Angiotensin I–Converting Enzyme Antisense Prevents Altered Renal Vascular Reactivity, but Not High Blood Pressure, in Spontaneously Hypertensive Rats
Abstract—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 F1 offspring of AS-treated spontaneously hypertensive rats (SHR). A single bolus intracardiac injection of ACE-AS (2×108 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 F1 generation offspring (n=7 to 9). Alterations in renal vascular reactivity, electrophysiology, and [Ca2+]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 Ca2+ 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 [Ca2+]i (n=142, P<0.01). Similar effects of ACE-AS treatment were observed in the F1 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.
Hypertension is a complex disease that is manifested as chronically high blood pressure (BP) and is a major risk factor in many cardiovascular pathophysiological states, including arteriosclerosis, stroke, heart failure, coronary artery disease, and progressive renal damage.1 2 3 Evidence has established that a dysfunctional renin-angiotensin system (RAS) is one of the many physiological alterations that contribute to the development and maintenance of hypertension.4 5 6 This is based on the fact that pharmacological interruption in the activity of the RAS has proven to be highly successful in the treatment and management of hypertension in a significant population of hypertensive patients.7 8 9 10 However, this pharmacological intervention has major limitations, which include compliance, side effects, and relatively short duration of antihypertensive effects.11 12 As a result of these limitations, we have begun to use an antisense (AS) gene therapy approach to determine whether targeting RAS at a genetic level would be a step forward in the long-term control of hypertension. These studies have revealed that a single intracardiac administration of retroviral vector containing angiotensin II type 1 receptor (AT1R) AS results in long-term prevention of high BP in the spontaneously hypertensive rat (SHR), which is an animal model associated with studies involving the prevention of renovascular and cardiac pathophysiological changes that are characteristic of hypertension.13 14 15 16 In view of these observations, we set out to investigate the following: First, we wanted to determine whether the targeting of another component of the RAS at a genetic level with a similar strategy (angiotensin I–converting enzyme [ACE]-AS) would produce long-lasting antihypertensive effects comparable to those seen with AT1R-AS. Second, we wanted to determine whether the alterations in Ca2+ and Kv channel activity as well as Ca2+ homeostasis, which occurs in vascular smooth muscle cells of renal resistance arterioles of the SHR, are prevented by use of ACE-AS.
Construction of LNSV Containing ACE-tS and ACE-AS
A retroviral vector, LNSV, was used to deliver ACE-AS and truncated ACE-sense (ACE-tS) into rats. Rat ACE cDNA was generated by reverse transcription–polymerase chain reaction (RT-PCR) with the use of ACE-specific primers (sense, 5′-GCGTCGACACCAACATCACGGAGGAGAA-3′; antisense, 5′-ATGTCGACCCGCGTGCACTTCTTAAT-3′) that corresponded to nucleotides 254 through 1181, as previously described.17
Preparation of Culture Media Containing ACE-tS and ACE-AS
Five-day-old Wistar-Kyoto rats (WKY) and SHR were divided into control, viral control (ACE-tS), and experimental (ACE-AS) groups. The animals were lightly anesthetized with methoxyflurane (Metophane, Pittman-Moore). They were injected with 10 μL, via cardiac route, of either physiological saline (control), 2×108 colony-forming units of viral particles containing ACE-tS (viral control), or ACE-AS (experimental), as described previously.13 14 15 16 WKY and SHR parents who were treated with either ACE-tS or ACE-AS at 5 days of age were used for breeding. A pair of 120-day-old ACE-tS–treated males were bred with a pair of ACE-tS–treated females of comparable age to generate ACE-tS offspring. Similarly, ACE-AS–treated males were mated with the ACE-AS–treated females to generate ACE-AS F1 offspring.
Carotid and jugular cannulations were carried out for the measurement of direct BPs in free-moving nonrestrained animals as described previously.13 14 15 16 Animals were euthanized, and the hearts and kidneys were excised in physiological saline. Renal arteriolar vasoreactivity, ion channel activity, and [Ca2+]i measurements were performed as previously described.15 16
All results are expressed as mean±SE. Direct mean BPs were analyzed by ANOVA. Vascular reactivity, ion channel activity, and [Ca2+]i measurements were analyzed by ANOVA and Student t test and were considered significant at P≤0.05.
ACE-AS and BP
Compared with ACE-tS–treated WKY, ACE-tS–treated SHR began to express significantly higher BP by 63 days of age. Therefore, direct mean BP of treated WKY and SHR was measured at 100 days of age (Table 1⇓). BPs in saline-treated control WKY and SHR were always comparable to those of respective controls (ACE-tS). As a result, only viral controls were used in subsequent experiments, unless indicated otherwise. No significant difference in BPs was observed between ACE-tS– and ACE-AS–treated WKY. In contrast to the direct mean BP in WKY, the direct mean BP in the ACE-AS–treated SHR was significantly lower (130± 4 mm Hg, n=7, P<0.01) than the mean direct BP of ACE-tS–treated SHR (147±4 mm Hg, n=7). F1 offspring from ACE-tS–treated SHR showed a significantly higher direct mean BP compared with that of their WKY controls (152±6 versus 124±6 mm Hg, n=9, P<0.01) at 100 days of age (Table 1⇓). Whereas offspring of ACE-AS–treated WKY showed no difference in mean BP compared with their ACE-tS controls, SHR offspring from the ACE-AS treatment group exhibited an average of 18±3 mm Hg lower direct mean BP compared with their ACE-tS controls (Table 1⇓). These observations establish the efficacy of the ACE viral vector and show that a single intracardiac administration of ACE-AS can result in a modest, yet long-term, attenuation of the development of high BP in the SHR and its offspring. In fact, we have previously shown that ACE-AS causes a reduction in ACE activity and expression in vitro,17 and we have also shown a decrease in ACE activity and expression with the use of ACE-AS gene delivery in vivo (authors’ unpublished observations, 1999).
ACE-AS and Renovascular Reactivity
Alterations in vascular contractile responses are known to exist in SHR, and our studies have shown that AT1R-AS gene therapy prevents these alterations in the renal artery.16 17 Because an increased renovascular resistance is an important underlining mechanism in hypertension,17 we investigated the possibility that ACE-AS treatment of the SHR parents would prevent this alteration in vascular tone in themselves and their offspring. The concentration-response relations for KCl and phenylephrine were shifted left as a result of an increase in EC50 compared with those for ACE-tS– or ACE-AS–treated WKY or ACE-AS–treated SHR (Table 2⇓). Data for the F1 generation offspring are similar. An impaired endothelium-dependent relaxation of precontracted renal arterioles was observed in the ACE-tS–treated SHR, which was manifested as a 62% decrease in the maximal responsiveness (10 mmol/L acetylcholine) compared with that in the WKY controls. This decrease was prevented in the ACE-AS–treated SHR such that the maximal responses in this group of rats were found to be similar to those of ACE-tS–treated WKY. A similar correction of endothelial dysfunction in response to acetylcholine was maintained in the offspring of ACE-AS–treated SHR (Table 2⇓). These data demonstrate that alterations in both the receptor- and voltage-mediated contractile responses as well as the endothelial dysfunction associated with hypertension were prevented by ACE-AS treatment of the SHR and that this prevention was maintained in the offspring.
ACE-AS and Renal Vascular Electrophysiology
It has been previously shown that cells from renal resistance arterioles of the SHR are ≈20 mV more depolarized than are vascular cells from the WKY.18 Table 3⇓ illustrates the resting membrane potentials of renal resistance arteriolar vascular smooth muscle cells of WKY and SHR treated with either ACE-tS or ACE-AS. The average resting membrane potentials were −54±9.3, −53±4.8, −55±2.7, and −33±2.5 mV in cells from the ACE-tS–treated WKY, ACE-AS–treated WKY, ACE-AS–treated SHR, and ACE-tS–treated SHR, respectively (n=38, P<0.01). Similar data are shown for the F1 generation offspring (Table 3⇓).
We have previously shown that Kv current density was decreased in vascular smooth muscle cells from the interlobar arteries of SHR compared with those of WKY controls.18 Figure 1⇓ illustrates the characteristics of Kv current in F0 and F1 SHR treated with either ACE-tS or ACE-AS and the current-voltage relation for all treatment groups in single cells isolated from renal resistance arterioles. Kv current was reduced in cells isolated from SHR treated with ACE-tS compared with cells from SHR treated with ACE-AS. This effect was also seen in the F1 generation. The mean current-voltage relation for the Kv current, which was normalized to cell capacitance, was significantly less in the cells from SHR treated with ACE-tS compared with cells obtained from WKY animals in either treatment group or SHR treated with ACE-AS.
We also studied L-type Ca2+ current in ACE-tS– and ACE-AS–treated WKY and SHR and their F1 generation. The rationale for this experiment was based on our previous observations that demonstrated that L-type Ca2+ current is increased in cells from renal arterioles of the SHR.16 Ca2+ current was significantly increased in both F0 and F1 generations of ACE-tS–treated SHR compared with ACE-AS–treated SHR (Figure 2⇓). The mean current-voltage relation for all 4 treatment groups is shown in Figure 2⇓. We next investigated the effects of KCl (30 mmol/L) and angiotensin II (Ang II, 100 nmol/L) on [Ca2+]i in renal arteriolar smooth muscle cells. Our previous studies have established that basal, KCl-induced, and Ang II–induced [Ca2+]i levels were significantly elevated in SHR compared with WKY.18 The resting levels of [Ca2+]i in the WKY, SHR, SHR+AS, and SHR+tS F0 were 95±7, 129±12, 90±4, and 125±5 nmol/L, respectively (n=310 cells). Data in Figure 3⇓ show that KCl-induced or Ang II–induced increases in [Ca2+]i in ACE-AS–treated SHR were significantly attenuated compared with the KCl and Ang II responses in the ACE-tS–treated SHR. Similar data were obtained in the F1 generation. These findings provide additional evidence that alterations in electrophysiological parameters and [Ca2+]i homeostasis in SHR renal arteriolar cells are permanently prevented by ACE-AS gene therapy.
Expression of ACE-AS in Offspring
RT-PCR followed by Southern analysis was carried out to determine whether the above-described long-term antihypertensive responses that are transferred from parents to offspring are a result of integration of ACE-AS into the genome of parents and its subsequent transmission into the offspring. Figure 4⇓ shows that retroviral vector containing ACE-AS was expressed in various angiotensin target tissues in the F1 offspring of parents who were injected with the ACE-AS viral particles. This was associated with a robust expression of ACE-AS.
The most significant finding of the present study is that a single administration of the retroviral vector containing ACE-AS results in long-term, yet modest, prevention of hypertension and its renovascular pathophysiologies and that this normotensive phenotype is transmitted from treated parents to their offspring. This suggests a possible germ-line transmission of the ACE-AS and its subsequent transduction/expression in the offspring. These observations support our concept and strengthen the hypothesis that inhibition of the RAS at a genetic level could be an important step toward long-term control of hypertension.13 14 15 16 In spite of this important similarity, there appears to be a major difference between the AT1R-AS and ACE-AS approaches. ACE-AS treatment produced only a modest decrease in high BP (≈15 mm Hg) compared with AT1R-AS gene therapy, in which the decrease was more pronounced (≈30 to 40 mm Hg).17 In spite of a modest BP response, there was a complete prevention of renovascular pathophysiology by the ACE-AS treatment. This observation supports clinical data indicating that low doses of ACE inhibitors are able to induce beneficial effects in remodeling and pathophysiology of many cardiovascular-relevant tissues.18 19 20 21 22 23 However, ACE-AS therapy could accomplish this by a single administration of the vector with long-term effects, whereas the traditional ACE therapy requires continuous treatment.
One fundamental reason for the increase in renal vascular resistance in hypertension is an altered ion channel function in vascular smooth muscle cells. Tonic changes in membrane potential and thus Ca2+ influx regulate the contractile tone of resistance arterioles. A number of reports show that membrane potential, Kv current density, and Ca2+ current density are altered in the SHR model of hypertension.23 However, most of these studies have been in conduit arteries, which play a less significant role in the regulation of peripheral resistance. In renal resistance arterioles, which play a major role in setting peripheral resistance (thus, blood pressure), we show not only that are there the above-described electrophysiological alterations in the SHR but also that they are prevented by ACE-AS gene therapy. The exact mechanism by which the changes in ion channel activity occur is not known at this time, but one can speculate that changes in K+/Ca2+ channel protein or the regulation of these channels could occur in hypertension. It is also possible that an increase in Ca2+ influx could tend to load the sarcoplasmic reticulum with more Ca2+, enabling more to be released when challenged by physiological stimuli. In the present study, we show that this altered Ca2+ handling by the sarcoplasmic reticulum can be prevented by use of ACE-AS. This would be important in the pharmacomechanical coupling of vascular smooth muscle.
A unique feature of the present study is that it is established, for the first time, that a normotensive phenotype can be transmitted from virus-treated parents to their offspring by using ACE-AS gene therapy. In fact, all of the pathophysiology that is observed in the SHR offspring is corrected in the offspring of ACE-AS–treated SHR. The mechanism of such a profound transmission remains to be completely elucidated, although our evidence supports the notion that the normotensive phenotype could be a result of transmission of ACE-AS cDNA from the parents to their offspring (Figure 4⇑). Lack of a blood-gonadal barrier and the presence of significant numbers of undifferentiated germ cells in the neonatal rat at the time of viral delivery could account for such a high efficiency of transduction.
Finally, we believe that ACE-AS gene therapy is a significant advancement over traditional hypertension therapies. In the SHR, an animal model for primary human hypertension, a single injection offers the possibility of a permanent prevention of hypertension. However, the permanent nature of this antihypertensive effect may not be appropriate in various physiological situations in which one might need to terminate therapy. Therefore, we believe that for the future of this type of gene therapy to be successful, a regulatable expression system must be developed in which exogenous agents control the expression of ACE-AS at demand and, in turn, regulate its therapeutic potential. In conclusion, ACE-AS produced a permanent antihypertensive action and prevention of renovascular pathophysiology in the SHR, and this effect is present in their offspring.
This study was supported by National Institutes of Health grants HL-52189 (to Dr Gelband) and HL-56921 (to Drs Raizada and Katovich) and a grant-in-aid from the American Heart Association, Florida affiliate (to Dr Gelband).
- Received September 13, 1999.
- Revision received October 19, 1999.
- Accepted October 26, 1999.
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