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Vasomotor dysfunction in the thoracic aorta of Marfan syndrome is associated with accumulation of oxidative stress
Vascular Pharmacology 52 (2010) 37–45
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Vascular Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / v p h
Vasomotor dysfunction in the thoracic aorta of Marfan syndrome is associated with accumulation of oxidative stress H.H. Clarice Yang, Cornelis van Breemen, Ada W.Y. Chung ⁎ Department of Cardiovascular Science, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada
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Article history: Received 4 June 2009 Received in revised form 5 October 2009 Accepted 14 October 2009 Keywords: Marfan syndrome Oxidative stress Thoracic aorta Contractile function Endothelium-dependent relaxation
a b s t r a c t We have described that the progression of thoracic aortic aneurysm in Marfan syndrome is accompanied with aortic vascular dysfunction. In the present study, we hypothesized that the impaired contractile function and endothelial-dependent relaxation could be resulted from oxidative stress in the thoracic aorta. Adrenergic contraction and cholinergic relaxation of thoracic aortae from mice (n = 40; age = 3, 6, 9 months) heterozygous for FBN1 allele (Fbn1C1039G/+), a well-defined model of Marfan syndrome, were compared with those from control (n = 40). The aortic 8-isoprostane level, an oxidative stress marker, was 32–50% greater in the Marfan group than in the control. Pre-incubation with superoxide dismutase (SOD) improved the phenylephrine-induced contraction and the sensitivity to acetylcholine in Marfan aortae, but not in controls. The phenylephrine-contraction in Marfan aortae was potentiated by 1400 W, an inducible nitric oxide synthase (iNOS) inhibitor, and allopurinol, a xanthine oxidase inhibitor. Acetylcholine-induced relaxation was restored by apocynin, an inhibitor of NAD(P)H oxidase. Protein expression of SOD-1 and SOD-2 was decreased in Marfan aortae, whereas that of xanthine oxidase, iNOS, and the enzymatic subunits of NAD(P)H oxidase was increased. The vasomotor dysfunction in Marfan thoracic aortae could be associated with accumulation of oxidative stress due to unbalanced protein expression of superoxide-producing and superoxide-eliminating enzymes. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Thoracic aortic aneurysm leading to dissection and rupture is the most life-threatening complication of Marfan syndrome (Judge and Dietz, 2005). We have demonstrated clearly that the progression of thoracic aortic aneurysm is associated with a pronounced impairment of aortic contractile function and reduction of nitric oxide (NO)mediated endothelial-dependent relaxation (Chung et al., 2007a,b,c, 2008a,b). Such vasomotor dysfunction might determine the susceptibility of aneurysm formation (Chew et al., 2004). Vasomotor function is tightly regulated by reactive oxygen species (ROS) (Lounsbury et al., 2000; Gutterman et al., 2005; Lee and Griendling, 2008). At low concentration, ROS regulates vascular tone, proliferation, and cell signaling (Lee and Griendling, 2008; Faraci and Didion, 2004). However, excessive amount of ROS, termed oxidative stress, is associated with the pathogenesis of cardiovascular diseases including hypertension, atherosclerosis, diabetes, and chronic kidney disease (Lee and Griendling, 2008; Faraci and Didion, 2004; Cai and Harrison, 2000). The elevation of ROS is caused by an imbalance
⁎ Corresponding author. Cardiovascular Science, Room 2099, 950 28th W Ave, Vancouver, British Columbia, Canada V5Z 4H4. Tel.: +1 604 875 3852; fax: +1 604 875 3120. E-mail address: [email protected] (A.W.Y. Chung). 1537-1891/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2009.10.005
between the production and neutralization of ROS (Cai and Harrison, 2000). All cell types in the vasculature contain enzymes that generate ROS (Schulz et al., 2004). Among the many potential pro-oxidant enzymes, NAD(P)H oxidase, xanthine oxidase, and nitric oxide synthase (NOS) are the most well-studied and are also believed to play a dominant role in vascular diseases (Cai and Harrison, 2000). Superoxide dismutase (SOD) is believed to be the main endogenous antioxidant responsible for superoxide removal (Faraci and Didion, 2004; Didion et al., 2002). It has been well-recognized that oxidative stress is associated with endothelial dysfunction in cardiac and vascular diseases (Johnstone et al., 1993; Panza et al., 1995). Endothelial dysfunction is commonly described as the impairment of endothelium-dependent vasorelaxation caused by a loss of NO bioavailability in the vasculature (Cai and Harrison, 2000; Schulz et al., 2004). We have shown that in the aorta of a mouse model of Marfan syndrome, NO mediated endotheliumdependent relaxation was impaired (Chung et al., 2007a). However, it is unclear whether the impairment of relaxation could result from oxidative stress. ROS has been reported to impede calcium signaling, which consequently leads to a reduction in vascular contractility (Lounsbury et al., 2000; Sener et al., 2004). It was reported that preservation of contraction affords protection against aneurysm formation in the abdominal aorta (Chew et al., 2004). The compromised contractile function in the Marfan thoracic aorta (Chung et al., 2007a,b, 2008a),
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which likely increases their susceptibility to aneurysm formation, might be the result of the excessive oxidative stress. In the present study, we hypothesized that the impaired contractile function and endothelial-dependent relaxation observed in the thoracic aorta of Marfan syndrome would be resulted from the elevation of oxidative stress during the disease progression. We demonstrated that the increase in oxidative stress could be associated with the unbalanced protein expression of superoxide-producing and superoxide-eliminating enzymes.
2. Methods 2.1. Experimental animals and tissue preparation Heterozygous (Fbn1C1039G/+) mice were mated to C57BL/6 mice to produce equal numbers of Fbn1C1039G/+ ‘Marfan’ subjects (n = 40) and wild-type ‘control’ (n = 40) (Chung et al., 2007a,b,c, 2008a,b). Both strains were housed in the institutional animal facility (University of British Columbia, Child and Family Research Institute) under standard animal room conditions, and all animal procedures were approved by the institutional Animal Ethics Board. Mice at age 3 (n = 30), 6 (n = 30) and 9 (n = 30) months were anesthetized with a mixture of ketamine hydrochloride (80 mg kg− 1) and xylazine hydrochloride (12 mg kg− 1) intraperitoneally. Given that severe aneurysm is found mainly in the ascending thoracic aorta and the aortic arch, both parts were dissected and examined in this study (Chung et al., 2007a,b, 2008a,b, Habashi et al., 2006).
2.2. Measurement of isoprostanes (8-isoprostane) Whole blood was collected via cardiac puncture and plasma was separated by centrifugation. 8-Isoprostane (8-epi-PGF2α) level was determined in plasma and aortic homogenate (ascending aorta = 1.2 mm; arch = 5 mm in length) using an enzyme immunoassay kit according to the manufacturer's procedures. 2.3. Measurement of isometric force Aortic arch segment (1.8 mm in length) was mounted isometrically in a small vessel myograph (A/S Danish Myotechnology, Aarhus N, Denmark) for force generation measurement (Chung et al., 2007a,b,c). Aortic segment was stretched to the resting tension (6.0 mN) for 20 min and challenged twice with 60 mM KCl before experiments were continued. To assess whether removal of superoxide affected contractile function, aortic segments were pre-incubated with SOD (150 U mL− 1) or SOD plus catalase (1000 U mL− 1) for 30 min before the addition of phenylephrine (1 nM–3 µM). After sustained pre-contraction was obtained, cumulative concentrations of acetylcholine (ACh; 1 nM– 100 µM) were added. Aortic segments were also pre-incubated with three inhibitors that block the potential superoxide-generating enzymes: the xanthine oxidase inhibitor allopurinol (300 µM), the NAD(P)H oxidase inhibitor apocynin (100 µM), and the inducible NOS (iNOS) inhibitor 1400 W (1 µM). They have all been shown to be selective towards the targeted proteins. Specifically, apocynin blocks the translocation of the regulatory unit of NADPH oxidase to the catalytic component; allopurinol is an allosteric inhibitor of xanthine oxidase; 1400 W is a tightly bound competitive inhibitor.
Fig. 1. Bar graph presenting the levels of isoprostane 8-epi-PGF2α in the (A) aortic homogenate and (B) plasma from control and Marfan group. (n = 4, ⁎p < 0.05, compared with the age-matched control. #p < 0.05, compared between 3 and 9 months control). (C) Western immunoblots showing the protein expression of SOD-1, SOD-2, xanthine oxidase, gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, iNOS, and GADPH in the Marfan and control aorta during aging (3, 6, and 9 months old). The densitometric analysis is shown in Table 1.
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2.4. Western immunoblotting
Table 1 Desitometric analysis of western immunoblots. Age (months)
3
6
9
Marfan SOD-1 SOD-2 Xanthine oxidase gp91phox p47phox p67phox iNOS
0.524 ± 0.062⁎ 0.187 ± 0.017⁎ 0.280 ± 0.031 0.125 ± 0.015 1.259 ± 0.113⁎ 0.558 ± 0.049⁎ 0.207 ± 0.018⁎
0.410 ± 0.029⁎ 0.155 ± 0.025 0.829 ± 0.094⁎ 1.045 ± 0.149⁎ 0.468 ± 0.055 0.477 ± 0.042⁎ 0.142 ± 0.010⁎
0.622 ± 0.059 0.120 ± 0.013⁎ 0.350 ± 0.041 0.967 ± 0.058 0.502 ± 0.042⁎ 0.444 ± 0.035⁎ 0.185 ± 0.012⁎
Control SOD-1 SOD-2 Xanthine oxidase gp91phox p47phox p67phox iNOS
0.946 ± 0.085 0.352 ± 0.026 0.231 ± 0.018 0.108 ± 0.007 0.465 ± 0.026 0.407 ± 0.035 0.052 ± 0.002
0.655 ± 0.079 0.220 ± 0.032 0.224 ± 0.017 0.699 ± 0.048 0.420 ± 0.036 0.331 ± 0.030 0.094 ± 0.008
0.569 ± 0.046 0.433 ± 0.039 0.319 ± 0.025 0.822 ± 0.075 1.187 ± 0.103 1.476 ± 0.117 0.095 ± 0.006
Data are expressed as ratio of interested protein to GADPH in the Marfan (n = 6) and control aortae (n = 10) during aging (3, 6 and 9 months). Data were reported as mean ± s.e.mean. Difference between control and Marfan mice at the same age group was analyzed by Student's t-test. ⁎ p ≤ 0.05, compared with the age-matched control.
To verify the importance of pharmacological agents acting on oxidative stress pathway, we also incubated vessels with irrelevant proteins, for example, purified matrix metalloproteinase-1 and angiostatin, as a control.
The procedures of protein homogenization and western immunoblotting were previously described (Chung et al., 2007a,b,c, 2008a,b). In brief, 40 µg of protein samples was separated on 6% (for iNOS and xanthine oxidase), 9% (for p67phox and gp91phox) or 13% (for p47phox, SOD-1 and -2) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were incubated with primary antibodies (dilution 1:200–1:800), then IgG peroxidase-conjugated secondary antibodies (dilution 1:2500). To ensure equal loading, membranes were stripped and blotted with anti-GADPH antibody (dilution 1:1000). Densitometric analysis was performed and normalized to the expression of GADPH of the same sample. 2.5. Materials Ketamine hydrochloride and xylazine hydrochloride (Research Biochemicals International, Natick, MA); phenylephrine, acetylcholine, catalase, allopurinol, 1400 W, potassium chloride, chemicals for preparing Krebs solution, rabbit anti-SOD1 and SOD-2 primary antibodies, antirabbit and mouse IgG peroxidase-conjugated secondary antibodies (Sigma-Aldrich, Oakville, ON); apocynin, SOD, matrix metalloproteinase-1 and angiostatin (Calbiochem, San Diego, CA); rabbit anti-xanthine oxidase, anti-p47phox, anti-p67phox, and mouse anti-iNOS primary antibody (Santa Cruz, Santa Cruz, CA); mouse anti-gp91phox primary antibody (BD Biosciences, Mississauga, ON); rabbit polyclonal antiGADPH antibody (Immunechem, BC, Canada); ECL western blotting
Fig. 2. Effect of superoxide dismutase (SOD, 150 U mL− 1) and SOD plus catalase (1000 U mL− 1) on phenylephrine-stimulated contraction in aortic arch. Upper panels showed (A) Emax and (B) pEC50 in response to phenylephrine in the presence and absence of SOD or SOD-plus-catalase pre-incubation at the age of 9 months. (C) and (D) are the concentration–response curves. (n = 5–7, ⁎p < 0.05, compared with control in the absence of the enzymes, #p < 0.05, compared with the responses in the absence of the enzymes in their respective groups).
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detection kit (Amersham Life Sciences, Arlington Heights, IL); 8isoprostane enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). 2.6. Statistics Percent of maximum contraction was determined by normalizing the increase in force from baseline at each concentration of phenylephrine with the maximal contractile force (Emax). Percent relaxation was calculated as the percent decrease in force with respect to the initial phenylephrine-induced pre-contraction, and the percent relaxation was used to construct the concentration–response curves of ACh-induced relaxation. To compare the sensitivities of the vessels to the agonists in the presence of different inhibitors, the negative logarithm (pD2) of the concentration of phenylephrine or ACh giving 50% of maximum response (pEC50) was determined by linear interpolation on the semilogarithm concentration–response curve [pD2 = −Log(EC50)]. Maximal contraction and relaxation responses as well as pEC50 values were compared by Student's un-paired t-test. Statistical significance was defined as p < 0.05. Data were reported as mean± s.e.mean. Statistical analysis and construction of concentration response curves were performed using GraphPad Prism software (San Diego, CA). 3. Results
age was 50% higher than that in the control (p ≤ 0.001). This difference disappeared at 9 months (Fig. 1B). To elucidate the effect of aging, in the control at 9 months of age, we found that the plasma and aortic homogenate isoprostane 8-epi-PGF2α level was significantly (p = 0.03) increased by 70% and 50%, respectively, compared with that at 3 months. 3.2. Imbalanced protein expression of superoxide-generating and degrading enzymes in the Marfan aorta The expression of SOD-1 (copper–zinc SOD or cytosolic SOD) in the Marfan aorta was comparable to that of the age-matched control at 9 months, but was significantly less at 3 and 6 months. SOD-2 (manganese SOD or mitochondrial SOD) expression was similar between groups at 6 months, but was lower in the Marfan group at 3 and 9 months (Fig. 1C, Table 1). In the Marfan aorta, iNOS protein expression was 298, 51, and 95% higher than that of the control at 3, 6, and 9 months, respectively. Xanthine oxidase is also increased by 270% at 6 months Marfan aorta (Fig. 1C, Table 1). The gp91phox, the catalytic subunit of NAD(P)H oxidase, was markedly increased in the Marfan aorta at 6 months old. With regard to the regulatory subunits, the expression of p47phox was elevated at 3 months and that of p67phox was elevated at 3 and 6 months in the Marfan group. However, at 9 months, the expression of both subunits was higher in the control (Fig. 1C).
3.1. Increased isoprostane 8-epi-PGF2α level in Marfan mice 3.3. Pre-incubation with SOD and catalase From 3 months on, Marfan aorta had elevated isoprostane 8-epiPGF2α compared with the age-matched control by 32–50% (Fig. 1A). The plasma isoprostane 8-epi-PGF2α level in Marfan mice 3 and 6 months of
To reveal the influence of superoxide on phenylephrine-induced contraction, we pre-incubated the aortic arch with SOD, an enzyme
Fig. 3. Effect of superoxide dismutase (SOD, 150 U mL− 1) and SOD plus catalase (1000 U mL− 1) on acetylcholine (ACh)-induced relaxation in aortic arch. Upper panels showed (A) Emax and (B) pEC50 at 6 months in response to ACh in the presence and absence of SOD or SOD plus catalase. (C) and (D) are the concentration–response curves. (n = 7, ⁎p < 0.05, compared with control in the absence of the enzymes, #p < 0.05, compared with the responses in the absence of the enzymes in their respective groups).
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that converts superoxide to H2O2, before the addition of phenylephrine. We previously showed that control and Marfan aorta demonstrated a difference in the phenylephrine-contraction only at 9 months of age (Chung et al., 2007a), and thus the effect of superoxide removal on contraction was examined in this age group. Pretreatment of SOD potentiated the contraction in the Marfan aorta and increased the Emax and the pEC50, returning both values to the control level. In contrast, SOD had no effect on phenylephrine-induced contraction in the control aorta (Fig. 2). The participation of H2O2, produced by SOD, in the alteration of phenylephrine response was further assessed with the co-incubation of SOD and catalase. In physiological conditions, SOD converts superoxide to H2O2, which is subsequently converted to oxygen and water by catalase. In Marfan aorta, the effect of SOD plus catalase was not different from that of SOD alone; like SOD, the combination treatment also significantly improved the Emax and pEC50 of phenylephrine on the Marfan aorta (Fig. 2). Impaired endothelium-dependent ACh-stimulated relaxation was previously observed in the Marfan aorta at 3 and 6 months of age (Chung et al., 2007a); therefore, in this study, the effect of oxidative stress on endothelium-dependent relaxation was assessed at these two ages. At 6 months of age, SOD and SOD-plus-catalase combination treatment in the Marfan aorta restored the ACh-maximal relaxation and ACh-pEC50 to the control levels (Fig. 3). At 3 (data now shown) and 6 months, both SOD and SOD-plus-catalase combination did not significantly alter the ACh-response in the control aorta. 3.4. Blockade of xanthine oxidase with allopurinol improved AChinduced relaxation in the Marfan aorta Allopurinol, a xanthine oxidase inhibitor, did not significantly alter phenylephrine-contraction in control aortic arch at 9 months of age. A trend of improvement was observed in the Marfan aorta although it did not reach statistical significance (Fig. 4A,B). With regard to ACh-
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mediated relaxation, allopurinol had no effect on both control and Marfan aorta at 3 months (data not shown). At 6 months old, although allopurinol had no significant effect on the ACh-Emax, it increased the pEC50 values from 6.34 to 7.65 (Fig. 4D). Allopurinol did not cause significant changes in the ACh-relaxation (Fig. 4C) in the control aortic arch.
3.5. Blockade of NAD(P)H oxidase with apocynin improved ACh-induced relaxation in the Marfan aorta Apocynin, an NAD(P)H oxidase inhibitor, did not cause significant changes in phenylephrine-Emax and pEC50 at 9 months old in both control and Marfan aortic arch (Fig. 5A,B). For the ACh-stimulated relaxation, apocynin did not have an effect on both Emax and pEC50 at 3 months in both groups (data not shown). However, at 6 months, apocynin increased the Emax from 46 to 77% and pEC50 from 6.34 to 7.49 in the Marfan arch whereas in the control, no significant changes were observed (Fig. 5C,D).
3.6. Blockade of iNOS with 1400 W improved vasomotor function in the Marfan aorta The specific iNOS inhibitor, 1400 W, altered phenylephrine and ACh responses in both control and Marfan aorta. At 9 months of age, 1400 W increased phenylephrine-Emax in both control and Marfan arch by 52.0 and 150%, respectively (Fig. 6A,B); however, the pEC50 values were not significantly changed in both groups. With respect to ACh-induced relaxation, 1400 W had no effect on control aorta at 3 months but increased the Emax from 60.5 to 90.2% in the Marfan arch (data not shown). At 6 months, 1400 W did not alter the AChrelaxation significantly in the control whereas ACh-Emax was increased from 57 to 86% in the Marfan arch (Fig. 6C,D).
Fig. 4. Effects of allopurinol (a xanthine oxidase inhibitor; 300 µM) on (A, B) phenylephrine-mediated contraction at 9 months, and (C, D) ACh-induced relaxation at 6 months in control and Marfan aortic arch. (n = 7; ⁎p < 0.05 vs no treatment).
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Fig. 5. Effects of apocynin (an NAD(P)H oxidase inhibitor; 100 µM) on (A, B) phenylephrine-mediated contraction at 9 months, and (C, D) ACh-induced relaxation at 6 months in control and Marfan aortic arch (n = 7; ⁎p < 0.05 vs no treatment).
Fig. 6. Effects of 1400 W (an iNOS inhibitor; 1 µM) on (A, B) phenylephrine-induced contraction at 9 months, and (C, D) ACh-mediated relaxation at 6 months in control and Marfan aortic arch (n = 8, ⁎p < 0.05 vs no treatment).
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Fig. 7. Concentration–response curves showing the effects of incubating with purified 0.5 µg/mL matrix metalloproteinase-1, and 1.5 µg/mL angiostatin, on (A, B) phenylephrinemediated contraction and (C, D) ACh-induced relaxation in control and Marfan aorta at 6 months of age.
Note that the incubation with irrelevant proteins matrix metalloproteinase-1 and angiostatin did not modify vasomotor function of both strains (Fig. 7). 4. Discussion The current study is the first to show the elevation of oxidative stress in the thoracic aorta of a mouse model of Marfan syndrome. The presence of oxidative stress, may be attributed to the downregulation of SOD and upregulation of superoxide-producing enzymes and could impair vasomotor function in Marfan aortae. However, it could be improved by the pre-incubation of the superoxide-eliminating enzymes (i.e. SOD, catalase) and the blockade of the superoxidegenerating enzymes (i.e. apocynin, allopurinol, 1400 W). Therefore, the present study suggests that by reducing oxidative stress level by pharmacological strategies, it could possibly improve aortic vasocontractile function and endothelium-dependent relaxation, the two crucial factors determining the susceptibility of aneurysm formation. The isoprostane 8-epi-PGF2α assay is a well-established method to provide a reliable assessment of oxidative injury in vivo (Hoffman et al., 1996; Patrono and FitzGerald, 1997; Delanty et al., 1996). Increased formation of F2 isoprostanes has been reported to be associated with vascular diseases such as vascular reperfusion, diabetes mellitus and hypercholesterolemia (Patrono and FitzGerald, 1997; Delanty et al., 1996). In the plasma and aortic homogenate of Marfan mice, significantly increased levels of isoprostane 8-epi-PGF2α were observed compared with the control, suggesting that oxidative stress was elevated by the disorder. The isoprostane 8-epi-PGF2α level was also found to be increased with age. It has been well-established that oxidative stress is associated with aging in the vasculature (Donato et al., 2007; Hamilton et al., 2001; Taddei et al., 2001). In healthy people, it was found that endothelial dysfunction and increased oxidative stress biomarkers in the aged subjects could be
linked to increased NAD(P)H oxidase expression (Donato et al., 2007). In the control mice, we also observed a significant increase in the expression of NAD(P)H oxidase subunits at 9 months, which may contribute to the increased aortic homogenate 8-epi-PGF2α level. We have shown that Marfan syndrome and the progression of thoracic aortic aneurysm were accompanied by a pronounced impairment of aortic contractile function (Chung et al., 2007a,b). The pre-incubation of SOD alone or a combination of SOD and catalase normalized the contractile response of Marfan aorta to the control level. This further reconfirmed the presence of oxidative stress, and demonstrated the impact of excess superoxide on vasoconstriction in Marfan syndrome. Oxidative stress has been reported to be involved in the pathogenesis of various cardiovascular diseases. SOD treatment was shown to reverse the hypersensitivity of the arteries in diabetic and hypertensive animal models and normalize the agonist-induced contraction to that of the control animals (Kanie and Kamata, 2000; Alvarez et al., 2008). In the rat model of chronic renal failure, similar to the present study, phenylephrine-induced contraction was compromised in the aorta but could be restored to the control level with an antioxidant, melatonin (Sener et al., 2004). Although the mechanism of action of ROS on smooth muscle cell contractility is still unclear (Lyle and Griendling, 2006), ROS has been proposed to have multiple effects on calcium signaling in both vascular endothelial and smooth muscle cells (Lounsbury et al., 2000; Elmoselhi et al., 1996; Walia et al., 2000). The impairment of the calcium signaling pathway caused by oxidative stress may consequently lead to the alteration of vascular reactivity (Lyle and Griendling, 2006). Therefore, the removal of superoxide with SOD and catalase may restore calcium signaling and thereby the contractile responses. In the Marfan aorta, reduced endothelial-dependent relaxation could also be reversed by addition of SOD or SOD-plus-catalase. The similar effects caused by SOD treatment alone or the combination of
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SOD and catalase suggest that the improvement of endothelial function was not due to the vaso-relaxant effect of the H2O2, but rather the removal of superoxide anions. Oxidative stress may cause endothelial dysfunction through several direct and indirect pathways, the most well-known of which is the scavenging of NO by superoxide. Superoxide radicals bind to NO at a rate three times faster than they bind to SOD; therefore, excess superoxide production would increase the rate of NO degradation (Cai and Harrison, 2000; Schulz et al., 2004). In addition, peroxynitrite, the product of the reaction between superoxide and NO, could cause the uncoupling of eNOS by oxidizing tetrahydrobiopterin. The uncoupled eNOS not only reduces the production of NO, but is also another important source of superoxide (Schulz et al., 2004). In hypercholesterolemia, diabetes, heart failure, hypertension and chronic renal disease, endothelial function has been shown to be improved by antioxidants, such as SOD, melatonin, and vitamins C and E (Schulz et al., 2004; Sener et al., 2004; Miyagawa et al., 2007). Therefore, in addition to reduced NO production caused by downregulation of eNOS and reduction of Akt phosphorylation (Chung et al., 2007a), the present study indicated that oxidative stress may be another contributor to endothelial dysfunction in Marfan syndrome. Oxidative stress could be associated with reduced superoxide neutralization, which could result from the downregulation of SOD-1 and -2. SOD is one of the most important endogenous superoxideneutralizing enzymes in the vasculature (Faraci and Didion, 2004; Cai and Harrison, 2000; Didion et al., 2002). In the mouse aorta, among the three isoforms of SOD, SOD-1 and SOD-2 account for 50–80% and 2–12% of SOD composition, respectively (Faraci and Didion, 2004). Therefore, the decreased expression of these two isoforms in Marfan aortae would likely cause a reduction in superoxide removal. In the streptozotocin-induced diabetic rat, decreased SOD expression has been shown to be associated with vascular dysfunction caused by superoxide anion (Kamata and Kobayashi, 1996). In the mouse models of both SOD-1 and SOD-2 deficient mice, basal superoxide level in the vasculature was elevated and ACh-mediated relaxation was impaired (Didion et al., 2002; Brown et al., 2007). Therefore, the impairment of ACh-induced relaxation and phenylephrine-mediated contraction in Marfan aortae may be attributed to the downregulation of SOD. We used various pharmacological inhibitors of superoxidegenerating enzymes to determine the mechanisms through which superoxide level was elevated in the Marfan aorta. Xanthine oxidase is an enzyme that catalyses the oxidation of hypoxanthine and xanthine and produces O2− and H2O2 during purine metabolism (Schulz et al., 2004). Inhibition of the upregulated enzyme with allopurinol improved both contraction and relaxation in the Marfan aorta. In patients with hypercholesterolemia and chronic heart failure, similar observations have been made where the administration of oxypurinol and allopurinol reduced oxidative stress and improved endothelial function (Cardillo et al., 1997; George et al., 2006). NAD(P)H oxidase, a membrane-associated enzyme which catalyzes reduction of oxygen with electrons from NADH or NADPH, is the major contributor of superoxide anions in the vasculature (Jiang et al., 2004; Yokoyama et al., 2000). Upregulation of NAD(P)H oxidase is found to be associated with various animal models of hypertension and to cause impairment of NO-dependent vaso-dilatation in patients with coronary artery disease (Jiang et al., 2004). Apocynin, an NAD(P)H oxidase inhibitor, normalized ACh-mediated relaxation in the Marfan aorta, but had no effect on contraction. This can be related to the temporal changes of the protein expression of different NAD(P)H oxidase subunits. The selective iNOS inhibitor, 1400 W, greatly improved the responses to phenylephrine and acetylcholine in the Marfan aorta. It is believed that induction of iNOS produces excessive NO as well as ROS (Liu et al., 2005). Thus, the upregulated iNOS expression in the Marfan aorta at all ages, which may be a compensatory mechanism for the reduced NO bioavailability (Vaziri
et al., 2000; Chung et al., 2007a), may be another contributor to oxidative stress. We acknowledge limitations in this study. First, all functional experiments were performed on the aortic arch but not the ascending part due to its very short length. Most of the aneurysm in human with Marfan syndrome is evidenced in the aortic root and ascending aorta. The differences between two strains in oxidative stress level, enzyme expression as well as aberrant vasomotor function presented herein might be more pronounced if we used the ascending aorta. Therefore, our results raise interesting questions for further testing using more sophisticated and appropriate methodology, or in bigger animal models. Second, in the present study, endothelial dysfunction is identified as impaired vasodilation in response to acetylcholine. However, a broader understanding of the term should also include the proinflammatory and prothrombic state. Third, there are over 600 mutations identified to cause Marfan syndrome. While missense mutations account for 60% of the mutations, 78% of the point mutations locate in the cbEGF modules. A further 12% of these mutations are recurrent and affect a mutation hotspot, CpG., for a cysteine residue (Judge and Dietz, 2005). This type of mutation is the basis of the mouse model used in this study, and represents the most common mutation in classic Marfan syndrome (Habashi et al., 2006). We therefore find that although our model is useful to investigate the general pathogenesis of Marfan syndrome, it may not be representative of all cases of Marfan syndrome in human. 5. Conclusion We used various pharmacological agonists and inhibitors to demonstrate the impact of oxidative stress on the thoracic aorta of Marfan syndrome. The elevated oxidative stress could be due to reduced expression of SOD and increased expression of iNOS, NAD(P) H oxidase, and xanthine oxidase at different ages. Since vasomotor function in part determines the susceptibility to aneurysm formation, normalization of vasoconstriction and relaxation with the use of antioxidants could suggest a novel pharmacological strategy in the treatment of thoracic aortic aneurysm in Marfan syndrome. Acknowledgements This study was supported by an operating grant from the Canadian Institutes of Health Research. H.H.C.Y. is the recipient of Michael Smith Foundation for Health Research Trainee Award and NSERC Canada Graduate Scholarship. A.W.Y.C. is the recipient of Michael Smith Foundation for Health Research/St Paul's Hospital Foundation Trainee Award. References Alvarez, Y., Briones, A.M., Hernanz, R., Perez-Giron, J.V., Alonso, M.J., Salaices, M., 2008. Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats. Br. J. Pharmacol. 153, 926–935. Brown, K.A., Didion, S.P., Andresen, J.J., Faraci, F.M., 2007. Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency. Arterioscler. Thromb. Vasc. Biol. 27, 1941–1946. Cai, H., Harrison, D.G., 2000. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ. Res. 87, 840–844. Cardillo, C., Kilcoyne, C.M., Cannon III, R.O., Quyyumi, A.A., Panza, J.A., 1997. Xanthine oxidase inhibition with oxypurinol improves endothelial vasodilator function in hypercholesterolemic but not in hypertensive patients. Hypertension 30, 57–63. Chew, D.K., Conte, M.S., Khalil, R.A., 2004. Matrix metalloproteinase-specific inhibition of Ca2+ entry mechanisms of vascular contraction. J. Vasc. Surg. 40, 1001–1010. Chung, A.W., Au Yeung, K., Cortes, S.F., Sandor, G.S., Judge, D.P., Dietz, H.C., van Breemen, C., 2007a. Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome. Br. J. Pharmacol. 150, 1075–1083. Chung, A.W., Au Yeung, K., Sandor, G.G., Judge, D.P., Dietz, H.C., van Breemen, C., 2007b. Loss of elastic fiber integrity and reduction of vascular smooth muscle contraction resulting from the upregulated activities of matrix metalloproteinase-2 and -9 in the thoracic aortic aneurysm in Marfan syndrome. Circ. Res. 101, 512–522.
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Johnstone, M.T., Creager, S.J., Scales, K.M., Cusco, J.A., Lee, B.K., Creager, M.A., 1993. Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. Circulation 88, 2510–2516. Judge, D.P., Dietz, H.C., 2005. Marfan's syndrome. Lancet 366, 1965–1976. Kamata, K., Kobayashi, T., 1996. Changes in superoxide dismutase mRNA expression by streptozotocin-induced diabetes. Br. J. Pharmacol. 119, 583–589. Kanie, N., Kamata, K., 2000. Contractile responses in spontaneously diabetic mice. I. Involvement of superoxide anion in enhanced contractile response of aorta to norepinephrine in C57BL/KsJ(db/db) mice. Gen. Pharmacol. 35, 311–318. Lee, M.Y., Griendling, K.K., 2008. Redox signaling, vascular function, and hypertension. Antioxid. Redox Signal. 10, 1045–1059. Liu, Y.H., Carretero, O.A., Cingolani, O.H., Liao, T.D., Sun, Y., Xu, J., Li, L.Y., Pagano, P.J., Yang, J.J., Yang, X.P., 2005. Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction. Am. J. Physiol., Heart Circ. Physiol. 289, H2616–H2623. Lounsbury, K.M., Hu, Q., Ziegelstein, R.C., 2000. Calcium signaling and oxidant stress in the vasculature. Free Radic. Biol. Med. 28, 1362–1369. Lyle, A.N., Griendling, K.K., 2006. Modulation of vascular smooth muscle signaling by reactive oxygen species. Physiology (Bethesda) 21, 269–280. Miyagawa, K., Ohashi, M., Yamashita, S., Kojima, M., Sato, K., Ueda, R., Dohi, Y., 2007. Increased oxidative stress impairs endothelial modulation of contractions in arteries from spontaneously hypertensive rats. J. Hypertens. 25, 415–421. Panza, J.A., Garcia, C.E., Kilcoyne, C.M., Quyyumi, A.A., Cannon III, R.O., 1995. Impaired endothelium-dependent vasodilation in patients with essential hypertension. evidence that nitric oxide abnormality is not localized to a single signal transduction pathway. Circulation 91, 1732–1738. Patrono, C., FitzGerald, G.A., 1997. Isoprostanes: potential markers of oxidant stress in atherothrombotic disease. Arterioscler. Thromb. Vasc. Biol. 17, 2309–2315. Schulz, E., Anter, E., Keaney Jr, J.F., 2004. Oxidative stress, antioxidants, and endothelial function. Curr. Med. Chem. 11, 1093–1104. Sener, G., Paskaloglu, K., Toklu, H., Kapucu, C., Ayanoglu-Dulger, G., Kacmaz, A., Sakarcan, A., 2004. Melatonin ameliorates chronic renal failure-induced oxidative organ damage in rats. J. Pineal Res. 36, 232–241. Taddei, S., Virdis, A., Ghiadoni, L., Salvetti, G., Bernini, G., Magagna, A., Salvetti, A., 2001. Agerelated reduction of NO availability and oxidative stress in humans. Hypertension 38, 274–279. Vaziri, N.D., Ni, Z., Oveisi, F., Trnavsky-Hobbs, D.L., 2000. Effect of antioxidant therapy on blood pressure and NO synthase expression in hypertensive rats. Hypertension 36, 957–964. Walia, M., Sormaz, L., Samson, S.E., Lee, R.M., Grover, A.K., 2000. Effects of hydrogen peroxide on pig coronary artery endothelium. Eur. J. Pharmacol. 400, 249–253. Yokoyama, M., Inoue, N., Kawashima, S., 2000. Role of the vascular NADH/NADPH oxidase system in atherosclerosis. Ann. N.Y. Acad. Sci. 902, 241–247.
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Vascular Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / v p h
Vasomotor dysfunction in the thoracic aorta of Marfan syndrome is associated with accumulation of oxidative stress H.H. Clarice Yang, Cornelis van Breemen, Ada W.Y. Chung ⁎ Department of Cardiovascular Science, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada
a r t i c l e
i n f o
Article history: Received 4 June 2009 Received in revised form 5 October 2009 Accepted 14 October 2009 Keywords: Marfan syndrome Oxidative stress Thoracic aorta Contractile function Endothelium-dependent relaxation
a b s t r a c t We have described that the progression of thoracic aortic aneurysm in Marfan syndrome is accompanied with aortic vascular dysfunction. In the present study, we hypothesized that the impaired contractile function and endothelial-dependent relaxation could be resulted from oxidative stress in the thoracic aorta. Adrenergic contraction and cholinergic relaxation of thoracic aortae from mice (n = 40; age = 3, 6, 9 months) heterozygous for FBN1 allele (Fbn1C1039G/+), a well-defined model of Marfan syndrome, were compared with those from control (n = 40). The aortic 8-isoprostane level, an oxidative stress marker, was 32–50% greater in the Marfan group than in the control. Pre-incubation with superoxide dismutase (SOD) improved the phenylephrine-induced contraction and the sensitivity to acetylcholine in Marfan aortae, but not in controls. The phenylephrine-contraction in Marfan aortae was potentiated by 1400 W, an inducible nitric oxide synthase (iNOS) inhibitor, and allopurinol, a xanthine oxidase inhibitor. Acetylcholine-induced relaxation was restored by apocynin, an inhibitor of NAD(P)H oxidase. Protein expression of SOD-1 and SOD-2 was decreased in Marfan aortae, whereas that of xanthine oxidase, iNOS, and the enzymatic subunits of NAD(P)H oxidase was increased. The vasomotor dysfunction in Marfan thoracic aortae could be associated with accumulation of oxidative stress due to unbalanced protein expression of superoxide-producing and superoxide-eliminating enzymes. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Thoracic aortic aneurysm leading to dissection and rupture is the most life-threatening complication of Marfan syndrome (Judge and Dietz, 2005). We have demonstrated clearly that the progression of thoracic aortic aneurysm is associated with a pronounced impairment of aortic contractile function and reduction of nitric oxide (NO)mediated endothelial-dependent relaxation (Chung et al., 2007a,b,c, 2008a,b). Such vasomotor dysfunction might determine the susceptibility of aneurysm formation (Chew et al., 2004). Vasomotor function is tightly regulated by reactive oxygen species (ROS) (Lounsbury et al., 2000; Gutterman et al., 2005; Lee and Griendling, 2008). At low concentration, ROS regulates vascular tone, proliferation, and cell signaling (Lee and Griendling, 2008; Faraci and Didion, 2004). However, excessive amount of ROS, termed oxidative stress, is associated with the pathogenesis of cardiovascular diseases including hypertension, atherosclerosis, diabetes, and chronic kidney disease (Lee and Griendling, 2008; Faraci and Didion, 2004; Cai and Harrison, 2000). The elevation of ROS is caused by an imbalance
⁎ Corresponding author. Cardiovascular Science, Room 2099, 950 28th W Ave, Vancouver, British Columbia, Canada V5Z 4H4. Tel.: +1 604 875 3852; fax: +1 604 875 3120. E-mail address: [email protected] (A.W.Y. Chung). 1537-1891/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2009.10.005
between the production and neutralization of ROS (Cai and Harrison, 2000). All cell types in the vasculature contain enzymes that generate ROS (Schulz et al., 2004). Among the many potential pro-oxidant enzymes, NAD(P)H oxidase, xanthine oxidase, and nitric oxide synthase (NOS) are the most well-studied and are also believed to play a dominant role in vascular diseases (Cai and Harrison, 2000). Superoxide dismutase (SOD) is believed to be the main endogenous antioxidant responsible for superoxide removal (Faraci and Didion, 2004; Didion et al., 2002). It has been well-recognized that oxidative stress is associated with endothelial dysfunction in cardiac and vascular diseases (Johnstone et al., 1993; Panza et al., 1995). Endothelial dysfunction is commonly described as the impairment of endothelium-dependent vasorelaxation caused by a loss of NO bioavailability in the vasculature (Cai and Harrison, 2000; Schulz et al., 2004). We have shown that in the aorta of a mouse model of Marfan syndrome, NO mediated endotheliumdependent relaxation was impaired (Chung et al., 2007a). However, it is unclear whether the impairment of relaxation could result from oxidative stress. ROS has been reported to impede calcium signaling, which consequently leads to a reduction in vascular contractility (Lounsbury et al., 2000; Sener et al., 2004). It was reported that preservation of contraction affords protection against aneurysm formation in the abdominal aorta (Chew et al., 2004). The compromised contractile function in the Marfan thoracic aorta (Chung et al., 2007a,b, 2008a),
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which likely increases their susceptibility to aneurysm formation, might be the result of the excessive oxidative stress. In the present study, we hypothesized that the impaired contractile function and endothelial-dependent relaxation observed in the thoracic aorta of Marfan syndrome would be resulted from the elevation of oxidative stress during the disease progression. We demonstrated that the increase in oxidative stress could be associated with the unbalanced protein expression of superoxide-producing and superoxide-eliminating enzymes.
2. Methods 2.1. Experimental animals and tissue preparation Heterozygous (Fbn1C1039G/+) mice were mated to C57BL/6 mice to produce equal numbers of Fbn1C1039G/+ ‘Marfan’ subjects (n = 40) and wild-type ‘control’ (n = 40) (Chung et al., 2007a,b,c, 2008a,b). Both strains were housed in the institutional animal facility (University of British Columbia, Child and Family Research Institute) under standard animal room conditions, and all animal procedures were approved by the institutional Animal Ethics Board. Mice at age 3 (n = 30), 6 (n = 30) and 9 (n = 30) months were anesthetized with a mixture of ketamine hydrochloride (80 mg kg− 1) and xylazine hydrochloride (12 mg kg− 1) intraperitoneally. Given that severe aneurysm is found mainly in the ascending thoracic aorta and the aortic arch, both parts were dissected and examined in this study (Chung et al., 2007a,b, 2008a,b, Habashi et al., 2006).
2.2. Measurement of isoprostanes (8-isoprostane) Whole blood was collected via cardiac puncture and plasma was separated by centrifugation. 8-Isoprostane (8-epi-PGF2α) level was determined in plasma and aortic homogenate (ascending aorta = 1.2 mm; arch = 5 mm in length) using an enzyme immunoassay kit according to the manufacturer's procedures. 2.3. Measurement of isometric force Aortic arch segment (1.8 mm in length) was mounted isometrically in a small vessel myograph (A/S Danish Myotechnology, Aarhus N, Denmark) for force generation measurement (Chung et al., 2007a,b,c). Aortic segment was stretched to the resting tension (6.0 mN) for 20 min and challenged twice with 60 mM KCl before experiments were continued. To assess whether removal of superoxide affected contractile function, aortic segments were pre-incubated with SOD (150 U mL− 1) or SOD plus catalase (1000 U mL− 1) for 30 min before the addition of phenylephrine (1 nM–3 µM). After sustained pre-contraction was obtained, cumulative concentrations of acetylcholine (ACh; 1 nM– 100 µM) were added. Aortic segments were also pre-incubated with three inhibitors that block the potential superoxide-generating enzymes: the xanthine oxidase inhibitor allopurinol (300 µM), the NAD(P)H oxidase inhibitor apocynin (100 µM), and the inducible NOS (iNOS) inhibitor 1400 W (1 µM). They have all been shown to be selective towards the targeted proteins. Specifically, apocynin blocks the translocation of the regulatory unit of NADPH oxidase to the catalytic component; allopurinol is an allosteric inhibitor of xanthine oxidase; 1400 W is a tightly bound competitive inhibitor.
Fig. 1. Bar graph presenting the levels of isoprostane 8-epi-PGF2α in the (A) aortic homogenate and (B) plasma from control and Marfan group. (n = 4, ⁎p < 0.05, compared with the age-matched control. #p < 0.05, compared between 3 and 9 months control). (C) Western immunoblots showing the protein expression of SOD-1, SOD-2, xanthine oxidase, gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, iNOS, and GADPH in the Marfan and control aorta during aging (3, 6, and 9 months old). The densitometric analysis is shown in Table 1.
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2.4. Western immunoblotting
Table 1 Desitometric analysis of western immunoblots. Age (months)
3
6
9
Marfan SOD-1 SOD-2 Xanthine oxidase gp91phox p47phox p67phox iNOS
0.524 ± 0.062⁎ 0.187 ± 0.017⁎ 0.280 ± 0.031 0.125 ± 0.015 1.259 ± 0.113⁎ 0.558 ± 0.049⁎ 0.207 ± 0.018⁎
0.410 ± 0.029⁎ 0.155 ± 0.025 0.829 ± 0.094⁎ 1.045 ± 0.149⁎ 0.468 ± 0.055 0.477 ± 0.042⁎ 0.142 ± 0.010⁎
0.622 ± 0.059 0.120 ± 0.013⁎ 0.350 ± 0.041 0.967 ± 0.058 0.502 ± 0.042⁎ 0.444 ± 0.035⁎ 0.185 ± 0.012⁎
Control SOD-1 SOD-2 Xanthine oxidase gp91phox p47phox p67phox iNOS
0.946 ± 0.085 0.352 ± 0.026 0.231 ± 0.018 0.108 ± 0.007 0.465 ± 0.026 0.407 ± 0.035 0.052 ± 0.002
0.655 ± 0.079 0.220 ± 0.032 0.224 ± 0.017 0.699 ± 0.048 0.420 ± 0.036 0.331 ± 0.030 0.094 ± 0.008
0.569 ± 0.046 0.433 ± 0.039 0.319 ± 0.025 0.822 ± 0.075 1.187 ± 0.103 1.476 ± 0.117 0.095 ± 0.006
Data are expressed as ratio of interested protein to GADPH in the Marfan (n = 6) and control aortae (n = 10) during aging (3, 6 and 9 months). Data were reported as mean ± s.e.mean. Difference between control and Marfan mice at the same age group was analyzed by Student's t-test. ⁎ p ≤ 0.05, compared with the age-matched control.
To verify the importance of pharmacological agents acting on oxidative stress pathway, we also incubated vessels with irrelevant proteins, for example, purified matrix metalloproteinase-1 and angiostatin, as a control.
The procedures of protein homogenization and western immunoblotting were previously described (Chung et al., 2007a,b,c, 2008a,b). In brief, 40 µg of protein samples was separated on 6% (for iNOS and xanthine oxidase), 9% (for p67phox and gp91phox) or 13% (for p47phox, SOD-1 and -2) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were incubated with primary antibodies (dilution 1:200–1:800), then IgG peroxidase-conjugated secondary antibodies (dilution 1:2500). To ensure equal loading, membranes were stripped and blotted with anti-GADPH antibody (dilution 1:1000). Densitometric analysis was performed and normalized to the expression of GADPH of the same sample. 2.5. Materials Ketamine hydrochloride and xylazine hydrochloride (Research Biochemicals International, Natick, MA); phenylephrine, acetylcholine, catalase, allopurinol, 1400 W, potassium chloride, chemicals for preparing Krebs solution, rabbit anti-SOD1 and SOD-2 primary antibodies, antirabbit and mouse IgG peroxidase-conjugated secondary antibodies (Sigma-Aldrich, Oakville, ON); apocynin, SOD, matrix metalloproteinase-1 and angiostatin (Calbiochem, San Diego, CA); rabbit anti-xanthine oxidase, anti-p47phox, anti-p67phox, and mouse anti-iNOS primary antibody (Santa Cruz, Santa Cruz, CA); mouse anti-gp91phox primary antibody (BD Biosciences, Mississauga, ON); rabbit polyclonal antiGADPH antibody (Immunechem, BC, Canada); ECL western blotting
Fig. 2. Effect of superoxide dismutase (SOD, 150 U mL− 1) and SOD plus catalase (1000 U mL− 1) on phenylephrine-stimulated contraction in aortic arch. Upper panels showed (A) Emax and (B) pEC50 in response to phenylephrine in the presence and absence of SOD or SOD-plus-catalase pre-incubation at the age of 9 months. (C) and (D) are the concentration–response curves. (n = 5–7, ⁎p < 0.05, compared with control in the absence of the enzymes, #p < 0.05, compared with the responses in the absence of the enzymes in their respective groups).
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detection kit (Amersham Life Sciences, Arlington Heights, IL); 8isoprostane enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). 2.6. Statistics Percent of maximum contraction was determined by normalizing the increase in force from baseline at each concentration of phenylephrine with the maximal contractile force (Emax). Percent relaxation was calculated as the percent decrease in force with respect to the initial phenylephrine-induced pre-contraction, and the percent relaxation was used to construct the concentration–response curves of ACh-induced relaxation. To compare the sensitivities of the vessels to the agonists in the presence of different inhibitors, the negative logarithm (pD2) of the concentration of phenylephrine or ACh giving 50% of maximum response (pEC50) was determined by linear interpolation on the semilogarithm concentration–response curve [pD2 = −Log(EC50)]. Maximal contraction and relaxation responses as well as pEC50 values were compared by Student's un-paired t-test. Statistical significance was defined as p < 0.05. Data were reported as mean± s.e.mean. Statistical analysis and construction of concentration response curves were performed using GraphPad Prism software (San Diego, CA). 3. Results
age was 50% higher than that in the control (p ≤ 0.001). This difference disappeared at 9 months (Fig. 1B). To elucidate the effect of aging, in the control at 9 months of age, we found that the plasma and aortic homogenate isoprostane 8-epi-PGF2α level was significantly (p = 0.03) increased by 70% and 50%, respectively, compared with that at 3 months. 3.2. Imbalanced protein expression of superoxide-generating and degrading enzymes in the Marfan aorta The expression of SOD-1 (copper–zinc SOD or cytosolic SOD) in the Marfan aorta was comparable to that of the age-matched control at 9 months, but was significantly less at 3 and 6 months. SOD-2 (manganese SOD or mitochondrial SOD) expression was similar between groups at 6 months, but was lower in the Marfan group at 3 and 9 months (Fig. 1C, Table 1). In the Marfan aorta, iNOS protein expression was 298, 51, and 95% higher than that of the control at 3, 6, and 9 months, respectively. Xanthine oxidase is also increased by 270% at 6 months Marfan aorta (Fig. 1C, Table 1). The gp91phox, the catalytic subunit of NAD(P)H oxidase, was markedly increased in the Marfan aorta at 6 months old. With regard to the regulatory subunits, the expression of p47phox was elevated at 3 months and that of p67phox was elevated at 3 and 6 months in the Marfan group. However, at 9 months, the expression of both subunits was higher in the control (Fig. 1C).
3.1. Increased isoprostane 8-epi-PGF2α level in Marfan mice 3.3. Pre-incubation with SOD and catalase From 3 months on, Marfan aorta had elevated isoprostane 8-epiPGF2α compared with the age-matched control by 32–50% (Fig. 1A). The plasma isoprostane 8-epi-PGF2α level in Marfan mice 3 and 6 months of
To reveal the influence of superoxide on phenylephrine-induced contraction, we pre-incubated the aortic arch with SOD, an enzyme
Fig. 3. Effect of superoxide dismutase (SOD, 150 U mL− 1) and SOD plus catalase (1000 U mL− 1) on acetylcholine (ACh)-induced relaxation in aortic arch. Upper panels showed (A) Emax and (B) pEC50 at 6 months in response to ACh in the presence and absence of SOD or SOD plus catalase. (C) and (D) are the concentration–response curves. (n = 7, ⁎p < 0.05, compared with control in the absence of the enzymes, #p < 0.05, compared with the responses in the absence of the enzymes in their respective groups).
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that converts superoxide to H2O2, before the addition of phenylephrine. We previously showed that control and Marfan aorta demonstrated a difference in the phenylephrine-contraction only at 9 months of age (Chung et al., 2007a), and thus the effect of superoxide removal on contraction was examined in this age group. Pretreatment of SOD potentiated the contraction in the Marfan aorta and increased the Emax and the pEC50, returning both values to the control level. In contrast, SOD had no effect on phenylephrine-induced contraction in the control aorta (Fig. 2). The participation of H2O2, produced by SOD, in the alteration of phenylephrine response was further assessed with the co-incubation of SOD and catalase. In physiological conditions, SOD converts superoxide to H2O2, which is subsequently converted to oxygen and water by catalase. In Marfan aorta, the effect of SOD plus catalase was not different from that of SOD alone; like SOD, the combination treatment also significantly improved the Emax and pEC50 of phenylephrine on the Marfan aorta (Fig. 2). Impaired endothelium-dependent ACh-stimulated relaxation was previously observed in the Marfan aorta at 3 and 6 months of age (Chung et al., 2007a); therefore, in this study, the effect of oxidative stress on endothelium-dependent relaxation was assessed at these two ages. At 6 months of age, SOD and SOD-plus-catalase combination treatment in the Marfan aorta restored the ACh-maximal relaxation and ACh-pEC50 to the control levels (Fig. 3). At 3 (data now shown) and 6 months, both SOD and SOD-plus-catalase combination did not significantly alter the ACh-response in the control aorta. 3.4. Blockade of xanthine oxidase with allopurinol improved AChinduced relaxation in the Marfan aorta Allopurinol, a xanthine oxidase inhibitor, did not significantly alter phenylephrine-contraction in control aortic arch at 9 months of age. A trend of improvement was observed in the Marfan aorta although it did not reach statistical significance (Fig. 4A,B). With regard to ACh-
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mediated relaxation, allopurinol had no effect on both control and Marfan aorta at 3 months (data not shown). At 6 months old, although allopurinol had no significant effect on the ACh-Emax, it increased the pEC50 values from 6.34 to 7.65 (Fig. 4D). Allopurinol did not cause significant changes in the ACh-relaxation (Fig. 4C) in the control aortic arch.
3.5. Blockade of NAD(P)H oxidase with apocynin improved ACh-induced relaxation in the Marfan aorta Apocynin, an NAD(P)H oxidase inhibitor, did not cause significant changes in phenylephrine-Emax and pEC50 at 9 months old in both control and Marfan aortic arch (Fig. 5A,B). For the ACh-stimulated relaxation, apocynin did not have an effect on both Emax and pEC50 at 3 months in both groups (data not shown). However, at 6 months, apocynin increased the Emax from 46 to 77% and pEC50 from 6.34 to 7.49 in the Marfan arch whereas in the control, no significant changes were observed (Fig. 5C,D).
3.6. Blockade of iNOS with 1400 W improved vasomotor function in the Marfan aorta The specific iNOS inhibitor, 1400 W, altered phenylephrine and ACh responses in both control and Marfan aorta. At 9 months of age, 1400 W increased phenylephrine-Emax in both control and Marfan arch by 52.0 and 150%, respectively (Fig. 6A,B); however, the pEC50 values were not significantly changed in both groups. With respect to ACh-induced relaxation, 1400 W had no effect on control aorta at 3 months but increased the Emax from 60.5 to 90.2% in the Marfan arch (data not shown). At 6 months, 1400 W did not alter the AChrelaxation significantly in the control whereas ACh-Emax was increased from 57 to 86% in the Marfan arch (Fig. 6C,D).
Fig. 4. Effects of allopurinol (a xanthine oxidase inhibitor; 300 µM) on (A, B) phenylephrine-mediated contraction at 9 months, and (C, D) ACh-induced relaxation at 6 months in control and Marfan aortic arch. (n = 7; ⁎p < 0.05 vs no treatment).
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Fig. 5. Effects of apocynin (an NAD(P)H oxidase inhibitor; 100 µM) on (A, B) phenylephrine-mediated contraction at 9 months, and (C, D) ACh-induced relaxation at 6 months in control and Marfan aortic arch (n = 7; ⁎p < 0.05 vs no treatment).
Fig. 6. Effects of 1400 W (an iNOS inhibitor; 1 µM) on (A, B) phenylephrine-induced contraction at 9 months, and (C, D) ACh-mediated relaxation at 6 months in control and Marfan aortic arch (n = 8, ⁎p < 0.05 vs no treatment).
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Fig. 7. Concentration–response curves showing the effects of incubating with purified 0.5 µg/mL matrix metalloproteinase-1, and 1.5 µg/mL angiostatin, on (A, B) phenylephrinemediated contraction and (C, D) ACh-induced relaxation in control and Marfan aorta at 6 months of age.
Note that the incubation with irrelevant proteins matrix metalloproteinase-1 and angiostatin did not modify vasomotor function of both strains (Fig. 7). 4. Discussion The current study is the first to show the elevation of oxidative stress in the thoracic aorta of a mouse model of Marfan syndrome. The presence of oxidative stress, may be attributed to the downregulation of SOD and upregulation of superoxide-producing enzymes and could impair vasomotor function in Marfan aortae. However, it could be improved by the pre-incubation of the superoxide-eliminating enzymes (i.e. SOD, catalase) and the blockade of the superoxidegenerating enzymes (i.e. apocynin, allopurinol, 1400 W). Therefore, the present study suggests that by reducing oxidative stress level by pharmacological strategies, it could possibly improve aortic vasocontractile function and endothelium-dependent relaxation, the two crucial factors determining the susceptibility of aneurysm formation. The isoprostane 8-epi-PGF2α assay is a well-established method to provide a reliable assessment of oxidative injury in vivo (Hoffman et al., 1996; Patrono and FitzGerald, 1997; Delanty et al., 1996). Increased formation of F2 isoprostanes has been reported to be associated with vascular diseases such as vascular reperfusion, diabetes mellitus and hypercholesterolemia (Patrono and FitzGerald, 1997; Delanty et al., 1996). In the plasma and aortic homogenate of Marfan mice, significantly increased levels of isoprostane 8-epi-PGF2α were observed compared with the control, suggesting that oxidative stress was elevated by the disorder. The isoprostane 8-epi-PGF2α level was also found to be increased with age. It has been well-established that oxidative stress is associated with aging in the vasculature (Donato et al., 2007; Hamilton et al., 2001; Taddei et al., 2001). In healthy people, it was found that endothelial dysfunction and increased oxidative stress biomarkers in the aged subjects could be
linked to increased NAD(P)H oxidase expression (Donato et al., 2007). In the control mice, we also observed a significant increase in the expression of NAD(P)H oxidase subunits at 9 months, which may contribute to the increased aortic homogenate 8-epi-PGF2α level. We have shown that Marfan syndrome and the progression of thoracic aortic aneurysm were accompanied by a pronounced impairment of aortic contractile function (Chung et al., 2007a,b). The pre-incubation of SOD alone or a combination of SOD and catalase normalized the contractile response of Marfan aorta to the control level. This further reconfirmed the presence of oxidative stress, and demonstrated the impact of excess superoxide on vasoconstriction in Marfan syndrome. Oxidative stress has been reported to be involved in the pathogenesis of various cardiovascular diseases. SOD treatment was shown to reverse the hypersensitivity of the arteries in diabetic and hypertensive animal models and normalize the agonist-induced contraction to that of the control animals (Kanie and Kamata, 2000; Alvarez et al., 2008). In the rat model of chronic renal failure, similar to the present study, phenylephrine-induced contraction was compromised in the aorta but could be restored to the control level with an antioxidant, melatonin (Sener et al., 2004). Although the mechanism of action of ROS on smooth muscle cell contractility is still unclear (Lyle and Griendling, 2006), ROS has been proposed to have multiple effects on calcium signaling in both vascular endothelial and smooth muscle cells (Lounsbury et al., 2000; Elmoselhi et al., 1996; Walia et al., 2000). The impairment of the calcium signaling pathway caused by oxidative stress may consequently lead to the alteration of vascular reactivity (Lyle and Griendling, 2006). Therefore, the removal of superoxide with SOD and catalase may restore calcium signaling and thereby the contractile responses. In the Marfan aorta, reduced endothelial-dependent relaxation could also be reversed by addition of SOD or SOD-plus-catalase. The similar effects caused by SOD treatment alone or the combination of
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SOD and catalase suggest that the improvement of endothelial function was not due to the vaso-relaxant effect of the H2O2, but rather the removal of superoxide anions. Oxidative stress may cause endothelial dysfunction through several direct and indirect pathways, the most well-known of which is the scavenging of NO by superoxide. Superoxide radicals bind to NO at a rate three times faster than they bind to SOD; therefore, excess superoxide production would increase the rate of NO degradation (Cai and Harrison, 2000; Schulz et al., 2004). In addition, peroxynitrite, the product of the reaction between superoxide and NO, could cause the uncoupling of eNOS by oxidizing tetrahydrobiopterin. The uncoupled eNOS not only reduces the production of NO, but is also another important source of superoxide (Schulz et al., 2004). In hypercholesterolemia, diabetes, heart failure, hypertension and chronic renal disease, endothelial function has been shown to be improved by antioxidants, such as SOD, melatonin, and vitamins C and E (Schulz et al., 2004; Sener et al., 2004; Miyagawa et al., 2007). Therefore, in addition to reduced NO production caused by downregulation of eNOS and reduction of Akt phosphorylation (Chung et al., 2007a), the present study indicated that oxidative stress may be another contributor to endothelial dysfunction in Marfan syndrome. Oxidative stress could be associated with reduced superoxide neutralization, which could result from the downregulation of SOD-1 and -2. SOD is one of the most important endogenous superoxideneutralizing enzymes in the vasculature (Faraci and Didion, 2004; Cai and Harrison, 2000; Didion et al., 2002). In the mouse aorta, among the three isoforms of SOD, SOD-1 and SOD-2 account for 50–80% and 2–12% of SOD composition, respectively (Faraci and Didion, 2004). Therefore, the decreased expression of these two isoforms in Marfan aortae would likely cause a reduction in superoxide removal. In the streptozotocin-induced diabetic rat, decreased SOD expression has been shown to be associated with vascular dysfunction caused by superoxide anion (Kamata and Kobayashi, 1996). In the mouse models of both SOD-1 and SOD-2 deficient mice, basal superoxide level in the vasculature was elevated and ACh-mediated relaxation was impaired (Didion et al., 2002; Brown et al., 2007). Therefore, the impairment of ACh-induced relaxation and phenylephrine-mediated contraction in Marfan aortae may be attributed to the downregulation of SOD. We used various pharmacological inhibitors of superoxidegenerating enzymes to determine the mechanisms through which superoxide level was elevated in the Marfan aorta. Xanthine oxidase is an enzyme that catalyses the oxidation of hypoxanthine and xanthine and produces O2− and H2O2 during purine metabolism (Schulz et al., 2004). Inhibition of the upregulated enzyme with allopurinol improved both contraction and relaxation in the Marfan aorta. In patients with hypercholesterolemia and chronic heart failure, similar observations have been made where the administration of oxypurinol and allopurinol reduced oxidative stress and improved endothelial function (Cardillo et al., 1997; George et al., 2006). NAD(P)H oxidase, a membrane-associated enzyme which catalyzes reduction of oxygen with electrons from NADH or NADPH, is the major contributor of superoxide anions in the vasculature (Jiang et al., 2004; Yokoyama et al., 2000). Upregulation of NAD(P)H oxidase is found to be associated with various animal models of hypertension and to cause impairment of NO-dependent vaso-dilatation in patients with coronary artery disease (Jiang et al., 2004). Apocynin, an NAD(P)H oxidase inhibitor, normalized ACh-mediated relaxation in the Marfan aorta, but had no effect on contraction. This can be related to the temporal changes of the protein expression of different NAD(P)H oxidase subunits. The selective iNOS inhibitor, 1400 W, greatly improved the responses to phenylephrine and acetylcholine in the Marfan aorta. It is believed that induction of iNOS produces excessive NO as well as ROS (Liu et al., 2005). Thus, the upregulated iNOS expression in the Marfan aorta at all ages, which may be a compensatory mechanism for the reduced NO bioavailability (Vaziri
et al., 2000; Chung et al., 2007a), may be another contributor to oxidative stress. We acknowledge limitations in this study. First, all functional experiments were performed on the aortic arch but not the ascending part due to its very short length. Most of the aneurysm in human with Marfan syndrome is evidenced in the aortic root and ascending aorta. The differences between two strains in oxidative stress level, enzyme expression as well as aberrant vasomotor function presented herein might be more pronounced if we used the ascending aorta. Therefore, our results raise interesting questions for further testing using more sophisticated and appropriate methodology, or in bigger animal models. Second, in the present study, endothelial dysfunction is identified as impaired vasodilation in response to acetylcholine. However, a broader understanding of the term should also include the proinflammatory and prothrombic state. Third, there are over 600 mutations identified to cause Marfan syndrome. While missense mutations account for 60% of the mutations, 78% of the point mutations locate in the cbEGF modules. A further 12% of these mutations are recurrent and affect a mutation hotspot, CpG., for a cysteine residue (Judge and Dietz, 2005). This type of mutation is the basis of the mouse model used in this study, and represents the most common mutation in classic Marfan syndrome (Habashi et al., 2006). We therefore find that although our model is useful to investigate the general pathogenesis of Marfan syndrome, it may not be representative of all cases of Marfan syndrome in human. 5. Conclusion We used various pharmacological agonists and inhibitors to demonstrate the impact of oxidative stress on the thoracic aorta of Marfan syndrome. The elevated oxidative stress could be due to reduced expression of SOD and increased expression of iNOS, NAD(P) H oxidase, and xanthine oxidase at different ages. Since vasomotor function in part determines the susceptibility to aneurysm formation, normalization of vasoconstriction and relaxation with the use of antioxidants could suggest a novel pharmacological strategy in the treatment of thoracic aortic aneurysm in Marfan syndrome. Acknowledgements This study was supported by an operating grant from the Canadian Institutes of Health Research. H.H.C.Y. is the recipient of Michael Smith Foundation for Health Research Trainee Award and NSERC Canada Graduate Scholarship. A.W.Y.C. is the recipient of Michael Smith Foundation for Health Research/St Paul's Hospital Foundation Trainee Award. References Alvarez, Y., Briones, A.M., Hernanz, R., Perez-Giron, J.V., Alonso, M.J., Salaices, M., 2008. Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats. Br. J. Pharmacol. 153, 926–935. Brown, K.A., Didion, S.P., Andresen, J.J., Faraci, F.M., 2007. Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency. Arterioscler. Thromb. Vasc. Biol. 27, 1941–1946. Cai, H., Harrison, D.G., 2000. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ. Res. 87, 840–844. Cardillo, C., Kilcoyne, C.M., Cannon III, R.O., Quyyumi, A.A., Panza, J.A., 1997. Xanthine oxidase inhibition with oxypurinol improves endothelial vasodilator function in hypercholesterolemic but not in hypertensive patients. Hypertension 30, 57–63. Chew, D.K., Conte, M.S., Khalil, R.A., 2004. Matrix metalloproteinase-specific inhibition of Ca2+ entry mechanisms of vascular contraction. J. Vasc. Surg. 40, 1001–1010. Chung, A.W., Au Yeung, K., Cortes, S.F., Sandor, G.S., Judge, D.P., Dietz, H.C., van Breemen, C., 2007a. Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome. Br. J. Pharmacol. 150, 1075–1083. Chung, A.W., Au Yeung, K., Sandor, G.G., Judge, D.P., Dietz, H.C., van Breemen, C., 2007b. Loss of elastic fiber integrity and reduction of vascular smooth muscle contraction resulting from the upregulated activities of matrix metalloproteinase-2 and -9 in the thoracic aortic aneurysm in Marfan syndrome. Circ. Res. 101, 512–522.
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