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CARDIOVASCULAR
Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, Parkville, Victoria, Australia
Received February 28, 2006; accepted June 1, 2006.
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Abstract |
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), and this proliferative response is blunted in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) (Nolan et al., 2003). An increase in IGF-I mRNA expression, however, is seen in rat models of hypertrophying aorta, portal vein, or urinary bladder (Khorsandi et al., 1992; Chen et al., 1994). IGF-I also causes direct vasodilation via a nitric oxide-dependent pathway (Pete et al., 1996; Walsh et al., 1996) and relaxes aortic rings precontracted with phenylephrine, an effect also impaired in SHR (Vecchione et al., 2001). Thus, in the vasculature, IGF-I has both pressor-inducing (VSMC mitogenic effect) and depressor effects, and both are altered in hypertensive models, whereas the expression of both the ligand and its receptor may increase in such circumstances.
There are profound interactions between IGF-I and the renin-angiotensin system with respect to vascular resistance. In VSMC, angiotensin II has been shown to potentiate IGF-I and IGF-IR expression, and this potentiation has been found to be important in the vascular growth-promoting effects of angiotensin II (Delafontaine and Lou, 1993). A similar effect of angiotensin II on IGF-I and IGF-IR gene expression was also seen in cardiac myocytes (Brink et al., 1999). Furthermore, potentiation of angiotensinogen production and AT1R expression was seen in VSMC in response to IGF-I (Kamide et al., 2000; Muller et al., 2000). On the other hand, in transgenic and diabetic wild-type mice, overexpression of IGF-I was found to reduce angiotensin II and AT1R expression (Leri et al., 1999; Kajstura et al., 2001).
We recently found that IGF-IR antisense reduced the pressor response to angiotensin II and decreased AT1R expression in normotensive rats (Nguyen and White, 2005). In normotensive rats, IGF-I causes nitric oxide-mediated vasodilation, an effect that is in opposition to the increase in angiotensin receptor expression and function mediated by IGF-I. Given that in hypertensive rats the vasodilator effects of IGF-I are reduced compared with normotensive controls, the effects of IGF-I on angiotensin receptor expression and function may be of greater functional significance in these animals. We therefore investigated the impact of IGF-IR knockdown by IGF-IR AS on angiotensin receptor expression, resting blood pressure, and responses to noradrenaline and angiotensin II in a systolic hypertension model, the spontaneously hypertensive rat.
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Materials and Methods |
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Receptor Autoradiography in Spontaneously Hypertensive Rat Arteries. To quantitatively determine the effects of the treatments on IGF-IR, AT1R, and AT2R expression, autoradiography was performed on tissue sections obtained from rats treated for 4 days with 0.4 mg/kg antisense oligonucleotide, mismatch oligonucleotide, or vehicle control (four serial sections per tissue per incubation; three rats per each treatment group). IGF-IR autoradiography was performed as described previously (Sidawy et al., 1990). Frozen sections (20 µm) were incubated at room temperature for 2 h in 50 mM Tris-HCl buffer with 0.1% bovine serum albumin, 1 mg/ml bacitracin, and 10 mM MgCl2 containing 40 pM 125I-IGF-I (Auspep, Parkville, VIC Australia; iodination performed by ProSearch, Sydney, NSW, Australia). Nonspecific binding was determined by incubating sequential tissue sections in buffer containing radioligand and excess unlabeled IGF-I (0.1 µM). Angiotensin receptor autoradiography was conducted as described previously (McDougall et al., 2000) using buffer containing 50 nM Sar1-Ile8-125I angiotensin II (Auspep; iodination performed by ProSearch); AT1 receptor levels were determined using the addition of the AT2R antagonist PD123319 (10 µM; Sigma, Castle Hill, NSW, Australia), whereas AT2R levels were determined using the AT1R antagonist losartan (10 µM; DuPont, Wilmington, DE); nonspecific binding was determined in the presence of 20 µM angiotensin II (Auspep). Sections were incubated in the above solutions for 60 min followed by four successive 3-min washes in ice-cold buffer.
After incubations, sections were washed in ice-cold buffer (2 x 2 min), dried overnight, and exposed to Kodak Biomax MR film (Eastman Kodak, Rochester, NY) for 5 days. Optical densities were determined using a computer-assisted digitization system (Scion Corporation, Frederick, MD) and were converted to disintegrations per millimeter2 via the use of 14C microscale standards. A low level of nonspecific binding was consistent across the different treatment groups and was subtracted from total binding. Nonspecific binding was therefore subtracted from total binding to determine specific binding densities for both IGF-IR and angiotensin receptor binding assays.
Surgical Procedure. Rats were anesthetized with amylobarbital sodium (0.1 g kg-1 i.p.). The left jugular vein and left carotid artery were cannulated with polyethylene (PE 50) tubing. The jugular vein was rinsed with physiological saline, and the carotid artery was rinsed with heparinized saline solution (100 U ml-1). The cannulas were tunneled subcutaneously to exit at the back of the neck.
In Vivo Effects of Antisense Treatments. Rats received one of the following treatments via the jugular vein cannula: 1) antisense oligonucleotide targeting the IGF-IR [antisense: 5'-UCC-CAC-AGC-TGC-UGC-AAG-3' with a modification of 1-6 2'-OMe RNA, 7-12 thioate DNA, 13-18 2'-OMe RNA (Sigma Genesis, Sydney, NSW, Australia), 2.5 µg/injection/rat, via cannulated jugular vein] to the same region as an IGF-IR antisense used previously to specifically reduce IGF-IR in psoriatic epidermis (Wraight et al., 2000); 2) complete mismatch of IGF-IR antisense (mismatch: 5'-CAC-ACU-CAG-CTG-GCG-CCA-3' with the same modification as antisense) (2.5 µg/injection/rat); or 3) vehicle every 2nd day for 2 weeks. Blood pressure from the carotid artery was recorded using a Gould Statham Physiological pressure transducer (Gould Instruments, Oxnard, CA) connected to a Power Lab System (AD Instruments, Sydney, Australia). Hypertensive responses to noradrenaline (10 ng kg-1-30 µgkg-1) and angiotensin II (1.0 ng kg-1-30 µgkg-1) were recorded at 7, 9, 11, and 14 days postsurgery.
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Data Analysis and Statistics. Emax, ED50, and associated confidence intervals were calculated using a computer program, Graph-Pad Prism 4 (GraphPad Software, San Diego, CA). Sigmoidal dose-response curves were generated from nonlinear regression using the equation Y = Bottom + (Top - Bottom)/(1 + 10log EC50 - X), with no constraints or weighting of values. Emax and ED50 were determined from the resultant curve fit. The effects of IGF-IR antisense on hypertensive responses to angiotensin II and noradrenaline and on other parameters were determined using one-way ANOVA, followed by Bonferroni's test for multiple comparisons. A P value of less than 0.05 was considered to indicate statistical significance. Data in graphs are presented as mean ± standard error of the mean (S.E.M.), and n indicates the number of animals being studied.
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Results |
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IGF-IR antisense treatment resulted in a significant, specific reduction in IGF-IR levels in both conductance and resistance vessels. Aortic IGF-IR density was reduced by 67.4 ± 6.0% in antisense-treated SHR compared with untreated animals, whereas mismatch treatment had no effect (ANOVA, n = 3, P < 0.01 comparing antisense-treated aortae with both untreated and mismatch-treated aortae; Fig. 1e). In tail arteries, antisense treatment resulted in a 52.9 ± 3.0% reduction in IGF-I binding compared with untreated vessels; mismatch treatment again had no effect (ANOVA, n = 3, P < 0.01 comparing antisense-treated tail arteries to both untreated and mismatch-treated tail arteries; Fig. 1f).
IGF-IR Antisense Treatment Reduced AT1R Expression in SHR. AT1R expression (determined in the presence of the AT2R ligand PD123319) was 2- to 3-fold greater than AT2R expression (determined in the presence of the AT1R antagonist losartan) in untreated SHR vessels. AT1R expression was significantly reduced by IGF-IR antisense treatment (Fig. 2). IGF-IR antisense treatment significantly reduced AT1R binding in both aorta (52.2 ± 8.9% reduction in AT1R binding compared with untreated rats; ANOVA n = 3, P < 0.01 comparing antisense-treated aortae with both untreated and mismatch-treated aortae) and tail arteries (48.1% reduction in AT1R binding compared with untreated rats; ANOVA n = 3, P < 0.01 compared with untreated tail arteries). Mismatch treatment resulted in a trend toward reduction in AT1R expression; however, there was no significant difference in expression between mismatch and untreated animals (ANOVA, n = 3, P = 0.09). There were no significant effects on AT2R levels in either antisense or mismatch oligonucleotide-treated animals compared with aortae or tail arteries from untreated animals (ANOVA, n = 3, P > 0.05). IGF-IR Antisense Reduced Responses to Vasocon-
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IGF-IR Antisense Treatment Had No Significant Effect on Aortic Medial Cross-Sectional Area or Left Ventricle/Body Weight Ratio. Figure 4 shows the effect of IGF-IR antisense treatment on SHR left ventricle/body weight ratio (Fig. 4a) and heart/body weight ratio (Fig. 4b). Although it does seem that there is a trend toward a reduction in cardiac parameters in the IGF-IR antisense-treated group, there were no significant differences between the treatment groups (n = 4-5, p > 0.05). Figure 4c shows that antisense treatment had no effect on aortic medial crosssectional area.
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Discussion |
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IGF-IR antisense treatment produced a specific reduction in the binding of 125I-IGF-I to both aortae and tail arteries of treated animals, whereas the mismatch-control oligonucleotide had no effect. Thus, systemic administration of chimeric oligonucleotide successfully diminished the expression of the IGF-IR in vasculature of SHR. The level of reduction in target expression is significant, particularly because we used microgram per kilogram doses rather than milligram per kilogram, and suggests that proteins expressed in blood vessel walls may be particularly suited to antisense intervention. We are currently investigating whether this high level of target reduction is due to the ease of access of the oligonucleotide to the target cells when the drug is given intravenously or whether vascular smooth muscle cells are particularly amenable to antisense intervention.
The reduction in IGF-IR seemed to result in a reduction in vascular AT1R (but not AT2R) expression in these animals. 125I-Sar1-Ile8 angiotensin II binding in the presence of PD123319, but not losartan, was reduced in antisense-treated rats. Antisense oligonucleotides do have nonsequence-specific and off-target effects, and in the current study, AT1R levels were lower in mismatch-treated rats than untreated rats. Thus, there may have been some small degree of nonspecific effect of oligonucleotide treatment on AT1R levels in this study, which a greater sample size could have revealed as statistically significant. However, there was indeed a significant difference between AT1R levels in antisense-treated rats compared with both forms of control—untreated and mismatch-treated rats. We can therefore conclude that IGF-I receptor knockdown does reduce AT1 receptor levels compared with relevant controls.
We were unable to accurately determine whether there was a greater antisense-mediated reduction in AT1 receptor levels in SHR compared with normotensive strains. The trend toward a reduction in AT1 receptor expression in the mismatch control oligonucleotide-treated SHR made a quantitative comparison invalid.
This reduction in the density of the angiotensin receptors responsible for vasoconstrictor effects of angiotensin II seemed to have important functional consequences in the treated animals. IGF-IR antisense treatment (and not mismatch or vehicle treatment) produced a significant reduction in vascular response to angiotensin II in SHR. The deficit in both AT1R density and in the constrictor effects of angiotensin after IGF-IR antisense treatment may provide insight into the functional relevance of previous work indicating that IGF-I acts to increase AT1R expression in vascular smooth muscle cells at the transcriptional level (Muller et al., 2000). The inhibitory effect of IGF-IR antisense on angiotensin II responses observed in this study may be due to the loss of the stimulatory effects of IGF-I in the expression of AT1R. These data suggest that there may be a powerful and ongoing role in SHR for IGF-I stimulation of AT1R expression. Despite evidence that AT2R expression is increased by IGF-I in vascular smooth muscle cells (Kambayashi et al., 1996), we saw no effect of IGF-IR antisense treatment on AT2R binding; we are currently investigating whether this is due to alterations in the effects of IGF-I on receptor expression in SHR. Changes in the level of radioligand binding to both the IGF-I receptor and AT receptors could reflect altered affinity rather than receptor number. However, in previous studies using antisense targeting the same region of the IGF-I receptor mRNA, we have exclusively observed changes in receptor number and not affinity (Wraight et al., 2000).
In the present study, we also found a reduction in vascular responses to noradrenaline in SHR treated with IGF-IR antisense. IGF-I has been found to up-regulate VSMC 
1-adrenoceptor expression (Hu et al., 1996). The activities of IGF-I are predominantly mediated through binding to the IGF-IR; hence, a decrease in aortic IGF-IR expression induced by IGF-IR antisense would probably attenuate any effect of IGF-I on 1-adrenoceptor expression, which may contribute to the in vivo decrease in noradrenaline response. The focus of this study was the interaction between IGF-I and angiotensin receptor function; however, we are currently investigating the effects of IGF-IR antisense on other signaling pathways in vitro using tissues from antisense-treated animals.
It might be expected that IGF-IR antisense treatment would increase resting blood pressure, since IGF-I causes nitric oxide release (Fryburg, 1996; Pete et al., 1996; Walsh et al., 1996), and blood pressure has been shown to be elevated in IGF-I knockout mice (Tivesten et al., 2002). We saw no major change in basal blood pressure during the 14-day antisense treatment period in any of the rat strains. SHR blood pressure was significantly lower at one time point (after 14 days) in the antisense-treated rats than the mismatch or vehicle controls; however, this was mainly due to a rise in the vehicle-treated rats pressure from day 11 to day 14. The lack of increased pressure in IGF-IR antisense-treated animals may be due to the blunting of IGF-I effects on vascular resistance in hypertensive animals (Vecchione et al., 2001; McCallum et al., 2005), or perhaps this simply reflects the relatively minor role of IGF-I in regulation of vascular resistance—blood pressure only drops 5 to 10 mm Hg after IGF-I administration in rats (Walsh et al., 1996; P. J. White, unpublished observations).
Although we observed a significant reduction in the vascular medial cross-sectional area in Hooded Wistar rats (Nguyen and White, 2005), there was no such effect in SHR in the present study. As mentioned above, the effects of IGF-I are altered in SHR, and this applies to the proliferative effects as well as those related to vascular resistance. Nolan et al. (2003) found that there was an impairment in IGF-I-induced proliferation in aortic vascular smooth muscle cells from SHR compared with Wistar Kyoto control, and our observations support this—we found that although IGF-IR expression was profoundly reduced, there was no consequent change in vascular thickness in SHR in vivo. Because vascular structure was unaffected in IGF-IR antisense-treated SHR, we suggest that the observed decrease in vasoconstrictor responses to angiotensin II may be due to changes in AT1 receptor expression rather than changes in tissue contractility in these animals.
We saw a greater inhibitory effect of AS treatment on the response to angiotensin II in particular and noradrenaline to a lesser extent in SHR compared with Hooded Wistar (where a moderate reduction in potency was observed) and Wistar Kyoto rats (where no effect was observed). It is possible that the efficacy of the antisense was greater in SHR than normotensive rats and that this is the reason for the greater effects of antisense treatment on responses to angiotensin II. We consider this unlikely, however, given that we find a similar level of knockdown in SHR and Hooded Wistar rats using immunohistochemistry of treated aortae (data not shown) and that autoradiography of normotensive hearts shows a similar (
50%, data not shown) reduction in IGF-I receptor levels to that found in the present study (52-67%). Therefore, the greater antisense effect is likely to be due to a greater involvement of IGF-I in these responses in SHR. Both renin-angiotensin-aldosterone system activity and sympathetic nervous system activity are elevated in SHR (Grisk, 2005), and therefore, the efficacy of the antisense may have been greater in rats where these systems are more "basally" active.
The results of this study show that IGF-IR knockdown induces a profound inhibitory effect on vascular response to vasoconstrictor agents, possibly through effects on AT1Rreceptor signaling, alone or in combination with downstream effects on receptors for other vasoactive signaling molecules in the SHR vascular system. We are currently investigating these possibilities.
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Acknowledgements |
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Footnotes |
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Address correspondence to: Dr. Paul White, Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, 381 Royal Pde, Parkville, VIC, Australia 3052. E-mail: paul.white{at}vcp.monash.edu.au
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References |
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