- Email: [email protected]
Over-expression of angiotensin II type 2 receptor (agtr2) reduces atherogenesis and modulates LOX-1, endothelial nitric oxide synthase and heme-oxygenase-1 expression
Atherosclerosis 199 (2008) 288–294
Over-expression of angiotensin II type 2 receptor (agtr2) reduces atherogenesis and modulates LOX-1, endothelial nitric oxide synthase and heme-oxygenase-1 expression Changping Hu a,b,1 , Abhijit Dandapat a,1 , Jiawei Chen a , Yong Liu a , Paul L. Hermonat a , Robert M. Carey c , Jawahar L. Mehta a,∗ a
b
Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and VA Medical Center, Little Rock, AR 72205, United States Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, China c Department of Medicine, University of Virginia, Charlottesville, VA, United States Received 10 June 2007; received in revised form 9 September 2007; accepted 7 November 2007 Available online 21 December 2007
Abstract Atherogenesis is associated with inflammation and oxidative stress. Activation of renin-angiotensin system with generation of angiotensin II and type 1 receptor (AT1R) stimulation has been amply reported in atherosclerosis. Since angiotensin II type 2 receptor (AT2R) activity is purported to oppose the effects of AT1R, we hypothesized that AT2R (agtr2) over-expression would inhibit atherogenesis. We prepared recombinant adeno-associated virus type-2 (AAV) carrying AT2R cDNA (AAV/AT2R), and homozygous LDLR-deficient (KO) mice were given AAV/AT2R, AAV/Neo or saline. All mice were placed on a high cholesterol diet. After 18 weeks, AT2R was found to be over-expressed systemically in AAV/AT2R-treated mice. Atherogenesis in aorta was reduced in the AAV/AT2R group by ≈50% compared to other LDLR KO mice groups. Expression of NADPH oxidase, nitrotyrosine and NF-B was increased in aortic tissues of the LDLR KO mice given saline or AAV/Neo, but not in mice with AT2R upregulation. Expression of endothelial nitric oxide synthase (eNOS) and heme-oxygenase-1 (HO-1) was decreased and that of the lectin-like oxidized-LDL receptor (LOX-1) increased in the LDLR KO mice, but not in the mice with AT2R over-expression. Further, Akt-1 phosphorylation was reduced in the LDLR KO mice, but not in the mice with AT2R over-expression. Thus, AT2R upregulation can reduce atherogenesis, possibly by modulating oxidative stress and the pro-inflammatory cascade, mediated via Akt-1. Over-expression of AT2R may be an important therapeutic approach in atherosclerosis. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Akt-1; Angiotensin; Atherosclerosis; Heme-oxygenase-1; Lectin-like oxidized-LDL receptor; Nitric oxide synthase; Oxidative stress
1. Introduction Atherosclerosis, at least in part, is an inflammatory/ immune disorder accompanied by oxidative stress [1]. Angiotensin (Ang) II type 1 receptor (AT1R) expression is upregulated in atherosclerosis and plays an important role in atherogenesis [2,3]. Actually, atherosclerosis and its sequelae are inhibited by AT1R inhibition via inhibition of inflammation and oxidative stress [4,5]. It has been postulated that ∗ 1
Corresponding author. Tel.: +1 501 296 1410. E-mail address: [email protected] (J.L. Mehta). These two authors contributed equally.
0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.11.006
AT1R blockers exert their effect not just by blocking AT1R activity, but also by redirecting Ang II towards unblocked type 2 receptors (AT2R) whereby AT2R activity increases [6]. It is generally assumed that AT2R activation exerts effects opposite to those of AT1R activation [2,6]. Interestingly, AT2R deletion was shown to exaggerate atherosclerosis in apolipoprotein E-null mice [7]. However, if upregulation of AT2R expression will inhibit atherosclerosis is not known. LOX-1, a lectin-like receptor for oxidized LDL (ox-LDL), is the major receptor responsible for binding, internalization and degradation of ox-LDL in endothelial cells and plays a critical role in atherogenesis [8,9]. Ang II upregulates LOX-1 expression via AT1R activation [10]. There is also evidence
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
289
for a reduction in the expression and activity of endothelial nitric oxide synthase (eNOS) in atherosclerosis [11]. Hemeoxygenase-1 (HO-1), an enzyme involved in the genesis of the potent vasodilator species carbon monoxide, is also altered in atherosclerosis [12,13]. Both eNOS and HO-1 are regulated by Ang II [11,12]. This study was designed to test the hypothesis that AT2R cDNA delivered with adeno-associated virus type 2 (AAV) will result in sustained over-expression of AT2R in vascular tissues, and will inhibit atherogenesis in LDL receptor-deficient (LDLR KO) mice. Further, we examined the hypothesis that AT2R over-expression will modulate LOX-1, eNOS and HO-1 expression.
2.3. Analysis of transgene vector DNA
2. Materials and methods
2.5. Measurement of plasma lipids
2.1. Construction of AAV/AT2R vector and generation of AAV/AT2R stocks
Plasma levels of total cholesterol, HDL-cholesterol, triglycerides, were measured by clinical laboratory.
Mouse AT2R (agtr2) cDNA was generated by PCR amplification. Total RNA was isolated from aortic tissues of C57BL/6 mice using Trizol reagent (Invitrogen) and treated with 2.0 U/g of RNase-free DNase I (Promega) at 37 ◦ C for 1 h. mRNA was separated using the Oligotex mRNA Mini Kit (QIAGEN). First-strand cDNA synthesis was performed using oligo(dT)15 primers. PCR amplification for the AT2R cDNA sequence was carried out using the following primer pair: 5 -GAGTTGCTGCAGTTCAAT-3 and 5 -GAACTGTATTATACGTATGCCAC-3 that amplify the sequence from nucleotides 149 to 1326 (NCBI Gene Bank: NM-007429). After AT2R cDNA was sequenced, it was ligated into an AAV vector, dl6-95, as described previously [14]. Hereafter, recombinant AAV (rAAV) vector is referred to as AAV/AT2R. The rAAV virus stocks were generated as described previously [14]. The titer of purified virus, in encapsidated genomes per milliliter (eg/ml), was calculated by dot-blot hybridization and determined to be about 1011 eg/ml. 2.2. Animal protocol Wild-type C57BL/6 mice and homozygous LDLR KO mice (on C57BL/6 background) were obtained from Jackson Laboratories. Group 1: Wild-type mice were injected with 100 l of saline via the tail vein (negative control group). Group 2: LDLR KO mice were injected with 100 l of saline via the tail vein (positive control group). Group 3: LDLR KO mice were injected with 100 l of AAV/AT2R virus (1010 eg) (experimental group). Group 4: LDLR KO mice were injected with 100 l of AAV/Neo virus (1010 eg) (AAV control group). All animals were kept on a high-cholesterol (4% cholesterol/10% cocoa butter) for 18 weeks. All experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Usage Committee.
Total DNA was isolated from the frozen tissue specimens from the AAV or saline-injected LDLR KO mice. PCR amplification was performed as described previously [14]. 2.4. Analysis of atherogenesis After harvesting the animals, aortic fatty deposits (index of atherosclerotic lesion formation) and intima thickness were quantitated by Sudan IV and H&E staining, respectively [9,14].
2.6. Analysis of protein by immunohistochemistry Aortic tissues were stained for ␣-actin and nitrotyrosine as described previously [14]. 2.7. Expression analysis Aortic specimens were derived from animals at 18 weeks of high cholesterol diet. The RNA isolation and RT-PCR amplification analysis of AT1 R and AT2R were carried out as described previously [10]. Protein expression of AT1 R, AT2R, NADPH oxidase p22phox , NADPH oxidase p47phox , NF-B p65, nitrotyrosine, Akt-1, phos-Akt-1, eNOS, phosS1177 eNOS, HO-1 and LOX-1 was carried out by Western blotting [9]. Band density relative to -actin was analyzed. 2.8. Data analysis Data are presented as mean ± S.E.M. All values were analyzed by using one way ANOVA and the Newman–KeulsStudent t-test. A P-value ≤ 0.05 was considered significant.
3. Results 3.1. Successful systemic delivery of transgene into LDLR KO mice The vector cDNA was found to be highly expressed in cardiovascular tissues of LDLR KO mice 18 weeks after administration of AAV/AT2R and high cholesterol diet feeding (Fig. 1A). We also established a marked increase in AT2R mRNA (Fig. 1B) and protein (Fig. 1C) in mice given AAV/ AT2R.
290
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
Fig. 1. Determination of vector DNA and AT2R mRNA and protein in various tissues. AT2R cDNA vector was highly expressed in the heart and aorta of the LDLR KO mice given AAV/AT2R (A). A single tail vein injection of AAV/AT2R markedly increased AT2R mRNA (B) and protein (C) in aortas of treated mice (n = 5).
3.2. Plasma lipid levels In keeping with our previous study [14], all LDLR KO mice on high-cholesterol diet had elevated total and LDLcholesterol levels in plasma (P < 0.01 vs. C57BL/6 mice) (data not shown). Administration of saline, AAV/AT2R or AAV/Neo had no effect on plasma lipid levels. 3.3. AT1R expression in atherosclerotic lesions In keeping with previous observations in the hypercholesterolemic and other models of atherosclerosis, AT1R
expression (mRNA and protein) was markedly increased in atherosclerotic aortas from all LDLR KO mice (Fig. 2A and B). Administration of saline, AAV/AT2R or AAV/Neo did not affect AT1R expression. 3.4. AT2R over-expression and inhibition of atherosclerotic lesion formation Wild-type mice had some areas of sudanophilia, perhaps an indication of very high cholesterol diet feeding. Salinetreated and AAV/Neo-treated LDLR KO mice had about 60% of aorta covered with areas of sudanophilia (P < 0.01 vs.
Fig. 2. AT1R mRNA and protein in aortas of different groups of mice. AT1R mRNA (A) and protein (B) were highly expressed in all LDLR KO mice groups. Administration of AAV/AT2R or AAV/Neo did not affect the enhanced AT1R expression (n = 5).
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
291
Fig. 3. AT2R and atherosclerotic lesion formation. (A) Representative aortas and quantitation of sudanophilic areas (n = 5–7 in each group). (B) Upregulation of AT2R in the LDLR KO mice markedly reduced intimal thickening and accumulation of macrophages (magnification 20×). Representative aortic sections are shown in panel B (left). Arrows show regions of intimal thickening. Bar graphs show intima thickness in each group (n = 5–7). (C) Representative cross-sectional areas of aorta stained with ␣-actin for vascular smooth muscle cells (SMCs). The inset (magnification 40×) shows the migration of SMCs in the proliferating intima. Upregulation of AT2R reduced SMCs accumulation and migration.
wild-type mice). In contrast, AAV/AT2R-treated LDLR KO mice displayed dramatically smaller areas of sudanophilia (Fig. 3A). The data on the extent of sudanophilic areas were complemented by the data on cross-sections of aorta wherein intimal area was examined. The intima was severalfold thicker in saline-treated and AAV/Neo-treated LDLR KO mice compared to wild-type mice and contained a large number of macrophages. The over-expression of AT2R in LDLR KO mice resulted in a consistent reduction in intimal thickness and there were many fewer macrophages (Fig. 3B). Further, there was extensive disarrayed SMC accumulation in the proliferating intima in saline-treated and AAV/Neotreated LDLR KO mice, characteristic of atherosclerosis. In contrast, AAV/AT2R-treated LDLR KO mice did not show excessive SMC accumulation (Fig. 3C). 3.5. Mechanisms of inhibition of atherosclerosis by AT2R over-expression To determine the potential mechanisms of the AT2R overexpression in atherogenesis, expression of NADPH oxidase subunits p47phox and p22phox and the redox-sensitive transcription factor NF-B was examined in this study. Both p47phox and p22phox subunits of NADPH oxidase were
markedly increased in saline-treated and AAV/Neo-treated LDLR KO mice (vs. wild-type mice) (Fig. 4A). The upregulation of both subunits of NADPH oxidase was reduced by AT2R over-expression. Expression of NF-B was also higher in saline-treated and AAV/Neo-treated LDLR KO mice (vs. the wild-type mice), and over-expression of AT2R limited the increase in NF-B expression (Fig. 4B). To confirm the increased production of ROS in aorta, we examined the presence of nitrotyrosine as an indirect marker of oxidative stress by immunohistochemistry and western blot analysis [14]. Expression of nitrotyrosine was found to be higher in saline-treated and AAV/Neo-treated LDLR KO mice (vs. the wild-type mice), and over-expression of AT2R limited the increase in nitrotyrosine expression (Fig. 4C and D). Next, we studied the expression of Akt-1 protein and its phosphorylation in the aortic tissues. While basal Akt-1 protein expression was similar in all mice, Akt-1 phosphorylation was reduced in the LDLR KO mice given saline or AAV Neo (vs. wild-type mice), and AT2R over-expression markedly increased Akt-1 phosphorylation (Fig. 5A). Akt-1 activation regulates the expression of eNOS and its activity. We, therefore, examined eNOS protein and phos-S1177 eNOS expression in aortic tissues. As shown in Fig. 5B, the expression of eNOS protein as well as its activity was
292
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
Fig. 4. Expression of NADPH oxidase (p47phox and p22phox subunits) (A), NF-B p65 (B) and nitrotyrosine (western analysis-C; immunostaining-D). The expression of NADPH oxidase, nitrotyrosine and NF-B was significantly increased in the LDLR KO mice given saline or AAV/Neo compared to the wild-type mice. The over-expression of AT2R in LDLR KO mice markedly inhibited the expression of NADPH oxidase, nitrotyrosine and NF-B. Data from three to five mice in each group.
reduced in the saline-treated and AAV/Neo-treated LDLR KO mice compared to the wild-type mice. On the other hand, AAV/AT2R upregulation enhanced eNOS protein expression and its activity.
We also examined the expression of HO-1, another vasodilator species that is relevant in atherogenesis [12–13]. As shown in Fig. 5C, HO-1 expression was reduced in the aortas from saline-treated and AAV/Neo-treated LDLR KO
Fig. 5. AT2R upregulation and signaling. AT2R upregulation increased Akt-1 phosphorylation (A) as well as the expression/activation of eNOS (B) and the expression of HO-1 (C), and reduced the expression LOX-1 (D). Data from three to five mice in each group.
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
mice (vs. wild-type mice). On the other hand, delivery of AAV/AT2R enhanced its expression. In keeping with recent observations [9], LOX-1 expression was markedly increased in the aortas from saline-treated and AAV/Neo-treated LDLR KO mice. On the other hand, AAV/AT2R upregulation reduced the enhanced LOX-1 expression (Fig. 5D).
4. Discussion AT1R in adult tissues mediates the major pathological effects of Ang II. The density of AT2R in cardiovascular tissues gradually decreases and that of AT1R increases with age [2,15]. The reason for this phenomenon is not clear, but is possible that reduction in the number of AT2R is related to aging and atherogenesis. Iwai et al. [7] reported that deletion of AT2R exaggerated atherosclerosis in the Apo-E KO mice. We, therefore, speculated that AT2R upregulation might have protective effect against atherogenesis. Our study indeed demonstrates that delivery of AT2R gene by AAV can reduce atherosclerotic lesion formation. Further, this study provides strong evidence that AT2R over-expression modulates NADPH oxidase and downstream signals which may have a bearing on the inhibition of atherogenesis. 4.1. Systemic delivery of AT2R gene by AAV AAV is a very desirable vector to use when long-term expression is needed. Our recent studies have demonstrated that AAV can successfully deliver TGF1 [14] and interleukin-10 [16] genes in the mice. We now show that administration of AT2R with AAV by a single tail vein injection can induce adequate and sustained high levels of the AT2R transgene in vascular tissues. 4.2. AT2R over-expression and atherosclerosis inhibition AT1R is highly expressed in atherosclerotic lesions [2]. This phenomenon was confirmed in the present study. Ang II via AT1R causes activation of signaling pathways such as NADPH oxidase, protein kinases and NF-B [13], which results in growth and proliferation of SMCs [2] and facilitates the cellular uptake of ox-LDL in endothelial cells [10]. Since AT2R activation exerts effects opposed to those of AT1R activation [17], we postulated that AT2R upregulation may alter AT1R/AT2R balance and thus inhibit many of these signals. We show that upregulation of AT2R reduces the accumulation and migration of SMCs, which are present in abundance in intima of the atherosclerotic regions in the LDLR KO mice. The signaling of AT2R activation remains enigmatic. Atherosclerotic tissues have an exaggerated redox state beyond the capability of endogenous anti-oxidants to regulate it [2]. A link between oxidative stress and AT1R
293
activation, mediated via NADPH oxidase, in atherosclerosis has been known for a while [2]. The pro-oxidant state in turn stimulates transcription factors such NF-B, which plays a critical role in Ang II-mediated pro-apoptotic and SMC growth promoting effects [2]. Iwai et al. [7] showed that NADPH oxidase activity and translocation of its p47phox subunit induced by Ang II were inhibited by AT1R blockade and were enhanced by AT2R blockade. Thomas et al. [18] showed that the deletion of p47phox subunit inhibited the adverse effect of Ang II on aortic aneurysm formation in the hyperlipidemic mice. We now show that AT2R upregulation reduces the expression of NADPH oxidase (both p47phox and p22phox subunits), nitrotyrosine and NF-B in the LDLR KO mice, so that these indicators of oxidant stress become comparable to those in the wild-type mice. Some investigators [19] have suggested that AT2R stimulation produces a kinin-dependent stimulation of NO generation and release. NO released locally may decrease oxidative stress and subsequent monocyte accumulation, collagen synthesis and cell proliferation. Akt-1 is important in downstream targeting of extracellular PI-3 kinase signaling, and alterations in its activity may be important in the phosphorylation of eNOS [20]. In keeping with this concept, we observed a reduction in Akt-1 phosphorylation and expression of eNOS as well as its phosphorylation in the LDLR KO mice given high cholesterol diet. Importantly, AT2R over-expression enhanced Akt-1 phosphorylation and expression of eNOS as well as its activity, even higher than that in the wild-type mice. It is of note that a link between Akt-1 and NF-B activation has also been clearly shown [21]. There is emerging evidence that HO-1 functions as an adaptive molecule against oxidative insult [12,13]. HO1 upregulation in turn reduces NADPH oxidase activity [22]. It appears that persistent oxidant stress may reduce the expression of HO-1 [23]. There is also evidence that Akt-1 activation upregulates HO-1 [24]. Yet et al. [25] found that the absence of HO-1 exacerbates atherosclerotic lesion formation, suggesting a protective role for HO-1 against atherogenesis. In keeping with these observations, we found that atherosclerotic aortas from LDLR KO mice had reduced expression of HO-1. With AT2R over-expression, there was a marked increase in Akt-1 and HO-1 expression and a decrease in NADPH oxidase expression. Whether these events are related or not is not clear, but our observations strongly suggest that oxidant stress and HO-1 are inter-twined, and Akt-1 phosphorylation may relate to these alterations. Importantly, a recent study from our laboratory showed that LOX-1 deletion reduces atherogenesis in LDLR KO mice [9]. Our previous work showed that Ang II via AT1R enhances ox-LDL uptake in endothelial cells, a process in which pro-oxidant signals play a key role [10]. In the present study, we show a marked upregulation of LOX-1 in the aortas of LDLR KO mice, and AT2R upregulation reduced the enhanced LOX-1 expression.
294
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
4.3. Differences from previous studies This study clearly documents that systemic delivery of AT2R gene (agtr2) inhibits atherosclerotic lesion formation, possibly via anti-oxidant mechanisms which reduce LOX-1 expression and promote eNOS and HO-1 expression. These results are supported by the observations of Iwai et al. [7] who showed that deletion of AT2R exaggerated atherosclerosis in Apo-E KO mice. Others, however, reported that AT2R deficiency had no effect on atherogenesis in LDLR KO [26] and Apo-E KO mice [27]; yet AT2R deficiency augmented the cellularity of atherosclerotic lesions. Johnasson et al. [28] found that administration of an AT2R antagonist had no effect on Ang II-accelerated atherosclerosis in Apo-E KO mice. These apparent discrepancies between different studies might be due to the differences in animal models, diet used for the induction of atherosclerosis and the study design. Future studies are necessary to clarify these differences. It is possible that AT1R antagonists would have a greater effect in the AT2R over-expression model based on the observation that AT1R activation contributes to atherosclerosis. Further, it would be interesting to examine the effects of AT2R over-expression in AT2R deficient LDLR KO background mice.
[9]
[10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements This study was funded by grants from the Department of Veterans Affairs (JLM) and American Heart Association (PLH).
[19]
[20] [21]
References [22] [1] Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74. [2] Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 2007;292:C82–97. [3] Chen J, Li D, Schaefer R, Mehta JL. Cross-talk between dyslipidemia and renin-angiotensin system and the role of LOX-1 and MAPK in atherogenesis studies with the combined use of rosuvastatin and candesartan. Atherosclerosis 2006;184:295–301. [4] Morawietz H, Erbs S, Holtz J, et al. Endothelial protection, AT1 blockade and cholesterol-dependent oxidative stress: the EPAS trial. Circulation 2006;114:I296–301. [5] Navalkar S, Parthasarathy S, Santanam N, Khan BV. Irbesartan, an angiotensin type 1 receptor inhibitor, regulates markers of inflammation in patients with premature atherosclerosis. J Am Coll Cardiol 2001;37:440–4. [6] Carey RM. Cardiovascular and renal regulation by the angiotensin type 2 receptor: the AT2 receptor comes of age. Hypertension 2005;45:840–4. [7] Iwai M, Chen R, Li Z, et al. Deletion of angiotensin II type 2 receptor exaggerated atherosclerosis in apolipoprotein E-null mice. Circulation 2005;112:1636–43. [8] Mehta JL, Chen J, Hermonat PL, Romeo F, Novelli G. Lectin-like oxidized-low density lipoprotein receptor-1 (LOX-1): a critical player
[23]
[24]
[25]
[26]
[27]
[28]
in the development of atherosclerosis and related disorders. Cardiovasc Res 2006;69:36–45. Mehta JL, Sanada N, Hu CP, et al. Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet. Circ Res 2007;100:1634–42. Li DY, Zhang YC, Philips MI, Sawamura T, Mehta JL. Upregulation of endothelial receptor for oxidized low-density lipoprotein (LOX-1) in cultured human coronary artery endothelial cells by angiotensin II type 1 receptor activation. Circ Res 1999;84:1043–9. Kawashima S, Yokoyama M. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler Thromb Vasc Biol 2004;24:998–1005. Morita T. Heme-oxygenase and atherosclerosis. Arterioscler Thromb Vasc Biol 2005;25:1786–95. Wu BJ, Kathir K, Witting PK, et al. Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging. J Exp Med 2006;203:1117–27. Li D, Liu Y, Chen J, et al. Suppression of atherogenesis by delivery of TGF1 ACT using adeno-associated virus type 2 in LDLR knockout mice. Biochem Biophys Res Commun 2006;344:701–7. Jones ES, Black MJ, Widdop RE. Angiotensin AT2 receptor contributes to cardiovascular remodeling of aged rats during chronic AT1 receptor blockade. J Mol Cell Cardiol 2004;37:1023–30. Liu Y, Li D, Chen J, et al. Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10. Atherosclerosis 2006;188:19–27. Li H, Gao Y, Grobe JL, et al. Potentiation of antihypertensive action of losartan by peripheral overexpression of Ang II type 2 receptor. Am J Physiol Heart Circ Physiol 2007;292:H727–35. Thomas M, Gavrila D, McCormick ML, et al. Deletion of p47phox attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-deficient mice. Circulation 2006;114:404–13. Kurisu S, Ozono R, Oshima T, et al. Cardiac angiotensin II type 2 receptor activates the kinin/NO system and inhibits fibrosis. Hypertension 2003;41:99–107. Michell BJ, Griffiths JE, Mitchelhill KI, et al. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999;9:845–8. Schmidt A, Geigenmuller S, Volker W, Buddecke E. The antiatherogenic and anti-inflammatory effect of HDL-associated lysosphingolipids operates via Akt → NF-kappaB signalling pathways in human vascular endothelial cells. Basic Res Cardiol 2006;101:109–16. Colombrita C, Lombardo G, Scapagnini G, Abraham NG. Heme oxygenase-1 expression levels are cell cycle dependent. Biochem Biophys Res Commun 2003;308:1001–8. Hoekstra KA, Godin DV, Kurtu J, Cheng KM. Effects of oxidantinduced injury on heme oxygenase and glutathione in cultured aortic endothelial cells from atherosclerosis-susceptible and -resistant Japanese quail. Mol Cell Biochem 2003;254:61–71. Hsu JT, Kan WH, Hsieh CH, et al. Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Aktdependent HO-1 up-regulation. J Leukoc Biol 2007. July 26. Yet SF, Layne MD, Liu X, et al. Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodelling. FASEB J 2003;17:1759–61. Daugherty A, Rateri DL, Lu H, Inagami T, Cassis LA. Hypercholesterolemia stimulates angiotensin peptide synthesis and contributes to atherosclerosis through the AT1A receptor. Circulation 2004;110:3849–57. Sales VL, Sukhova GK, Lopez-Ilasaca MA, et al. Angiotensin type 2 receptor is expressed in murine atherosclerotic lesions and modulates lesion evolution. Circulation 2005;112:3328–36. Johansson ME, Wickman A, Fitzgerald SM, Gan LM, Bergstrom G. Angiotensin II, type 2 receptor is not involved in the angiotensin II-mediated pro-atherogenic process in ApoE−/− mice. J Hypertens 2005;23:1541–9.
Over-expression of angiotensin II type 2 receptor (agtr2) reduces atherogenesis and modulates LOX-1, endothelial nitric oxide synthase and heme-oxygenase-1 expression Changping Hu a,b,1 , Abhijit Dandapat a,1 , Jiawei Chen a , Yong Liu a , Paul L. Hermonat a , Robert M. Carey c , Jawahar L. Mehta a,∗ a
b
Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and VA Medical Center, Little Rock, AR 72205, United States Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, China c Department of Medicine, University of Virginia, Charlottesville, VA, United States Received 10 June 2007; received in revised form 9 September 2007; accepted 7 November 2007 Available online 21 December 2007
Abstract Atherogenesis is associated with inflammation and oxidative stress. Activation of renin-angiotensin system with generation of angiotensin II and type 1 receptor (AT1R) stimulation has been amply reported in atherosclerosis. Since angiotensin II type 2 receptor (AT2R) activity is purported to oppose the effects of AT1R, we hypothesized that AT2R (agtr2) over-expression would inhibit atherogenesis. We prepared recombinant adeno-associated virus type-2 (AAV) carrying AT2R cDNA (AAV/AT2R), and homozygous LDLR-deficient (KO) mice were given AAV/AT2R, AAV/Neo or saline. All mice were placed on a high cholesterol diet. After 18 weeks, AT2R was found to be over-expressed systemically in AAV/AT2R-treated mice. Atherogenesis in aorta was reduced in the AAV/AT2R group by ≈50% compared to other LDLR KO mice groups. Expression of NADPH oxidase, nitrotyrosine and NF-B was increased in aortic tissues of the LDLR KO mice given saline or AAV/Neo, but not in mice with AT2R upregulation. Expression of endothelial nitric oxide synthase (eNOS) and heme-oxygenase-1 (HO-1) was decreased and that of the lectin-like oxidized-LDL receptor (LOX-1) increased in the LDLR KO mice, but not in the mice with AT2R over-expression. Further, Akt-1 phosphorylation was reduced in the LDLR KO mice, but not in the mice with AT2R over-expression. Thus, AT2R upregulation can reduce atherogenesis, possibly by modulating oxidative stress and the pro-inflammatory cascade, mediated via Akt-1. Over-expression of AT2R may be an important therapeutic approach in atherosclerosis. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Akt-1; Angiotensin; Atherosclerosis; Heme-oxygenase-1; Lectin-like oxidized-LDL receptor; Nitric oxide synthase; Oxidative stress
1. Introduction Atherosclerosis, at least in part, is an inflammatory/ immune disorder accompanied by oxidative stress [1]. Angiotensin (Ang) II type 1 receptor (AT1R) expression is upregulated in atherosclerosis and plays an important role in atherogenesis [2,3]. Actually, atherosclerosis and its sequelae are inhibited by AT1R inhibition via inhibition of inflammation and oxidative stress [4,5]. It has been postulated that ∗ 1
Corresponding author. Tel.: +1 501 296 1410. E-mail address: [email protected] (J.L. Mehta). These two authors contributed equally.
0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.11.006
AT1R blockers exert their effect not just by blocking AT1R activity, but also by redirecting Ang II towards unblocked type 2 receptors (AT2R) whereby AT2R activity increases [6]. It is generally assumed that AT2R activation exerts effects opposite to those of AT1R activation [2,6]. Interestingly, AT2R deletion was shown to exaggerate atherosclerosis in apolipoprotein E-null mice [7]. However, if upregulation of AT2R expression will inhibit atherosclerosis is not known. LOX-1, a lectin-like receptor for oxidized LDL (ox-LDL), is the major receptor responsible for binding, internalization and degradation of ox-LDL in endothelial cells and plays a critical role in atherogenesis [8,9]. Ang II upregulates LOX-1 expression via AT1R activation [10]. There is also evidence
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
289
for a reduction in the expression and activity of endothelial nitric oxide synthase (eNOS) in atherosclerosis [11]. Hemeoxygenase-1 (HO-1), an enzyme involved in the genesis of the potent vasodilator species carbon monoxide, is also altered in atherosclerosis [12,13]. Both eNOS and HO-1 are regulated by Ang II [11,12]. This study was designed to test the hypothesis that AT2R cDNA delivered with adeno-associated virus type 2 (AAV) will result in sustained over-expression of AT2R in vascular tissues, and will inhibit atherogenesis in LDL receptor-deficient (LDLR KO) mice. Further, we examined the hypothesis that AT2R over-expression will modulate LOX-1, eNOS and HO-1 expression.
2.3. Analysis of transgene vector DNA
2. Materials and methods
2.5. Measurement of plasma lipids
2.1. Construction of AAV/AT2R vector and generation of AAV/AT2R stocks
Plasma levels of total cholesterol, HDL-cholesterol, triglycerides, were measured by clinical laboratory.
Mouse AT2R (agtr2) cDNA was generated by PCR amplification. Total RNA was isolated from aortic tissues of C57BL/6 mice using Trizol reagent (Invitrogen) and treated with 2.0 U/g of RNase-free DNase I (Promega) at 37 ◦ C for 1 h. mRNA was separated using the Oligotex mRNA Mini Kit (QIAGEN). First-strand cDNA synthesis was performed using oligo(dT)15 primers. PCR amplification for the AT2R cDNA sequence was carried out using the following primer pair: 5 -GAGTTGCTGCAGTTCAAT-3 and 5 -GAACTGTATTATACGTATGCCAC-3 that amplify the sequence from nucleotides 149 to 1326 (NCBI Gene Bank: NM-007429). After AT2R cDNA was sequenced, it was ligated into an AAV vector, dl6-95, as described previously [14]. Hereafter, recombinant AAV (rAAV) vector is referred to as AAV/AT2R. The rAAV virus stocks were generated as described previously [14]. The titer of purified virus, in encapsidated genomes per milliliter (eg/ml), was calculated by dot-blot hybridization and determined to be about 1011 eg/ml. 2.2. Animal protocol Wild-type C57BL/6 mice and homozygous LDLR KO mice (on C57BL/6 background) were obtained from Jackson Laboratories. Group 1: Wild-type mice were injected with 100 l of saline via the tail vein (negative control group). Group 2: LDLR KO mice were injected with 100 l of saline via the tail vein (positive control group). Group 3: LDLR KO mice were injected with 100 l of AAV/AT2R virus (1010 eg) (experimental group). Group 4: LDLR KO mice were injected with 100 l of AAV/Neo virus (1010 eg) (AAV control group). All animals were kept on a high-cholesterol (4% cholesterol/10% cocoa butter) for 18 weeks. All experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Usage Committee.
Total DNA was isolated from the frozen tissue specimens from the AAV or saline-injected LDLR KO mice. PCR amplification was performed as described previously [14]. 2.4. Analysis of atherogenesis After harvesting the animals, aortic fatty deposits (index of atherosclerotic lesion formation) and intima thickness were quantitated by Sudan IV and H&E staining, respectively [9,14].
2.6. Analysis of protein by immunohistochemistry Aortic tissues were stained for ␣-actin and nitrotyrosine as described previously [14]. 2.7. Expression analysis Aortic specimens were derived from animals at 18 weeks of high cholesterol diet. The RNA isolation and RT-PCR amplification analysis of AT1 R and AT2R were carried out as described previously [10]. Protein expression of AT1 R, AT2R, NADPH oxidase p22phox , NADPH oxidase p47phox , NF-B p65, nitrotyrosine, Akt-1, phos-Akt-1, eNOS, phosS1177 eNOS, HO-1 and LOX-1 was carried out by Western blotting [9]. Band density relative to -actin was analyzed. 2.8. Data analysis Data are presented as mean ± S.E.M. All values were analyzed by using one way ANOVA and the Newman–KeulsStudent t-test. A P-value ≤ 0.05 was considered significant.
3. Results 3.1. Successful systemic delivery of transgene into LDLR KO mice The vector cDNA was found to be highly expressed in cardiovascular tissues of LDLR KO mice 18 weeks after administration of AAV/AT2R and high cholesterol diet feeding (Fig. 1A). We also established a marked increase in AT2R mRNA (Fig. 1B) and protein (Fig. 1C) in mice given AAV/ AT2R.
290
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
Fig. 1. Determination of vector DNA and AT2R mRNA and protein in various tissues. AT2R cDNA vector was highly expressed in the heart and aorta of the LDLR KO mice given AAV/AT2R (A). A single tail vein injection of AAV/AT2R markedly increased AT2R mRNA (B) and protein (C) in aortas of treated mice (n = 5).
3.2. Plasma lipid levels In keeping with our previous study [14], all LDLR KO mice on high-cholesterol diet had elevated total and LDLcholesterol levels in plasma (P < 0.01 vs. C57BL/6 mice) (data not shown). Administration of saline, AAV/AT2R or AAV/Neo had no effect on plasma lipid levels. 3.3. AT1R expression in atherosclerotic lesions In keeping with previous observations in the hypercholesterolemic and other models of atherosclerosis, AT1R
expression (mRNA and protein) was markedly increased in atherosclerotic aortas from all LDLR KO mice (Fig. 2A and B). Administration of saline, AAV/AT2R or AAV/Neo did not affect AT1R expression. 3.4. AT2R over-expression and inhibition of atherosclerotic lesion formation Wild-type mice had some areas of sudanophilia, perhaps an indication of very high cholesterol diet feeding. Salinetreated and AAV/Neo-treated LDLR KO mice had about 60% of aorta covered with areas of sudanophilia (P < 0.01 vs.
Fig. 2. AT1R mRNA and protein in aortas of different groups of mice. AT1R mRNA (A) and protein (B) were highly expressed in all LDLR KO mice groups. Administration of AAV/AT2R or AAV/Neo did not affect the enhanced AT1R expression (n = 5).
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
291
Fig. 3. AT2R and atherosclerotic lesion formation. (A) Representative aortas and quantitation of sudanophilic areas (n = 5–7 in each group). (B) Upregulation of AT2R in the LDLR KO mice markedly reduced intimal thickening and accumulation of macrophages (magnification 20×). Representative aortic sections are shown in panel B (left). Arrows show regions of intimal thickening. Bar graphs show intima thickness in each group (n = 5–7). (C) Representative cross-sectional areas of aorta stained with ␣-actin for vascular smooth muscle cells (SMCs). The inset (magnification 40×) shows the migration of SMCs in the proliferating intima. Upregulation of AT2R reduced SMCs accumulation and migration.
wild-type mice). In contrast, AAV/AT2R-treated LDLR KO mice displayed dramatically smaller areas of sudanophilia (Fig. 3A). The data on the extent of sudanophilic areas were complemented by the data on cross-sections of aorta wherein intimal area was examined. The intima was severalfold thicker in saline-treated and AAV/Neo-treated LDLR KO mice compared to wild-type mice and contained a large number of macrophages. The over-expression of AT2R in LDLR KO mice resulted in a consistent reduction in intimal thickness and there were many fewer macrophages (Fig. 3B). Further, there was extensive disarrayed SMC accumulation in the proliferating intima in saline-treated and AAV/Neotreated LDLR KO mice, characteristic of atherosclerosis. In contrast, AAV/AT2R-treated LDLR KO mice did not show excessive SMC accumulation (Fig. 3C). 3.5. Mechanisms of inhibition of atherosclerosis by AT2R over-expression To determine the potential mechanisms of the AT2R overexpression in atherogenesis, expression of NADPH oxidase subunits p47phox and p22phox and the redox-sensitive transcription factor NF-B was examined in this study. Both p47phox and p22phox subunits of NADPH oxidase were
markedly increased in saline-treated and AAV/Neo-treated LDLR KO mice (vs. wild-type mice) (Fig. 4A). The upregulation of both subunits of NADPH oxidase was reduced by AT2R over-expression. Expression of NF-B was also higher in saline-treated and AAV/Neo-treated LDLR KO mice (vs. the wild-type mice), and over-expression of AT2R limited the increase in NF-B expression (Fig. 4B). To confirm the increased production of ROS in aorta, we examined the presence of nitrotyrosine as an indirect marker of oxidative stress by immunohistochemistry and western blot analysis [14]. Expression of nitrotyrosine was found to be higher in saline-treated and AAV/Neo-treated LDLR KO mice (vs. the wild-type mice), and over-expression of AT2R limited the increase in nitrotyrosine expression (Fig. 4C and D). Next, we studied the expression of Akt-1 protein and its phosphorylation in the aortic tissues. While basal Akt-1 protein expression was similar in all mice, Akt-1 phosphorylation was reduced in the LDLR KO mice given saline or AAV Neo (vs. wild-type mice), and AT2R over-expression markedly increased Akt-1 phosphorylation (Fig. 5A). Akt-1 activation regulates the expression of eNOS and its activity. We, therefore, examined eNOS protein and phos-S1177 eNOS expression in aortic tissues. As shown in Fig. 5B, the expression of eNOS protein as well as its activity was
292
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
Fig. 4. Expression of NADPH oxidase (p47phox and p22phox subunits) (A), NF-B p65 (B) and nitrotyrosine (western analysis-C; immunostaining-D). The expression of NADPH oxidase, nitrotyrosine and NF-B was significantly increased in the LDLR KO mice given saline or AAV/Neo compared to the wild-type mice. The over-expression of AT2R in LDLR KO mice markedly inhibited the expression of NADPH oxidase, nitrotyrosine and NF-B. Data from three to five mice in each group.
reduced in the saline-treated and AAV/Neo-treated LDLR KO mice compared to the wild-type mice. On the other hand, AAV/AT2R upregulation enhanced eNOS protein expression and its activity.
We also examined the expression of HO-1, another vasodilator species that is relevant in atherogenesis [12–13]. As shown in Fig. 5C, HO-1 expression was reduced in the aortas from saline-treated and AAV/Neo-treated LDLR KO
Fig. 5. AT2R upregulation and signaling. AT2R upregulation increased Akt-1 phosphorylation (A) as well as the expression/activation of eNOS (B) and the expression of HO-1 (C), and reduced the expression LOX-1 (D). Data from three to five mice in each group.
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
mice (vs. wild-type mice). On the other hand, delivery of AAV/AT2R enhanced its expression. In keeping with recent observations [9], LOX-1 expression was markedly increased in the aortas from saline-treated and AAV/Neo-treated LDLR KO mice. On the other hand, AAV/AT2R upregulation reduced the enhanced LOX-1 expression (Fig. 5D).
4. Discussion AT1R in adult tissues mediates the major pathological effects of Ang II. The density of AT2R in cardiovascular tissues gradually decreases and that of AT1R increases with age [2,15]. The reason for this phenomenon is not clear, but is possible that reduction in the number of AT2R is related to aging and atherogenesis. Iwai et al. [7] reported that deletion of AT2R exaggerated atherosclerosis in the Apo-E KO mice. We, therefore, speculated that AT2R upregulation might have protective effect against atherogenesis. Our study indeed demonstrates that delivery of AT2R gene by AAV can reduce atherosclerotic lesion formation. Further, this study provides strong evidence that AT2R over-expression modulates NADPH oxidase and downstream signals which may have a bearing on the inhibition of atherogenesis. 4.1. Systemic delivery of AT2R gene by AAV AAV is a very desirable vector to use when long-term expression is needed. Our recent studies have demonstrated that AAV can successfully deliver TGF1 [14] and interleukin-10 [16] genes in the mice. We now show that administration of AT2R with AAV by a single tail vein injection can induce adequate and sustained high levels of the AT2R transgene in vascular tissues. 4.2. AT2R over-expression and atherosclerosis inhibition AT1R is highly expressed in atherosclerotic lesions [2]. This phenomenon was confirmed in the present study. Ang II via AT1R causes activation of signaling pathways such as NADPH oxidase, protein kinases and NF-B [13], which results in growth and proliferation of SMCs [2] and facilitates the cellular uptake of ox-LDL in endothelial cells [10]. Since AT2R activation exerts effects opposed to those of AT1R activation [17], we postulated that AT2R upregulation may alter AT1R/AT2R balance and thus inhibit many of these signals. We show that upregulation of AT2R reduces the accumulation and migration of SMCs, which are present in abundance in intima of the atherosclerotic regions in the LDLR KO mice. The signaling of AT2R activation remains enigmatic. Atherosclerotic tissues have an exaggerated redox state beyond the capability of endogenous anti-oxidants to regulate it [2]. A link between oxidative stress and AT1R
293
activation, mediated via NADPH oxidase, in atherosclerosis has been known for a while [2]. The pro-oxidant state in turn stimulates transcription factors such NF-B, which plays a critical role in Ang II-mediated pro-apoptotic and SMC growth promoting effects [2]. Iwai et al. [7] showed that NADPH oxidase activity and translocation of its p47phox subunit induced by Ang II were inhibited by AT1R blockade and were enhanced by AT2R blockade. Thomas et al. [18] showed that the deletion of p47phox subunit inhibited the adverse effect of Ang II on aortic aneurysm formation in the hyperlipidemic mice. We now show that AT2R upregulation reduces the expression of NADPH oxidase (both p47phox and p22phox subunits), nitrotyrosine and NF-B in the LDLR KO mice, so that these indicators of oxidant stress become comparable to those in the wild-type mice. Some investigators [19] have suggested that AT2R stimulation produces a kinin-dependent stimulation of NO generation and release. NO released locally may decrease oxidative stress and subsequent monocyte accumulation, collagen synthesis and cell proliferation. Akt-1 is important in downstream targeting of extracellular PI-3 kinase signaling, and alterations in its activity may be important in the phosphorylation of eNOS [20]. In keeping with this concept, we observed a reduction in Akt-1 phosphorylation and expression of eNOS as well as its phosphorylation in the LDLR KO mice given high cholesterol diet. Importantly, AT2R over-expression enhanced Akt-1 phosphorylation and expression of eNOS as well as its activity, even higher than that in the wild-type mice. It is of note that a link between Akt-1 and NF-B activation has also been clearly shown [21]. There is emerging evidence that HO-1 functions as an adaptive molecule against oxidative insult [12,13]. HO1 upregulation in turn reduces NADPH oxidase activity [22]. It appears that persistent oxidant stress may reduce the expression of HO-1 [23]. There is also evidence that Akt-1 activation upregulates HO-1 [24]. Yet et al. [25] found that the absence of HO-1 exacerbates atherosclerotic lesion formation, suggesting a protective role for HO-1 against atherogenesis. In keeping with these observations, we found that atherosclerotic aortas from LDLR KO mice had reduced expression of HO-1. With AT2R over-expression, there was a marked increase in Akt-1 and HO-1 expression and a decrease in NADPH oxidase expression. Whether these events are related or not is not clear, but our observations strongly suggest that oxidant stress and HO-1 are inter-twined, and Akt-1 phosphorylation may relate to these alterations. Importantly, a recent study from our laboratory showed that LOX-1 deletion reduces atherogenesis in LDLR KO mice [9]. Our previous work showed that Ang II via AT1R enhances ox-LDL uptake in endothelial cells, a process in which pro-oxidant signals play a key role [10]. In the present study, we show a marked upregulation of LOX-1 in the aortas of LDLR KO mice, and AT2R upregulation reduced the enhanced LOX-1 expression.
294
C. Hu et al. / Atherosclerosis 199 (2008) 288–294
4.3. Differences from previous studies This study clearly documents that systemic delivery of AT2R gene (agtr2) inhibits atherosclerotic lesion formation, possibly via anti-oxidant mechanisms which reduce LOX-1 expression and promote eNOS and HO-1 expression. These results are supported by the observations of Iwai et al. [7] who showed that deletion of AT2R exaggerated atherosclerosis in Apo-E KO mice. Others, however, reported that AT2R deficiency had no effect on atherogenesis in LDLR KO [26] and Apo-E KO mice [27]; yet AT2R deficiency augmented the cellularity of atherosclerotic lesions. Johnasson et al. [28] found that administration of an AT2R antagonist had no effect on Ang II-accelerated atherosclerosis in Apo-E KO mice. These apparent discrepancies between different studies might be due to the differences in animal models, diet used for the induction of atherosclerosis and the study design. Future studies are necessary to clarify these differences. It is possible that AT1R antagonists would have a greater effect in the AT2R over-expression model based on the observation that AT1R activation contributes to atherosclerosis. Further, it would be interesting to examine the effects of AT2R over-expression in AT2R deficient LDLR KO background mice.
[9]
[10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements This study was funded by grants from the Department of Veterans Affairs (JLM) and American Heart Association (PLH).
[19]
[20] [21]
References [22] [1] Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74. [2] Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 2007;292:C82–97. [3] Chen J, Li D, Schaefer R, Mehta JL. Cross-talk between dyslipidemia and renin-angiotensin system and the role of LOX-1 and MAPK in atherogenesis studies with the combined use of rosuvastatin and candesartan. Atherosclerosis 2006;184:295–301. [4] Morawietz H, Erbs S, Holtz J, et al. Endothelial protection, AT1 blockade and cholesterol-dependent oxidative stress: the EPAS trial. Circulation 2006;114:I296–301. [5] Navalkar S, Parthasarathy S, Santanam N, Khan BV. Irbesartan, an angiotensin type 1 receptor inhibitor, regulates markers of inflammation in patients with premature atherosclerosis. J Am Coll Cardiol 2001;37:440–4. [6] Carey RM. Cardiovascular and renal regulation by the angiotensin type 2 receptor: the AT2 receptor comes of age. Hypertension 2005;45:840–4. [7] Iwai M, Chen R, Li Z, et al. Deletion of angiotensin II type 2 receptor exaggerated atherosclerosis in apolipoprotein E-null mice. Circulation 2005;112:1636–43. [8] Mehta JL, Chen J, Hermonat PL, Romeo F, Novelli G. Lectin-like oxidized-low density lipoprotein receptor-1 (LOX-1): a critical player
[23]
[24]
[25]
[26]
[27]
[28]
in the development of atherosclerosis and related disorders. Cardiovasc Res 2006;69:36–45. Mehta JL, Sanada N, Hu CP, et al. Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet. Circ Res 2007;100:1634–42. Li DY, Zhang YC, Philips MI, Sawamura T, Mehta JL. Upregulation of endothelial receptor for oxidized low-density lipoprotein (LOX-1) in cultured human coronary artery endothelial cells by angiotensin II type 1 receptor activation. Circ Res 1999;84:1043–9. Kawashima S, Yokoyama M. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler Thromb Vasc Biol 2004;24:998–1005. Morita T. Heme-oxygenase and atherosclerosis. Arterioscler Thromb Vasc Biol 2005;25:1786–95. Wu BJ, Kathir K, Witting PK, et al. Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging. J Exp Med 2006;203:1117–27. Li D, Liu Y, Chen J, et al. Suppression of atherogenesis by delivery of TGF1 ACT using adeno-associated virus type 2 in LDLR knockout mice. Biochem Biophys Res Commun 2006;344:701–7. Jones ES, Black MJ, Widdop RE. Angiotensin AT2 receptor contributes to cardiovascular remodeling of aged rats during chronic AT1 receptor blockade. J Mol Cell Cardiol 2004;37:1023–30. Liu Y, Li D, Chen J, et al. Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10. Atherosclerosis 2006;188:19–27. Li H, Gao Y, Grobe JL, et al. Potentiation of antihypertensive action of losartan by peripheral overexpression of Ang II type 2 receptor. Am J Physiol Heart Circ Physiol 2007;292:H727–35. Thomas M, Gavrila D, McCormick ML, et al. Deletion of p47phox attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-deficient mice. Circulation 2006;114:404–13. Kurisu S, Ozono R, Oshima T, et al. Cardiac angiotensin II type 2 receptor activates the kinin/NO system and inhibits fibrosis. Hypertension 2003;41:99–107. Michell BJ, Griffiths JE, Mitchelhill KI, et al. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999;9:845–8. Schmidt A, Geigenmuller S, Volker W, Buddecke E. The antiatherogenic and anti-inflammatory effect of HDL-associated lysosphingolipids operates via Akt → NF-kappaB signalling pathways in human vascular endothelial cells. Basic Res Cardiol 2006;101:109–16. Colombrita C, Lombardo G, Scapagnini G, Abraham NG. Heme oxygenase-1 expression levels are cell cycle dependent. Biochem Biophys Res Commun 2003;308:1001–8. Hoekstra KA, Godin DV, Kurtu J, Cheng KM. Effects of oxidantinduced injury on heme oxygenase and glutathione in cultured aortic endothelial cells from atherosclerosis-susceptible and -resistant Japanese quail. Mol Cell Biochem 2003;254:61–71. Hsu JT, Kan WH, Hsieh CH, et al. Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Aktdependent HO-1 up-regulation. J Leukoc Biol 2007. July 26. Yet SF, Layne MD, Liu X, et al. Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodelling. FASEB J 2003;17:1759–61. Daugherty A, Rateri DL, Lu H, Inagami T, Cassis LA. Hypercholesterolemia stimulates angiotensin peptide synthesis and contributes to atherosclerosis through the AT1A receptor. Circulation 2004;110:3849–57. Sales VL, Sukhova GK, Lopez-Ilasaca MA, et al. Angiotensin type 2 receptor is expressed in murine atherosclerotic lesions and modulates lesion evolution. Circulation 2005;112:3328–36. Johansson ME, Wickman A, Fitzgerald SM, Gan LM, Bergstrom G. Angiotensin II, type 2 receptor is not involved in the angiotensin II-mediated pro-atherogenic process in ApoE−/− mice. J Hypertens 2005;23:1541–9.