiovascular function and that the mechanisms for the cardiovascular effects of central and peripheral relaxin are distinct.

Salt-induced hypertension in normotensive spontaneously hypertensive rats.

Lifetime treatment with oral captopril prevents the development of hypertension in spontaneously hypertensive rats (SHR). We tested the hypothesis that this treatment also prevents the hypertensive response that occurs when untreated NaCl-sensitive SHR are placed on a high NaCl diet. Female SHR were continuously treated with oral captopril before conception and throughout lactation, and the offspring were similarly treated with oral captopril throughout life. At 6 weeks of age, treated male SHR were placed on an 8% (or remained on a 1%) NaCl diet, and systolic arterial pressure, heart rate, and body weight were monitored for 2 weeks. The 8% NaCl diet caused a rapid increase in arterial pressure in the lifetime captopril-treated rats, and 18 days after the initiation of the diet, the mean arterial pressure of this group was 136 +/- 7 mm Hg compared with 100 +/- 2 mm Hg in the 1% NaCl diet rats. The results of a second experiment confirmed the hypertensive effect of the high NaCl diet in lifetime captopril-treated SHR and demonstrated that after 18 days on the diet the dietary NaCl-induced hypertensive response was greater in magnitude in lifetime captopril-treated compared with untreated SHR. The results also demonstrated that lifetime captopril-treated Wistar-Kyoto rats, which are normotensive irrespective of captopril treatment, display no significant increase in arterial pressure when given a high NaCl diet. A third experiment demonstrated that rapidly progressing NaCl sensitivity is also present in female lifetime captopril-treated SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in renal angiotensin II receptors in spontaneously hypertensive rats by early treatment with the angiotensin-converting enzyme inhibitor captopril.

We tested the hypothesis that in utero treatment with the angiotensin-converting enzyme inhibitor captopril could change the affinity, density, and/or subtypes of angiotensin II (Ang II) receptors in the kidneys of spontaneously hypertensive rats (SHR). Newborn, 7-day-old, and 4-month-old SHR and Wistar-Kyoto (WKY) rats were used. SHR and WKY rat breeders were treated with captopril (0.4 mg/mL, 100 mg/kg per day) in drinking water, and their pups were maintained on captopril treatment until experimentation. Control groups were untreated, age-matched SHR and WKY rats. The density, affinity, and subtypes of renal Ang II receptors were determined using radioligand binding techniques and receptor antagonists specific for Ang II receptor subtypes 1 and 2 (losartan, an AT1-specific antagonist, and CGP 42112B, an AT2-specific antagonist). AT1 receptor density in kidneys was higher than AT2 receptor density in both neonatal and adult rats. AT1 receptor density in kidneys increased approximately twofold from birth to 7 days of age in all groups. Newborn and 7-day-old SHR showed significantly greater Ang II receptor densities in kidneys than other rat groups because of significantly greater densities of both AT1 and AT2 receptors. At 4 months of age, there were no significant differences in Ang II receptor densities in kidneys between captopril-treated and control SHR. Our data indicate that the expression of AT1 and AT2 receptors in kidneys is differentially regulated during development. Enhanced activity of the renal renin-Ang II system in newborn and probably fetal SHR may be involved in the pathogenesis of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of anterior hypothalamic angiotensin II in the pathogenesis of salt sensitive hypertension in the spontaneously hypertensive rat.

Selective alterations in noradrenergic mechanisms in the anterior hypothalamic area (AHA) of NaCl-sensitive spontaneously hypertensive rats (SHR-S) have been demonstrated during dietary NaCl supplementation. To test the hypothesis that endogenous angiotensin II (Ang II) in the AHA also plays a role in blood pressure regulation and in NaCl sensitive hypertension in the SHR-S, Type 1 Ang II (AT1) receptors in the AHA were blocked by local microinjection of losartan, a selective nonpeptide AT1 receptor antagonist, and the effects of the intervention on blood pressure were observed. Microinjection of losartan into the AHA of conscious rats caused a significant dose-related decrease in mean arterial pressure in SHR-S but not in Wistar-Kyoto (WKY) rats. To test the hypothesis that the depressor response to AHA AT1 receptor blockade is enhanced by high (8%) NaCl feeding in SHR-S, losartan was microinjected into the AHA of conscious SHR-S and WKY rats that had been fed 1% or 8% NaCl diets for 3 weeks. The magnitude and duration of the depressor response to losartan were significantly greater in the 8% NaCl fed SHR-S than in the 1% NaCl fed rats. These findings, along with the observation that Ang II receptor numbers are increased in neurons isolated from brain of SHR compared with WKY rats, suggest that endogenous Ang II acting on AT1 receptors in the AHA participates in the tonic control of blood pressure in SHR-S but not in normotensive WKY rats. In addition, it is involved in the pathogenesis of NaCl sensitive hypertension in the SHR-S.

Prevention of genetic hypertension by early treatment of spontaneously hypertensive rats with the angiotensin converting enzyme inhibitor captopril.

Our purpose was to evaluate whether early treatment of spontaneously hypertensive rats (SHR) with the angiotensin converting enzyme inhibitor captopril could permanently alter the course of hypertension. Mating pairs of SHR were treated with captopril, and their pups were maintained on captopril until experimentation. Some captopril-treated rats were taken off treatment at 2 months of age, and then some of these rats were mated at 3 months of age. The mean arterial pressures of conscious captopril-treated rats, the rats removed from therapy, and the offspring of the rats removed from therapy were significantly smaller than control rats at 4 and 9 months of age. Central administration of angiotensin I or II induced significantly smaller increases in blood pressure and drinking in captopril-treated rats and the rats removed from therapy compared with control rats. The increase in blood pressure in response to intravenous injection of angiotensin I or II was similar among all groups, with the exception that captopril-treated rats showed lesser pressor responses to angiotensin I. Early administration of captopril, even after administration was stopped, prevented the subsequent development of hypertension in SHR and altered the course of development of hypertension in their progeny. This effect was associated with decreased central responses to angiotensin I and II. Our data suggest that captopril may permanently alter the development of hypertension in SHR through an alteration in the central renin-angiotensin system.