Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Adrenomedullin (AM) is a potent vasodilator and natriuretic peptide that plays an important role in cardiovascular function. In this study, we employed a somatic gene delivery approach to explore its potential protective role in renovascular hypertension. A single tail vein injection of adenovirus harboring the human AM gene significantly blunted a blood pressure increase that lasted for more than 3 wk in two-kidney one-clip (2K1C) hypertensive rats. The expression of human AM mRNA was detected in the kidney, adrenal gland, heart, lung, and liver, and immunoreactive human AM was detected in the plasma and urine of 2K1C rats after human AM gene delivery. A maximal blood pressure difference of 28 mmHg was observed 10 days after AM gene delivery, compared with that in rats injected with the control virus carrying the LacZ gene. Human AM gene delivery significantly attenuated increases in the ratio of left ventricular weight to heart weight, cardiomyocyte diameter, and fibrosis in the heart, as well as glomerular sclerosis, tubular injuries, and protein casts in the kidney. The beneficial effects of AM gene delivery were accompanied by increased urinary cAMP levels, indicating activation of AM receptors. These findings provide new insights into the role of AM in renovascular hypertension and may have significance in therapeutic applications in cardiovascular diseases.
adenovirus; gene delivery; blood pressure; hypertrophy; glomerular sclerosis
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INTRODUCTION |
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ADRENOMEDULLIN (AM) is a potent vasodilator peptide that was originally discovered in human pheochromocytoma (14). Human AM consists of 52 amino acids with a 6-member ring structure linked by a disulfide bond and amidated COOH terminal, which belongs to the calcitonin gene-related peptide (CGRP) family (24). Recent studies have shown that both AM and CGRP signal through a common receptor with seven transmembrane domains, the calcitonin receptor-like receptor (17). The ligand specificity of the receptor is modulated by its association with chaperone receptor activity-modifying proteins, and the signal transduction is mediated by CGRP receptor component protein (4, 17). AM has been implicated as an important regulator in the renal and cardiovascular systems (8, 24). AM increases glomerular filtration rate and natriuresis and suppresses aldosterone release in the renal system. In the cardiovascular system, AM produces a dose-dependent vasodilation and blood pressure reduction and an increase in cardiac index and stroke volume.
The AM gene is highly expressed in the adrenal gland, heart, kidney, and lung of both rats and humans (15, 23). Immunoreactive AM has been detected in numerous tissues, including adrenal cortex, kidney, lung, heart, endothelium, and vascular smooth muscle cells (10, 22). Although AM appears to act in a local paracrine and/or autocrine fashion within the tissue of origin, AM also circulates in plasma, and its plasma level is increased in various cardiorenal diseases, such as essential hypertension, chronic renal failure, and congestive heart failure (8). Administration of exogenous AM peptide and genetic manipulation of AM gene expression elicit significant pharmacological effects in experimental animals. AM also displays significant vasorelaxant activity in the isolated rat mesenteric vascular bed (21). A recent study showed that transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (25). Given that administration of AM elicited vascular relaxation and reduction of blood pressure and that human AM peptide is biologically active in the rat, these findings implicate potential beneficial effects of AM gene transfer in the pathogenesis of cardiovascular diseases.
Intravenous bolus injection of AM peptide caused a potent hypotensive effect in anesthetized rats in vivo (11); however, the effect was transient probably due to the fact that the peptide is cleared or inactivated via passage though the lung (20). Chronic infusion of a relatively low dose of human AM peptide in two-kidney one-clip (2K1C) renovascular hypertension led to a reduction of blood pressure (13). Both the plasma renin activity and aldosterone concentrations of the AM-infused 2K1C rats were significantly reduced. These results indicate that a continuous supply of AM might provide a long-term protective effect against hypertension. In our previous study, we have shown that somatic gene delivery of human AM via intravenous injection of the AM DNA reduced blood pressure in both young adult and adult spontaneously hypertensive rats (SHR) (2). Although human AM expression was detected in SHR after AM gene delivery, the efficiency of cellular uptake and the expression of foreign genes were quite low (30). In this study, we delivered the human AM gene in an adenoviral vector to achieve high-efficiency expression. Systemic gene delivery of human AM led to sustained blood pressure reduction and alleviation of cardiac hypertrophy and renal damage in 2K1C renovascular hypertensive rats.
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MATERIALS AND METHODS |
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Preparation of replication-deficient adenovirus vector.
Adenovirus Ad.CMV-AM, in which the expression of human AM cDNA is under
the control of cytomegalovirus (CMV) promoter, was generated by
employing a simplified system (7). Adenovirus encoding
-galactosidase under the control of CMV promoter (Ad.CMV-LacZ) was
prepared as previously described (29).
Animal treatment. Goldblatt hypertension was induced in 5-wk-old male Wistar rats obtained from Harlan Sprague Dawley (Indianapolis, IN). The rats were prepared by placing a constricting silver clip (internal gap 0.2 mm) around the left renal artery under anesthesia with pentobarbital sodium (50 mg/kg ip) (29). For sham-operated rats, an incision was made in the left flank to expose the kidney and the renal artery without clipping the vessel. One week after the surgery, 2K1C rats were injected with 4 × 1010 plaque-forming units of adenoviral particles of Ad.CMV-AM or Ad.CMV-LacZ via the tail vein. Procedures and protocols were in accordance with our institutional guidelines.
Urine collection. At 7 and 14 days after adenoviral injection, 2K1C rats were housed in metabolic cages for 24-h urine collection. The urine samples were subjected to measurements of immunoreactive human AM, cAMP, and cGMP levels.
Systolic blood pressure. Systolic blood pressure was measured with a photoelectric tail-cuff device (Natsume, Tokyo, Japan). This device requires minimal warming of rats (usually <15 min) before blood pressure measurement and a brief period of restraint in a plastic cage. For each animal, the systolic blood pressure was represented as the mean of eight recordings.
Morphological and histological analysis. Rats were anesthetized with pentobarbital (50 mg/kg body wt), and hearts and kidneys were removed, washed in saline, blotted, and weighed at 14 days after human AM gene delivery. Sections of the kidney and heart were preserved in 4% PBS-buffered formaldehyde solution and embedded in paraffin. Four micrometer-thick sections were cut and stained with hematoxylin-eosin (H&E), periodic acid-Schiff (PAS), Sirius red, and by Gordon and Sweets silver staining. The final glomerular sclerosis score was graded under PAS staining as previously described (27). The degree of sclerosis was scored as follows: 0, no changes; 1, lesions involving <25% of the capillary tuft; 2, lesions affecting 25-49% of the capillary tuft; 3, lesions involving 50-75% of the capillary tuft; and 4, lesions involving >75% of the capillary tuft. The resulting index in each animal was expressed as a mean of all scores obtained. Extracellular matrix (ECM) production was quantified on Sirius red-stained sections by using Adobe Photoshop 5.0. Cardiomyocytes stain yellow whereas collagen stains red with Sirius red staining. Fibrotic area pixels are calculated by selecting a red color and using the Histogram option. The percent fibrosis area is calculated by dividing fibrotic pixels by total tissue pixels and multiplying by 100. All sections were evaluated under blind conditions without prior knowledge as to which section belonged to which rat.
Heart and left ventricular weight. At 14 days after human AM gene delivery, the heart was removed and weighed. The right ventricular free wall and both atria were carefully dissected from the left ventricle. The intraventricular septum was included in the left ventricular weight.
Cardiomyocyte diameter. Rat hearts were harvested at 14 days after gene delivery. Tissue sections were stained by Gordon and Sweets silver staining, and transverse cardiomyocyte diameter (100 cardiomyocytes/rat, n = 3 rats) was measured as previously described (28).
RT-PCR Southern blot analysis of human AM. Total RNA was extracted from fresh rat tissues by using TRIzol reagent (BRL). RT-PCR Southern blot analysis specific for human AM (5'-primer, 5'-CGC TCG GTT GGA TGT CG-3'; 3'-primer, 5'-CCG TGT GCT TGT GGC TTA-3'; and probe, 5'-CAA CTT CCA GGG CCT CC-3') was performed as previously described (2).
RIA for human AM, cAMP, and cGMP. Rat plasma and urine were acidified immediately after collection by adding 0.1 × vol of 1 N HCl. After centrifugation, the supernatant was diluted in assay buffer containing 50 mM sodium phosphate, pH 7.4, 0.5% BSA, 0.5% Triton X-100, 25 mM EDTA, 80 mM NaCl, and 0.05% sodium azide. Immunoreactive human AM level in rat plasma and urine was determined by a RIA by using rabbit anti-human AM 1-52 antiserum (Peninsula Laboratories, San Carlos, CA) (2). Rat urinary cAMP and cGMP levels were determined by RIA as previously described (5, 6).
Statistical analysis. The statistical significance of the difference in systolic blood pressure between 2K1C rats receiving Ad.CMV-LacZ and 2K1C rats receiving Ad.CMV-AM was determined by ANOVA. In addition, we used an unpaired Student's t-test to assess the differences in physiological parameters between Ad.CMV-AM and Ad.CMV-LacZ groups after gene delivery. The results were expressed as means ± SE.
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RESULTS |
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Expression of human AM mRNA in 2K1C hypertensive rats.
After gene delivery, human AM mRNA in 2K1C Goldblatt hypertensive rats
was analyzed with RT-PCR followed by Southern blot analysis. Total RNAs
were prepared from rat tissues at 5 days after intravenous
injection of adenoviral vectors Ad.CMV-AM or Ad.CMV-LacZ. The
expression of human AM mRNA was detected in the heart, aorta, clipped
and unclipped kidney, adrenal gland, lung, spleen, and liver but was
not detected in 2K1C rats receiving Ad.CMV-LacZ (Fig.
1, top). Similar levels of
-actin mRNA were detected in tissues of both experimental and
control groups, indicating the integrity of RNA in these samples
(Fig. 1, bottom). These results showed that human AM is
expressed in tissues relevant to cardiovascular and renal function
after adenovirus-mediated gene transfer in 2K1C hypertensive rats.
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Immunoreactive human AM in 2K1C hypertensive rats.
Immunoreactive human AM levels in 2K1C rats receiving human AM gene
delivery were measured by RIA. Human AM in plasma and urine of rats
receiving human AM gene delivery displayed parallelism to the human AM
standard curve, indicating their immunological identity (Fig.
2A). Immunoreactive human AM
was detected in rat plasma from 3 to 25 days after human AM gene
delivery. Immunoreactive human AM in rat plasma reached the highest
level at 6 days after gene transfer (64.32 ± 10.63 ng/ml,
n = 3) and then gradually declined (Fig.
2B). Immunoreactive human AM was detected in urine (2.54 ± 0.38 ng · 24-h urine1 · 100 g body wt
1) at 7 days after gene delivery (Fig.
2B). Serial dilutions of control rat urine and plasma did
not show parallelism with the human AM standard (data not shown). These
results indicate that the rabbit anti-human AM antibody had some
cross-reactivity with endogenous rat AM. The expression levels of human
AM in this assay are only considered semiquantitative due to
cross-reactivity of rat AM with the human AM antibody.
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Adenovirus-mediated gene delivery of human AM blunts systolic blood
pressure increase in 2K1C hypertensive rats.
Figure 3 shows the effect of AM gene
delivery on systolic blood pressure of 2K1C Goldblatt hypertensive
rats receiving adenoviral vectors Ad.CMV-AM and Ad.CMV-LacZ.
Systolic blood pressure of 2K1C rats at 1 wk after renal artery
constriction was significantly higher than that of sham-operated rats
(159 ± 3 vs. 126 ± 2 mmHg, n = 5, P < 0.01). A single intravenous injection of the
recombinant adenovirus harboring the human AM gene caused a delay in
blood pressure increase that began 3 days after injection and continued for 24 days. A maximal blood pressure difference of 28 mmHg was observed 10 days after AM gene delivery compared with that of 2K1C rats
injected with Ad.CMV-LacZ (158 ± 4 vs. 186 ± 8 mmHg, n = 5, P < 0.05). At 28 days after
gene delivery, there was no significant difference in systolic blood
pressure of Ad.CMV-AM (192 ± 4 mmHg, n = 4) and
Ad.CMV-LacZ (196 ± 4 mmHg, n = 4). Body weight
was measured at 3, 7, 10, 14, 17, 21, 24, and 28 days after intravenous
delivery of the human AM gene. No statistical difference was found in
body weight between 2K1C rats receiving control or AM viruses.
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Human AM gene delivery attenuates cardiac hypertrophy and fibrosis.
The extent of cardiac damage was attenuated in 2K1C hypertensive rats
receiving AM gene delivery. Total heart weight-to-body weight ratio of
the 2K1C rats injected with the AM gene was significantly lower than
2K1C rats injected with the LacZ gene (3.53 ± 0.15 vs. 4.55 ± 0.14 mg/g, n = 3 or 4, P < 0.05).
AM gene delivery significantly attenuated left ventricular
weight-to-heart weight ratio compared with 2K1C rats injected with LacZ
gene (0.719 ± 0.016 vs. 0.758 ± 0.010 g/g,
n = 3 or 4, P < 0.05) (Fig.
4). Further morphological evaluation by
Gordon and Sweets silver staining showed that cardiomyocyte diameter
was significantly increased in 2K1C rats injected with LacZ gene
compared with sham-operated rats (25.6 ± 0.2 vs. 18.03 ± 0.37 µm, n = 300, P < 0.01), and AM
gene delivery significantly reduced cardiomyocyte diameter compared
with 2K1C rats injected with LacZ gene (20.4 ± 0.9 vs. 25.6 ± 0.2 µm, n = 300, P < 0.01).
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Effects of human AM gene delivery on renal morphology in 2K1C
hypertensive rats.
Morphological evaluation of the PAS staining of renal cortex (Fig.
6, top row) and medulla (Fig.
6, middle row) in the unclipped kidney showed the beneficial
effect of AM gene delivery on 2K1C hypertensive rats. PAS stained
glycoprotein moieties bright red, clearly delineating the brush border
and tubule epithelium. The cortex and medulla of sham-operated rats
appeared normal (Fig. 6, A and D), whereas 2K1C
rats injected with Ad.CMV-LacZ developed significant renal injury,
occurring in both the cortex and medulla (Fig. 6, B and
E). The damage in the cortex of 2K1C rats injected with
Ad.CMV-LacZ included tubular dilatation, loss of brush border in
proximal tubule, areas of inflammation, and glomerular sclerosis. Renal
damage in the cortex was attenuated by human AM gene delivery (Fig.
6C). In the medulla, 2K1C rats injected with the LacZ gene developed large colloidal casts within renal tubules (Fig.
6E). Human AM gene transfer greatly reduced the size and
number of protein casts present in the tubules (Fig. 6F). In
the unclipped kidney, 2K1C hypertensive rats injected with human AM
gene had a significant reduction in glomerular sclerosis compared with 2K1C rats receiving the LacZ gene (0.9 ± 0.2 vs. 2.2 ± 0.4 sclerosis score, n = 180, P < 0.05)
(Fig. 6G).
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Effects of human AM gene delivery on urinary cAMP and cGMP levels.
Figure 7 shows that urinary cAMP level
was significantly reduced in 2K1C rats injected with LacZ gene compared
with sham-operated rats (9.75 ± 1.51 vs. 31.96 ± 2.38 nmol · day1 · 100 g body
wt
1), and human AM gene delivery significantly increased
urinary cAMP level compared with 2K1C rats receiving the LacZ gene at 1-wk post-human AM gene delivery (34.17 ± 1.05 vs. 9.75 ± 1.51 nmol · day
1 · 100 g body
wt
1). However, urinary cGMP levels were not altered in
rats receiving human AM gene delivery vs. rats receiving control virus
(1.02 ± 0.16 vs. 1.10 ± 0.18 nmol · day
1 · 100 g body
wt
1). Similarly, at 2 wk postinjection of adenovirus, the
urinary cAMP level was significantly reduced in 2K1C rats injected with LacZ gene compared with sham-operated rats (17.98 ± 1.08 vs.
31.29 ± 3.37 nmol · day
1 · 100 g
body wt
1), and human AM gene transfer significantly
increased urinary cAMP levels compared with 2K1C rats receiving the
LacZ gene (27.27 ± 4.39 vs. 17.98 ± 1.08 nmol · day
1 · 100 g body
wt
1). Urinary cGMP level was not altered in rats
receiving human AM gene delivery vs. rats receiving the control virus
(1.03 ± 0.13 vs. 0.88 ± 0.06 nmol · day
1 · 100 g body
wt
1).
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DISCUSSION |
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In this study, we showed that adenovirus-mediated human AM gene delivery attenuated blood pressure increase, cardiac, and renal dysfunction in 2K1C hypertensive rats. Human AM mRNA was identified in the heart, aorta, and kidney of rats after AM gene delivery, which are important to cardiovascular and renal functions. Immunoreactive AM was also detected in the plasma and urine, indicating that AM was produced and secreted from the liver and kidney. This study demonstrated that human AM gene delivery is capable of protecting the kidney from renal damage and attenuating cardiac remodeling in 2K1C hypertensive rats. This protective ability provides significant insight regarding AM's ability to regulate blood pressure via a cAMP-dependent pathway, which may aid in the prevention of end-organ damage in pressure-overload hypertension.
The observed reduction in blood pressure in this study is in agreement with previous studies stating that AM is a potent vasodilator and natriuretic peptide. A bolus injection of AM produced a drastic and hypotensive effect, which was correlated with a decrease in total peripheral resistance (11). Chronic infusion of AM peptide significantly reduced pulmonary hypertension, right ventricular hypertrophy, and attenuated the medial thickening of the pulmonary artery (31). Somatic gene delivery of human AM plasmid DNA induced a significant and long-lasting reduction in blood pressure in spontaneously hypertensive rats (2). Plasma levels of AM peptide are increased in hypertensive animal models, which indicate that AM participates in the mechanism to counteract high blood pressure (1). Increased levels of locally synthesized and systemic plasma AM may also compensate for or prevent damage in target tissues, such as the kidney and heart, as well as in the vasculature. A recent study has shown that transgenic mice overexpressing AM developed significantly lower blood pressure compared with wild-type littermates and exhibited increased cAMP levels (25). Taken together, these data suggest that enhancement of systemic and/or local AM systems may play an important role in the attenuation of renal and cardiovascular damage.
Increased systemic blood pressure and glomerular capillary hypertension are important causative factors in glomerular sclerosis. Histological examination revealed marked renal damage within the unclipped kidney of the rats treated with the LacZ gene, which was significantly alleviated in 2K1C rats treated with the human AM gene. H&E staining showed increased general interstitial inflammation in the kidneys, and Sirius red staining detected increased ECM protein production (data not shown), whereas PAS staining showed marked glomerular sclerosis, glomerular basement membrane thickening, renal tubular dilation, disruption of the proximal tubular brush border, and lumenal protein cast accumulation in 2K1C rats receiving control virus. AM is immunohistochemically localized to the glomeruli (12) and is synthesized in and secreted by mesangial cells, endothelial cells, and vascular smooth muscle cells (18). Intrarenal infusion of AM increased renal blood flow, urine flow, and urinary sodium excretion in a dose-related fashion, without changes in heart rate or mean arterial blood pressure (3). These results indicate that exogenous AM may exert direct renovascular and tubular effects independently of a blood pressure-lowering effect.
Previous studies have suggested that AM plays a major role in cardiac function in addition to blood pressure regulation and renal function (8, 24). Production and secretion of AM have been documented in cultured neonatal rat cardiac myocytes and fibroblasts (19). Our results showed that human AM gene delivery caused a significant reduction in left ventricular weight and total heart weight, along with a reduction in cardiomyocyte diameter in 2K1C rats. These results are consistent with previous observations by Tsuruda and colleagues (26), who reported that AM inhibited protein synthesis in cultured neonatal rat cardiomyocytes and may thus attenuate hypertrophy in vivo. Our results showed that the AM-treated 2K1C rats had a marked reduction in both cardiac fibroblast activation and extracellular matrix accumulation compared with 2K1C rats treated with the LacZ gene as evidenced by staining with both H&E and Sirius red. Consistent with our present studies, in vitro cell culture study showed that an AM peptide acts directly to inhibit collagen synthesis in cardiac fibroblasts mediated via a cAMP pathway in an autocrine and/or paracrine fashion (9). In cardiovascular tissues AM increases cAMP, but not cGMP levels, possibly via receptors present in myocyte and nonmyocytes (19). The reduced damage observed in the heart is most likely due to a mixture of direct and indirect mechanisms. Indirect organ protection may be provided by significant reduction of blood pressure after human AM gene delivery.
Our results showed that the beneficial effect of AM gene transfer correlated with the expression of the AM transgene. The transient nature of adenovirus-mediated AM gene expression in 2K1C rats is most likely due to a host immune response to viral proteins and episomal expression. We have included 2K1C rats that did not receive adenovirus in this study. Blood pressure, kidney, and heart morphology were similar between 2K1C groups with or without receiving control adenovirus containing the LacZ gene. These results indicate that the contribution of adenovirus-induced inflammation to the development of renovascular hypertension and tissue damage was minimal. Another factor that may limit the efficiency of recombinant AM gene transfer is the degradation of AM by neutral endopeptidase. Endopeptidase 24.11 (NEP) is localized in greatest abundance in the kidney and cleaves endogenous peptides like atrial natriuretic peptide. NEP inhibition increases plasma AM levels and potentiates the natriuretic and diuretic responses to intrarenal infusion of AM (16). These results suggest that AM is a substrate for NEP, and human AM introduced by gene delivery may be degraded by NEP.
In this study, we have shown that adenovirus-mediated gene delivery of human AM is capable of not only significantly reducing blood pressure but also alleviating renal and cardiovascular damage in the 2K1C hypertensive rat. AM's ability to attenuate blood pressure-regulatory defects and safeguards against organ damage provide evidence for the potential use of AM as a candidate for the treatment of cardiovascular and renal diseases.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-29397 and HL-52196.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Chao, Dept. of Biochemistry and Molecular Biology, Medical Univ. of South Carolina, 173 Ashley Ave., PO Box 240509, Charleston, South Carolina 29425.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 August 2000; accepted in final form 6 February 2001.
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