D-4F Induces Heme Oxygenase-1 and Extracellular Superoxide Dismutase, Decreases Endothelial Cell Sloughing, and Improves Vascular Reactivity
http://www.100md.com
《循环学杂志》
the Departments of Medicine (A.L.K., S.P.), Pharmacology (S.T., F.F.Z., S.Q., N.G.A.)
Physiology (P.M.K., M.S.W.), New York Medical College, Valhalla, NY
The Rockefeller University (N.G.A.), New York, NY.
Abstract
Background— Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals.
Methods and Results— We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 μg/100 g of body weight, intraperitoneal injection) on superoxide (O2–), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2– levels were elevated in diabetic rats (74.8±8x103 cpm/10 mg protein) compared with controls (38.1±8x103 cpm/10 mg protein; P<0.01). D-4F decreased O2– levels to 13.23±1x103 (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50±6 cells/mL) than in controls (5±1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20±3 cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0±2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level.
Conclusions— We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.
Key Words: atherosclerosis ; antioxidants ; apolipoproteins ; lipids
Introduction
D-4F is a synthesized mimetic peptide of apolipoprotein A1, the major lipoprotein found in HDL. Prior studies have shown that oral administration of D-4F can reduce atherosclerotic disease independent of cholesterol levels and enhance the ability of HDL to protect against LDL oxidation.1–3 Navab et al1,2 have demonstrated that treatment with D-4F causes HDL to become antiinflammatory, stimulates HDL-mediated cholesterol efflux, and reverses cholesterol transport from macrophages. It has been shown previously that apolipoprotein A1 can restore the balance between nitric oxide (NO) and superoxide (O2–) anions in mice.4 Using a sickle cell disease model, Ou et al5 have shown that apolipoprotein A1 can improve vasoreactivity in LDL-receptor null mice. These early data indicate an antioxidant effect from the administration of apolipoprotein A1 and possibly D-4F.
The realization that heme oxygenase-1 (HO-1) is strongly induced by oxidant stress and its substrate, heme, in conjunction with the robust ability of HO-1 to guard against oxidative insult6–8 has led to examination of the antioxidant nature of HO-1 and HO-2.7,9 Antioxidant effects arise from the capacity of HO-1 to degrade the heme from destabilized heme proteins10 and from the elaboration of biliverdin and bilirubin, which are products of HO with potent antioxidant properties.11 Carbon monoxide (CO), an HO product, is not an antioxidant,12,13 but it has an antiapoptotic effect14 and vasodilator properties.14–16 Additionally, the induction of HO-1 prevents cell death, which is attributed to its augmentation of ion efflux and exportation of iron-binding proteins.17 The protective actions of HO-1 are not confined to overtly oxidant processes but rather extend widely to such disease processes as inflammation8,18 and atherosclerosis.19 Furthermore, upregulation of HO-1 increases the antioxidant system by decreasing O2–.20,21 However, the mechanism by which HO-1 decreases O2– is not clear.
Superoxide dismutase (SOD) is an antioxidant enzyme that converts superoxide (O2–) anions into hydrogen peroxide and molecular oxygen.22 All mammalian tissues contain 3 forms of SOD: copper/zinc SOD (Cu/Zn-SOD), manganese SOD (Mn-SOD), and extracellular SOD (EC-SOD). Under physiological conditions, the basal level of O2– is controlled by mitochondrial and cytosolic SOD.23 Under pathological conditions, such as heart ischemia or inflammatory blood cells, O2– is acted on by EC-SOD instead.24 The vascular concentration of EC-SOD is a key determinant of endothelial function; a decrease in EC-SOD is associated with a decrease in NO bioavailability and an increase in the reaction of NO with superoxide to form peroxynitrite, a potent cytotoxic oxidant.22 In the present study, we measured EC-SOD because HO-1 has been shown to attenuate the increased level of superoxide in diabetics and to increase reduced glutathione levels.21,25 We hypothesized that this effect might be due to an increase in another antioxidant gene such as vascular EC-SOD, which is responsible for metabolizing superoxide. Therefore, we examined the effect of HO-1 on the levels of EC-SOD.
Hyperglycemia-mediated cardiovascular complications and atherosclerosis contribute to the formation of O2–26 and reactive oxygen species, each of which contributes to endothelial dysfunction and apoptosis.27 It has been shown that these abnormalities are reversible with increased expression of antioxidant enzymes or administration of antioxidant agents.28 A reduction in antioxidant reserves has been related to endothelial cell dysfunction in diabetes, even in patients with well-controlled blood glucose levels.27,29 Similarly, it has been shown that individuals with moderately to well-controlled type 2 diabetes mellitus demonstrate an increase in LDL oxidation.30 Conditions associated with an elevation of oxidative stress have been shown to have an increased level of endothelial cell sloughing.31–33 In a recent study, diabetes was also found to be associated with increased endothelial apoptosis.20,21
The primary objective of the present study was to determine whether apolipoprotein D-4F causes an antioxidant effect as a result of increased HO-1–derived bilirubin and CO. Additional objectives were to investigate whether D-4F decreases endothelial cell sloughing and fragmentation or restores vascular reactivity in diabetic rats. The present results demonstrated that treatment with D-4F increased HO-1 protein and activity and EC-SOD. The increase in HO-1 and EC-SOD was associated with restoration in vascular reactivity and the amelioration of endothelial cell sloughing and fragmentation. These results indicate that the antioxidant effect of D-4F operates via an increase in both HO-1 and EC-SOD, both of which cause decreased oxidative stress and attenuate the vascular damage seen with diabetes.
Methods
Animal Treatment
Male Sprague-Dawley rats (Charles River Laboratory, Wilmington, Mass) weighing 170 to 190 g were maintained on standard rat diet and tap water ad libitum. After the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg body weight), diabetes was induced by a single injection, via the tail vein, of streptozotocin (STZ; Sigma; 60 mg/kg body weight) dissolved in 0.05 mol/L citrate buffer (pH 4.5). Age-matched control rats were injected with an equal volume of vehicle (sodium citrate buffer). Rats were divided into 3 groups: control, STZ, and STZ plus D-4F (daily 100 μg/100 g body weight intraperitoneal injections for 6 weeks starting the day after injection of STZ). Blood glucose level was measured 2 days after injection of STZ or vehicle. These rats were used 1 month after induction of diabetes. The Animal Care and Use Committee of New York Medical College approved all experiments.
Measurement of Vascular O2– Levels
Previously described methods34 were used in which control and diabetic vessels were placed in plastic scintillation minivans that contained 5 μm of lucigenin, for detection of O2– and other additions, in a final volume of 1 mL of air-equilibrated Krebs solution buffered with 10 mmol/L HEPES-NaOH (pH 7.4). Lucigenin chemiluminescence was measured in a liquid scintillation counter (LS6000IC, Beckman Instruments) at 37°C, and data are reported as counts/min per milligram of protein after background subtraction.
Tissue Preparation for HO and EC-SOD Measurement
Frozen thoracic aorta segments were pulverized under liquid nitrogen and placed in a homogenization buffer (10 mmol/L phosphate buffer, 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L PMSF, and 0.1% tergitol, pH 7.5). Homogenates were centrifuged at 27 000g for 10 minutes at 4°C. The supernatant was isolated, and protein levels were assayed (Bradford method). The supernatant was used for measurement of HO-1, HO-2, and EC-SOD–Cu/Zn-SOD protein expression and determination of HO activity.
Western Blot Analysis
These measurements were performed to determine HO-1, HO-2, EC-SOD, and Cu/Zn-SOD protein expression. Protein levels were visualized by immunoblotting with antibodies against rat HO-1, HO-2, EC-SOD, and Cu/Zn-SOD (Stressgen Biotechnologies Corp). Briefly, 20 μg of lysate supernatant was separated by 12% SDS/PAGE and transferred to a nitrocellulose membrane (Amersham) with a semidry transfer apparatus (Bio-Rad). The membranes were incubated with 10% milk in 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.05% Tween 20 (TBST) buffer at 4°C overnight. After they were washed with TBST, the membranes were incubated with anti-HO-1, anti-HO-2, anti-EC-SOD, and anti-Cu/Zn-SOD antibodies for 1 hour at room temperature with constant shaking. The filters were washed and subsequently probed with horseradish peroxidase–conjugated donkey anti-rabbit or anti-mouse IgG (Amersham). Chemiluminescence detection was performed with the Amersham ECL detection kit, according to the manufacturer’s instructions.
Measurement of HO Activity
HO activity was assayed as previously described20 in which bilirubin, the end product of heme degradation, was extracted with chloroform and its concentration determined spectrophotometrically (Perkin-Elmer Dual UV/VIS Beam Spectrophotometer Lambda 25) using the difference in absorbance at a wavelength from 460 to 530 nm with an absorption coefficient of 40 mmol–1 · cm–1.
Detection and Quantification of Circulating Endothelial Cells
For immunomagnetic isolation and quantification of endothelial cells, we used monodispersed magnetizable particles (Dynabeads CELLection Pan Mouse IgG kit) obtained from Dynal. The 4.5-μm diameter polystyrene beads are coated with affinity-purified pan-anti-mouse immunoglobulin G1 covalently bound to the surface. The beads were washed according to the manufacturer, with a strong magnet (MPC6, Dynal) used to remove sodium azide. Typically, 100 μL of bead suspension was coated noncovalently with 10 μg/mL RECA-1 (Novus Biologicals), a pan-rat endothelial cell–specific monoclonal antibody diluted 1:10 in PBS 0.1% BSA, by overnight incubation at 4°C with head-over-head agitation. After 3 washes with PBS-BSA to remove excess antibodies, the beads were resuspended in buffer until use. RECA-uncovered particles were used as a negative control. If the beads were stored for a long period of time, 0.1% sodium azide was added to the buffer. Beads and target cells were incubated for 1.5 hours at 4°C on a rotator. The amount of beads (4x108/mL) was calculated to be in great excess of target cells (>2000 beads per target cell). Separation of beads and rosetted cells from the blood samples required a minimum of 1 minute of exposure to the magnet. Three washes were performed to completely remove nonrosetted cells. After the third wash, rosetted cells were recovered in a 150 μL solution of acridine orange (a vital fluorescent dye at final concentration of 5 μg/mL in PBS), and observations were made in a hemacytometer under both white and fluorescent blue excitation with fluorescence microscopy (Olympus IX81 F).
Assessment of Vascular Reactivity
The thoracic aorta was removed and placed in cold Krebs-bicarbonate solution, cleaned of fat and loose connective tissue, and sectioned into 3-mm-long rings. Two rings per aorta were mounted on stainless steel hooks and suspended in 5-mL tissue baths. The bath was filled with Krebs solution at 37°C, pH 7.4, with 95% O2/5% CO2 as a gas phase. Aortic rings were stretched to a 2 g initial tension and were equilibrated for 60 minutes. Tension development was measured by isometric force transducers (GRASS FT03) connected to an amplifier. The Krebs buffer solution in the tissue bath was replaced every 15 minutes, and the tension was readjusted each time. At the end of the equilibration period, the maximal force generated by addition of a depolarizing solution of 60 mmol/L KCl was determined. To evaluate acetylcholine-induced vasodilation, the rings were preconstricted with phenylephrine (3.5x10–7 mol/L) to obtain a stable plateau, and then a cumulative dose-response curve to acetylcholine or NONOate was obtained.
Statistical Analyses
Data are presented as mean±SEM for the number of experiments. Statistical significance (P<0.05) between experimental groups was determined by the Fisher method of analysis of multiple comparisons. For comparison between treatment groups, the null hypothesis was tested by a single-factor ANOVA for multiple groups or unpaired t test for 2 groups.
Results
Effect of D-4F on HO Activity and HO-1 and HO-2 Protein
To define the effect of D-4F on vascular HO-1, we measured HO activity and the protein levels in control and diabetic rats. Compared with controls, the induction of diabetes had no significant effect on either HO-1 or HO-2 protein (Figure 1). Treatment with D-4F resulted in a significant increase in the amount of HO-1 protein compared with control (290%, P<0.001) and untreated diabetic rats (277%, P<0.05). Administration of D-4F had no effect on HO-2. Because HO-1 converts heme to equimolar amounts of CO and bilirubin,20 we measured HO activity by formation of bilirubin. HO activity in control vessels was 0.81±0.03 nmol bilirubin formed/mg protein and decreased to 0.59±0.02 nmol bilirubin formed/mg protein in diabetic vessels (P<0.05). The stimulatory effect of D-4F on HO-1 protein was associated with an increase in HO activity (Figure 1E) in both diabetic (1.14±0.04 nmol bilirubin) and control (01.11±0.02 nmol bilirubin) rats (P<0.0001).
Effect of D-4F on Superoxide Formation
Because D-4F increases HO-1-derived bilirubin formation, we compared the effect of D-4F on superoxide formation. A comparison was made between superoxide levels in the tissues of D-4F–treated diabetic rats, untreated diabetic rats, and controls. Compared with controls, diabetic rats showed an elevation in aorta O2– formation from 38.1±8 to 74.8±8x103 cpm/mg protein (P<0.01). D-4F was a very potent inhibitor of O2– formation. Administration of D-4F decreased O2– by 85% in diabetic rats (Figure 2). D-4F also decreased O2– compared with controls, although not significantly.
Endothelial Cell Sloughing
Endothelial Cell Fragmentation
Under microscopic examination, there was evidence of cellular fragmentation and debris, which was bound by the RECA-1–coated beads and fluoresced after staining with acridine but was too small to be considered as whole cells (Figure 5). Quantification of these fragments (Figure 6) yielded an average of 8.3±2 fragments/mL in controls and 38±4 fragments/mL in untreated diabetic rats (P=0.009). Administration of D-4F to diabetic rats prevented endothelial fragmentation by 80% in diabetic rats; only 6.8±1 fragments/mL was seen in D-4F–treated diabetic rats (P<0.0001 versus untreated diabetics). Treatment with D-4F also decreased endothelial cell fragmentation in control rats to 2.8±1 fragments/mL (P<0.05 versus untreated control). The level of decrease in cellular fragmentation was indicative of cellular protection secondary to the D-4F–mediated decrease in O2–.
Vascular Reactivity
It has been reported that acetylcholine-evoked endothelium-mediated relaxation is impaired in diabetes.35 The present study confirmed a significant impairment in diabetic rats (51±7% relaxation) compared with controls (77±3% relaxation). D-4F restored vascular reactivity in diabetic rats to 85±2% relaxation (Figure 7A). In the presence of the NO donor NONOate, there were no significant changes noted between controls and diabetic rats in response to acetylcholine (Figure 7B). Aorta from diabetic rats treated with D-4F showed an even higher response to NONOate than either control or diabetic vessels at a concentration of 10–6 mol/L (P<0.05).
Effect of HO-1 Expression on the Antioxidant System
In an attempt to elucidate the mechanism by which D-4F attenuates endothelial sloughing and fragmentation and decreases O2–, we examined the effect of D-4F on vascular SOD. As shown in Figure 8, the level of EC-SOD was significantly reduced in diabetic rats compared with controls (P<0.003); however, treatment with D-4F significantly restored EC-SOD protein levels (P<0.001). Treatment with D-4F also increased the amount of EC-SOD in control rats (P<0.02 versus control, P<0.0001 versus diabetic). The levels of Cu/Zn-SOD and Mn-SOD proteins were not affected by D-4F in either control or diabetic rats.
Discussion
This study demonstrates that D-4F participates in the attenuation of diabetic vascular complications, presumably by increases in bilirubin (an antioxidant) and CO (which is cytoprotective) secondary to increased HO-1 and/or by a robust increase in EC-SOD. This conclusion is supported by 4 important key findings.
First, D-4F was shown to decrease the formation of reactive oxygen species, namely, superoxide (O2–). Prior study has indicated elevated O2– in the hyperglycemic state associated with diabetes, which was attenuated with upregulation of HO-1.21,36 A similar decrease in O2– was seen with administration of D-4F. This confirms the antioxidant nature of D-4F and supports earlier studies.1,3 Treatment of diabetic rats with D-4F induced increases of HO-1 and significantly increased EC-SOD but had no effect on the other SOD isoforms (Cu/Zn or Mn). We believe that the action of D-4F is largely or solely through the induction of HO-1; however, the present results, which indicate that D-4F dramatically induces HO-1 and EC-SOD, are novel, and together with our findings that D-4F dramatically reduced endothelial sloughing and improved vasoreactivity in diabetic rats, they constitute important new information about the actions of apolipoprotein A1 mimetic peptides. Although rats have a diminished level of EC-SOD compared with other species, there is evidence of EC-SOD in the vascular tissue.22 There is potentially an important role for EC-SOD in the attenuation of reactive oxygen species and peroxynitrite generation from inducible NO synthase– and endothelial NO synthase–derived NO. Peroxynitrite oxidizes and inactivates the NOS cofactor tetrahydrobiopterin into inactive molecules, such as dihydrobiopterin.37 This uncouples the enzyme, which then preferentially increases O2– production over NO production.37,38 Therefore, the HO-1 gene expression–mediated increase in EC-SOD may protect endothelial NO synthase from uncoupling. This is of particular importance in diabetes, in which hyperglycemia causes increased endothelial NO synthase uncoupling.39 The mechanism by which the HO-1 products CO and/or bilirubin may directly regulate this uncoupled state remains unclear.
Second, administration of D-4F increased both the level and activity of HO-1. These results confirmed our previous study,36 which established that diabetes results in a downregulation of HO-1 level and activity. Administration of D-4F to diabetic rats led to a reversal of this trend and a resultant increase in both the level and activity of HO-1, which strongly suggests a role for HO in the antioxidant effect of D-4F; however, D-4F–mediated HO-1 may be considered as a signaling mechanism in decreasing inducible NO synthase and O2– with an increase in EC-SOD. Gene transfer of antisense HO-1, as a specific inhibitor of HO-1, has been shown to decrease EC-SOD and increase circulating endothelial cells and inducible NO synthase.36
The third major finding indicated the vascular protective effect of D-4F administration by decreasing endothelial cell sloughing and debris. Many of the significant clinical problems associated with diabetes can be traced to vascular damage induced by hyperglycemia.20,27 Unfortunately, even patients with well-controlled blood glucose levels are not totally protected from these side effects.27,29 The increased vascular damage with resultant sloughing and fragmentation of endothelial cells typically found with diabetes was lessened in D-4F–treated animals. This demonstrates a possible clinical role for D-4F in protecting the vasculature from the damage associated with diabetes mellitus.
Finally, D-4F was shown to have positive effects on vascular reactivity to stimulation with acetylcholine. Reactivity of the blood vessels has been shown to be diminished in diabetes.35 Vascular reactivity in control, untreated diabetic, and D-4F–treated diabetic rats did not differ significantly in the presence of NONOate, which indicates a dependence on the bioavailability of NO.
There is an ever-increasing body of evidence that indicates that inflammation and oxidative stress a play major role in the formation of atherosclerotic disease. The oxidative-modification hypothesis indicates that only after oxidation does LDL become atherogenic.40–42 After incorporation into foam cells, oxidized LDL acts to recruit inflammatory cells, including T lymphocytes and monocytes, via O2–.43,44 There is also evidence that oxidative stress plays a role in the mechanisms for the documented risk factors of atherosclerosis, including hypertension, diabetes, and smoking.45,46 In individuals with diabetes, there is increased LDL oxidation even in the face of adequate glucose control.30 Prior study has shown that patients with documented atherosclerosis had LDL that was highly proinflammatory, independent of their total cholesterol or HDL levels.2,3 Superoxide, a marker for oxidative stress, has been implicated in many cardiovascular diseases, including atherosclerosis, and has been shown to rapidly inactivate endothelium-derived NO.47,48 Peroxynitrite, formed from the reaction of superoxide with NO, is known to promote lipid peroxidation, contributing to formation of atherosclerotic lesions.49 Superoxide-mediated impairment of NO may result in vascular smooth muscle cell proliferation and migration and the expression of proinflammatory molecules, which contribute to atherosclerosis.47,50 It is hypothesized that the elevated superoxide seen in the diabetic animals in the present study was vascular in origin, because EC-SOD is primarily vascular in nature. This hypothesis is supported by prior study showing that the apolipoprotein A1 mimetic L-4F can attenuate O2– production in cultured endothelial cells.4,5
In light of these recent discoveries, there is a growing potential for therapeutic agents that can attenuate oxidative stress. Mounting evidence demonstrates that D-4F can protect against oxidative injury, which makes it a potential therapeutic agent in the fight against atherosclerosis. With administration of D-4F, HDL becomes antiinflammatory, which increases HDL-mediated cholesterol efflux and reverses cholesterol transport from macrophages.1 In LDL-receptor null or apolipoprotein E null experimental mice fed Western diets, D-4F administration resulted in a 79% and 75% reduction in atherosclerotic lesions, respectively.3 This is in agreement with the potency of D-4F on induction of the HO-1–mediated increase in EC-SOD and subsequent decrease in O2–. The previously unknown mechanism for the antioxidant role of D-4F has now been shown to be due primarily to HO, a known antioxidant enzyme. In summation, the present results when added to the previous findings about D-4F indicate a possible clinical benefit in preventing the cardiovascular complications of diabetes.
Acknowledgments
This work was supported by National Institutes of Health grants HL55601 and the Phillip Morris Management Group (Dr Abraham). We thank Jennifer Brown for her excellent secretarial assistance.
Footnotes
Presented in part at the 77th Scientific Sessions of the American Heart Association, November 7–10, 2004, and published in abstract form (Circulation. 2004;110[suppl III]:III-129).
References
Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR, Wagner AC, Frank JS, Datta G, Garber D, Fogelman AM. Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice. Circulation. 2004; 109: 3215–3220.
Navab M, Ananthramaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fonarow GC, Vahabzadeh K, Hama S, Hough G, Kamranpour N, Berliner JA, Lusis AJ, Fogelman AM. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J Lipid Res. 2004; 45: 993–1007.
Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Hough G, Wagner A, Nakamura K, Garber DW, Datta G, Segrest JP, Hama S, Fogelman AM. Human apolipoprotein AI mimetic peptides for the treatment of atherosclerosis. Curr Opin Investig Drugs. 2003; 4: 1100–1104.
Ou Z, Ou J, Ackerman AW, Oldham KT, Pritchard KA Jr. L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-treated endothelial cells. Circulation. 2003; 107: 1520–1524.
Ou J, Ou Z, Jones DW, Holzhauer S, Hatoum OA, Ackerman AW, Weihrauch DW, Gutterman DD, Guice K, Oldham KT, Hillery CA, Pritchard KA Jr. L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation. 2003; 107: 2337–2341.
Motterlini R, Foresti R, Intaglietta M, Winslow RM. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol. 1996; 270: H107–H114.
Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Easton JW, Vercellotti GM. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem. 1992; 267: 18148–18153.
Hayashi S, Takamiya R, Yamaguchi T, Matsumoto K, Tojo SJ, Tamatani T, Kitajima M, Makino N, Ishimura Y, Suematsu M. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme. Circ Res. 1999; 85: 663–671.
Platt JL, Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med. 1998; 4: 1364–1365.
Nath KA, Haggard JJ, Croatt AJ, Grande JP, Poss KD, Alam J. The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am J Pathol. 2000; 156: 1527–1535.
Stocker R, McDonagh AF, Glazer AN, Ames BN. Antioxidant activities of bile pigments: biliverdin and bilirubin3. Methods Enzymol. 1990; 186: 301–309.
Wiesel P, Patel AP, DiFonzo N, Marria PB, Sim CU, Pellacani A, Maemura K, LeBlanc BW, Marino K, Doerschuk CM, Yet SF, Lee ME, Perrella MA. Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice. Circulation. 2000; 102: 3015–3022.
Wu P, Phillips MI, Bui J, Terilliger EF. Adeno-associated viral vector mediated transgene integration in nondividing cells. J Virol. 1998; 72: 5919–5926.
Otterbein LE, Bach FH, Alam J, Soares M, Tao LH, Wysk M, Davis RJ, Flavell RA, Choi AM. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med. 2000; 6: 422–428.
Leffler CW, Nasjletti A, Yu C, Johnson RA, Fedinec AL, Walker N. Carbon monoxide and cerebral microvascular tone in newborn pigs. Am J Physiol. 1999; 276 (pt 2): H1641–H1646.
Kushida T, LiVolti G, Goodman AI, Abraham NG. TNF-alpha-mediated cell death is attenuated by retrovirus delivery of human heme oxygenase-1 gene into human microvessel endothelial cells. Transplant Proc. 2002; 34: 2973–2978.
Ferris CD, Jaffrey SR, Sawa A, Takahashi M, Brady SD, Barrow RK, Tysoe SA, Wolosker H, Baranano DE, Dore S, Poss KD, Snyder SH. Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol. 1999; 1: 152–157.
Laniado-Schwartzman M, Conners MS, Dunn MW, Levere RD, Kappas A, Abraham NG. Heme oxygenase induction with attenuation of experimentally-induced corneal inflammation. Biochem Pharmacol. 1997; 53: 1069–1075.
Ishikawa K, Navab M, Leitinger N, Fogelman AM, Lusis AJ. Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. J Clin Invest. 1997; 100: 1209–1216.
Abraham NG, Kushida T, McClung J, Weiss M, Quan S, Lafaro R, Darzynkiewicz Z, Wolin M. Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells. Circ Res. 2003; 93: 507–514.
Quan S, Kaminski PM, Yang L, Morita T, Inaba M, Ikehara S, Goodman AI, Wolin MS, Abraham NG. Heme oxygenase-1 prevents superoxide anion associated endothelial cell sloughing in diabetic rats. Biochem Biophys Res Commun. 2004; 315: 509–516.
Faraci FM, Didion SP. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol. 2004; 24: 1367–1373.
Fattman CL, Schaefer LM, Oury TD. Extracellular superoxide dismutase in biology and medicine. Free Radic Biol Med. 2003; 35: 236–256.
Takatsu H, Tasaki H, Kim HN, Ueda S, Tsutsui M, Yamashita K, Toyokawa T, Morimoto Y, Nakashima Y, Adachi T. Overexpression of EC-SOD suppresses endothelial-cell-mediated LDL oxidation. Biochem Biophys Res Commun. 2001; 285: 84–91.
Quan S, Yang L, Shenouda S, Schwartzman ML, Nasjletti A, Goodman AI, Abraham NG. Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin II-mediated increase in oxidative injury. Kidney Int. 2004; 65: 1628–1639.
Da Ros R, Assaloni R, Ceriello A. Antioxidant therapy in diabetic complications: what is new; Curr Vasc Pharmacol. 2004; 2: 335–341.
Zou MH, Shi C, Cohen RA. High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H2 receptor-mediated apoptosis and adhesion molecular expression in cultured human aortic endothelial cells. Diabetes. 2002; 51: 198–203.
Ceriello A, dello Russo P, Amstad P, Cerutti P. High glucose induces antioxidant enzymes in human endothelial cells in culture: evidence linking hyperglycemia and oxidative stress. Diabetes. 1996; 45: 471–477.
Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991; 40: 405–412.
Scheffer PG, Henry RM, Wever EJ, van Rooij GJ, Bos G, Heine RJ, Dekker JM, Diamant M, Stehouwer CD, Nijpels G, Blankenstein MA, Teerlink T. LDL oxidative modifications in well- or moderately controlled type 2 diabetes. Diabetes Metab Res Rev. 2004; 20: 298–304.
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1–E7.
Challah M, Nadaud S, Philippe M, Battle T, Soubrier F, Corman B, Michel JB. Circulating and cellular markers of endothelial dysfunction with aging in rats. Am J Physiol. 1997; 273 (pt 2): H1941–H1948.
Woywodt A, Streiber F, de Groot K, Regelsberger H, Haller H, Haubitz M. Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis. Lancet. 2003; 361: 206–210.
Mohazzab H, Kaminski PM, Fayngersh RP, Wolin MS. Oxygen-elicited responses in calf coronary arteries: role of H2O2 production via NADH-derived superoxide. Am J Physiol. 1996; 270: H1044–H1053.
Reyes-Toso CF, Roson MI, Albornoz LE, Damiano PF, Linares LM, Cardinali DP. Vascular reactivity in diabetic rats: effect of melatonin. J Pineal Res. 2002; 33: 81–86.
Abraham NG, Rezzani R, Rodella L, Quan S, Kruger A, Li Volti G, Goodman AI, Kappas A. Overexpression of heme-oxygenase-1 attenuates endothelial cells sloughing in experimental diabetes. Am J Physiol Heart Circ Physiol. 2004; 287: H2468–H2477.
Milstien S, Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun. 1999; 263: 681–684.
Wever RM, Luscher TF, Cosentino F, Rabelink TJ. Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation. 1998; 97: 108–112.
Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004; 11: 89–97.
Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004; 84: 1381–1478.Review.
Reddy ST, Grijalva V, Ng C, Hassan K, Hama S, Mottahedeh R, Wadleigh DJ, Navab M, Fogelman AM. Identification of genes induced by oxidized phospholipids in human aortic endothelial cells. Vascul Pharmacol. 2002; 38: 211–218.
Briand O, Nizard FM, David-Dufilho M, Six I, Lestavel S, Brunet A, Fruchart JC, Torpier G, Bordet R, Clavey V, Duriez P. Human free apolipoprotein A-I and artificial pre-beta-high-density lipoprotein inhibit eNOS activity and NO release. Biochim Biophys Acta. 2004; 1683: 69–77.
Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991; 88: 2039–2046.
Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A. 1987; 84: 2995–2998.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Morrow JD, Frei B, Longmire AW, Gaziano JM, Lynch SM, Shyr Y, Strauss WE, Oates JA, Roberts LJ. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers: smoking as a cause of oxidative damage. N Engl J Med. 1995; 332: 1198–1203.
Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.
Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, Hamilton CA. Superoxide anion production is increased in a model of genetic hypertension: role of the endothelium. Hypertension. 1999; 33: 1353–1358.
White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A. 1994; 91: 1044–1048.
Dusting GJ, Fennessy P, Yin ZL, Gurevich V. Nitric oxide in atherosclerosis: vascular protector or villain; Clin Exp Pharmacol Physiol Suppl. 1998; 25: S34–S41.(Adam L. Kruger, DO; Steph)
Physiology (P.M.K., M.S.W.), New York Medical College, Valhalla, NY
The Rockefeller University (N.G.A.), New York, NY.
Abstract
Background— Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals.
Methods and Results— We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 μg/100 g of body weight, intraperitoneal injection) on superoxide (O2–), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2– levels were elevated in diabetic rats (74.8±8x103 cpm/10 mg protein) compared with controls (38.1±8x103 cpm/10 mg protein; P<0.01). D-4F decreased O2– levels to 13.23±1x103 (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50±6 cells/mL) than in controls (5±1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20±3 cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0±2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level.
Conclusions— We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.
Key Words: atherosclerosis ; antioxidants ; apolipoproteins ; lipids
Introduction
D-4F is a synthesized mimetic peptide of apolipoprotein A1, the major lipoprotein found in HDL. Prior studies have shown that oral administration of D-4F can reduce atherosclerotic disease independent of cholesterol levels and enhance the ability of HDL to protect against LDL oxidation.1–3 Navab et al1,2 have demonstrated that treatment with D-4F causes HDL to become antiinflammatory, stimulates HDL-mediated cholesterol efflux, and reverses cholesterol transport from macrophages. It has been shown previously that apolipoprotein A1 can restore the balance between nitric oxide (NO) and superoxide (O2–) anions in mice.4 Using a sickle cell disease model, Ou et al5 have shown that apolipoprotein A1 can improve vasoreactivity in LDL-receptor null mice. These early data indicate an antioxidant effect from the administration of apolipoprotein A1 and possibly D-4F.
The realization that heme oxygenase-1 (HO-1) is strongly induced by oxidant stress and its substrate, heme, in conjunction with the robust ability of HO-1 to guard against oxidative insult6–8 has led to examination of the antioxidant nature of HO-1 and HO-2.7,9 Antioxidant effects arise from the capacity of HO-1 to degrade the heme from destabilized heme proteins10 and from the elaboration of biliverdin and bilirubin, which are products of HO with potent antioxidant properties.11 Carbon monoxide (CO), an HO product, is not an antioxidant,12,13 but it has an antiapoptotic effect14 and vasodilator properties.14–16 Additionally, the induction of HO-1 prevents cell death, which is attributed to its augmentation of ion efflux and exportation of iron-binding proteins.17 The protective actions of HO-1 are not confined to overtly oxidant processes but rather extend widely to such disease processes as inflammation8,18 and atherosclerosis.19 Furthermore, upregulation of HO-1 increases the antioxidant system by decreasing O2–.20,21 However, the mechanism by which HO-1 decreases O2– is not clear.
Superoxide dismutase (SOD) is an antioxidant enzyme that converts superoxide (O2–) anions into hydrogen peroxide and molecular oxygen.22 All mammalian tissues contain 3 forms of SOD: copper/zinc SOD (Cu/Zn-SOD), manganese SOD (Mn-SOD), and extracellular SOD (EC-SOD). Under physiological conditions, the basal level of O2– is controlled by mitochondrial and cytosolic SOD.23 Under pathological conditions, such as heart ischemia or inflammatory blood cells, O2– is acted on by EC-SOD instead.24 The vascular concentration of EC-SOD is a key determinant of endothelial function; a decrease in EC-SOD is associated with a decrease in NO bioavailability and an increase in the reaction of NO with superoxide to form peroxynitrite, a potent cytotoxic oxidant.22 In the present study, we measured EC-SOD because HO-1 has been shown to attenuate the increased level of superoxide in diabetics and to increase reduced glutathione levels.21,25 We hypothesized that this effect might be due to an increase in another antioxidant gene such as vascular EC-SOD, which is responsible for metabolizing superoxide. Therefore, we examined the effect of HO-1 on the levels of EC-SOD.
Hyperglycemia-mediated cardiovascular complications and atherosclerosis contribute to the formation of O2–26 and reactive oxygen species, each of which contributes to endothelial dysfunction and apoptosis.27 It has been shown that these abnormalities are reversible with increased expression of antioxidant enzymes or administration of antioxidant agents.28 A reduction in antioxidant reserves has been related to endothelial cell dysfunction in diabetes, even in patients with well-controlled blood glucose levels.27,29 Similarly, it has been shown that individuals with moderately to well-controlled type 2 diabetes mellitus demonstrate an increase in LDL oxidation.30 Conditions associated with an elevation of oxidative stress have been shown to have an increased level of endothelial cell sloughing.31–33 In a recent study, diabetes was also found to be associated with increased endothelial apoptosis.20,21
The primary objective of the present study was to determine whether apolipoprotein D-4F causes an antioxidant effect as a result of increased HO-1–derived bilirubin and CO. Additional objectives were to investigate whether D-4F decreases endothelial cell sloughing and fragmentation or restores vascular reactivity in diabetic rats. The present results demonstrated that treatment with D-4F increased HO-1 protein and activity and EC-SOD. The increase in HO-1 and EC-SOD was associated with restoration in vascular reactivity and the amelioration of endothelial cell sloughing and fragmentation. These results indicate that the antioxidant effect of D-4F operates via an increase in both HO-1 and EC-SOD, both of which cause decreased oxidative stress and attenuate the vascular damage seen with diabetes.
Methods
Animal Treatment
Male Sprague-Dawley rats (Charles River Laboratory, Wilmington, Mass) weighing 170 to 190 g were maintained on standard rat diet and tap water ad libitum. After the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg body weight), diabetes was induced by a single injection, via the tail vein, of streptozotocin (STZ; Sigma; 60 mg/kg body weight) dissolved in 0.05 mol/L citrate buffer (pH 4.5). Age-matched control rats were injected with an equal volume of vehicle (sodium citrate buffer). Rats were divided into 3 groups: control, STZ, and STZ plus D-4F (daily 100 μg/100 g body weight intraperitoneal injections for 6 weeks starting the day after injection of STZ). Blood glucose level was measured 2 days after injection of STZ or vehicle. These rats were used 1 month after induction of diabetes. The Animal Care and Use Committee of New York Medical College approved all experiments.
Measurement of Vascular O2– Levels
Previously described methods34 were used in which control and diabetic vessels were placed in plastic scintillation minivans that contained 5 μm of lucigenin, for detection of O2– and other additions, in a final volume of 1 mL of air-equilibrated Krebs solution buffered with 10 mmol/L HEPES-NaOH (pH 7.4). Lucigenin chemiluminescence was measured in a liquid scintillation counter (LS6000IC, Beckman Instruments) at 37°C, and data are reported as counts/min per milligram of protein after background subtraction.
Tissue Preparation for HO and EC-SOD Measurement
Frozen thoracic aorta segments were pulverized under liquid nitrogen and placed in a homogenization buffer (10 mmol/L phosphate buffer, 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L PMSF, and 0.1% tergitol, pH 7.5). Homogenates were centrifuged at 27 000g for 10 minutes at 4°C. The supernatant was isolated, and protein levels were assayed (Bradford method). The supernatant was used for measurement of HO-1, HO-2, and EC-SOD–Cu/Zn-SOD protein expression and determination of HO activity.
Western Blot Analysis
These measurements were performed to determine HO-1, HO-2, EC-SOD, and Cu/Zn-SOD protein expression. Protein levels were visualized by immunoblotting with antibodies against rat HO-1, HO-2, EC-SOD, and Cu/Zn-SOD (Stressgen Biotechnologies Corp). Briefly, 20 μg of lysate supernatant was separated by 12% SDS/PAGE and transferred to a nitrocellulose membrane (Amersham) with a semidry transfer apparatus (Bio-Rad). The membranes were incubated with 10% milk in 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.05% Tween 20 (TBST) buffer at 4°C overnight. After they were washed with TBST, the membranes were incubated with anti-HO-1, anti-HO-2, anti-EC-SOD, and anti-Cu/Zn-SOD antibodies for 1 hour at room temperature with constant shaking. The filters were washed and subsequently probed with horseradish peroxidase–conjugated donkey anti-rabbit or anti-mouse IgG (Amersham). Chemiluminescence detection was performed with the Amersham ECL detection kit, according to the manufacturer’s instructions.
Measurement of HO Activity
HO activity was assayed as previously described20 in which bilirubin, the end product of heme degradation, was extracted with chloroform and its concentration determined spectrophotometrically (Perkin-Elmer Dual UV/VIS Beam Spectrophotometer Lambda 25) using the difference in absorbance at a wavelength from 460 to 530 nm with an absorption coefficient of 40 mmol–1 · cm–1.
Detection and Quantification of Circulating Endothelial Cells
For immunomagnetic isolation and quantification of endothelial cells, we used monodispersed magnetizable particles (Dynabeads CELLection Pan Mouse IgG kit) obtained from Dynal. The 4.5-μm diameter polystyrene beads are coated with affinity-purified pan-anti-mouse immunoglobulin G1 covalently bound to the surface. The beads were washed according to the manufacturer, with a strong magnet (MPC6, Dynal) used to remove sodium azide. Typically, 100 μL of bead suspension was coated noncovalently with 10 μg/mL RECA-1 (Novus Biologicals), a pan-rat endothelial cell–specific monoclonal antibody diluted 1:10 in PBS 0.1% BSA, by overnight incubation at 4°C with head-over-head agitation. After 3 washes with PBS-BSA to remove excess antibodies, the beads were resuspended in buffer until use. RECA-uncovered particles were used as a negative control. If the beads were stored for a long period of time, 0.1% sodium azide was added to the buffer. Beads and target cells were incubated for 1.5 hours at 4°C on a rotator. The amount of beads (4x108/mL) was calculated to be in great excess of target cells (>2000 beads per target cell). Separation of beads and rosetted cells from the blood samples required a minimum of 1 minute of exposure to the magnet. Three washes were performed to completely remove nonrosetted cells. After the third wash, rosetted cells were recovered in a 150 μL solution of acridine orange (a vital fluorescent dye at final concentration of 5 μg/mL in PBS), and observations were made in a hemacytometer under both white and fluorescent blue excitation with fluorescence microscopy (Olympus IX81 F).
Assessment of Vascular Reactivity
The thoracic aorta was removed and placed in cold Krebs-bicarbonate solution, cleaned of fat and loose connective tissue, and sectioned into 3-mm-long rings. Two rings per aorta were mounted on stainless steel hooks and suspended in 5-mL tissue baths. The bath was filled with Krebs solution at 37°C, pH 7.4, with 95% O2/5% CO2 as a gas phase. Aortic rings were stretched to a 2 g initial tension and were equilibrated for 60 minutes. Tension development was measured by isometric force transducers (GRASS FT03) connected to an amplifier. The Krebs buffer solution in the tissue bath was replaced every 15 minutes, and the tension was readjusted each time. At the end of the equilibration period, the maximal force generated by addition of a depolarizing solution of 60 mmol/L KCl was determined. To evaluate acetylcholine-induced vasodilation, the rings were preconstricted with phenylephrine (3.5x10–7 mol/L) to obtain a stable plateau, and then a cumulative dose-response curve to acetylcholine or NONOate was obtained.
Statistical Analyses
Data are presented as mean±SEM for the number of experiments. Statistical significance (P<0.05) between experimental groups was determined by the Fisher method of analysis of multiple comparisons. For comparison between treatment groups, the null hypothesis was tested by a single-factor ANOVA for multiple groups or unpaired t test for 2 groups.
Results
Effect of D-4F on HO Activity and HO-1 and HO-2 Protein
To define the effect of D-4F on vascular HO-1, we measured HO activity and the protein levels in control and diabetic rats. Compared with controls, the induction of diabetes had no significant effect on either HO-1 or HO-2 protein (Figure 1). Treatment with D-4F resulted in a significant increase in the amount of HO-1 protein compared with control (290%, P<0.001) and untreated diabetic rats (277%, P<0.05). Administration of D-4F had no effect on HO-2. Because HO-1 converts heme to equimolar amounts of CO and bilirubin,20 we measured HO activity by formation of bilirubin. HO activity in control vessels was 0.81±0.03 nmol bilirubin formed/mg protein and decreased to 0.59±0.02 nmol bilirubin formed/mg protein in diabetic vessels (P<0.05). The stimulatory effect of D-4F on HO-1 protein was associated with an increase in HO activity (Figure 1E) in both diabetic (1.14±0.04 nmol bilirubin) and control (01.11±0.02 nmol bilirubin) rats (P<0.0001).
Effect of D-4F on Superoxide Formation
Because D-4F increases HO-1-derived bilirubin formation, we compared the effect of D-4F on superoxide formation. A comparison was made between superoxide levels in the tissues of D-4F–treated diabetic rats, untreated diabetic rats, and controls. Compared with controls, diabetic rats showed an elevation in aorta O2– formation from 38.1±8 to 74.8±8x103 cpm/mg protein (P<0.01). D-4F was a very potent inhibitor of O2– formation. Administration of D-4F decreased O2– by 85% in diabetic rats (Figure 2). D-4F also decreased O2– compared with controls, although not significantly.
Endothelial Cell Sloughing
Endothelial Cell Fragmentation
Under microscopic examination, there was evidence of cellular fragmentation and debris, which was bound by the RECA-1–coated beads and fluoresced after staining with acridine but was too small to be considered as whole cells (Figure 5). Quantification of these fragments (Figure 6) yielded an average of 8.3±2 fragments/mL in controls and 38±4 fragments/mL in untreated diabetic rats (P=0.009). Administration of D-4F to diabetic rats prevented endothelial fragmentation by 80% in diabetic rats; only 6.8±1 fragments/mL was seen in D-4F–treated diabetic rats (P<0.0001 versus untreated diabetics). Treatment with D-4F also decreased endothelial cell fragmentation in control rats to 2.8±1 fragments/mL (P<0.05 versus untreated control). The level of decrease in cellular fragmentation was indicative of cellular protection secondary to the D-4F–mediated decrease in O2–.
Vascular Reactivity
It has been reported that acetylcholine-evoked endothelium-mediated relaxation is impaired in diabetes.35 The present study confirmed a significant impairment in diabetic rats (51±7% relaxation) compared with controls (77±3% relaxation). D-4F restored vascular reactivity in diabetic rats to 85±2% relaxation (Figure 7A). In the presence of the NO donor NONOate, there were no significant changes noted between controls and diabetic rats in response to acetylcholine (Figure 7B). Aorta from diabetic rats treated with D-4F showed an even higher response to NONOate than either control or diabetic vessels at a concentration of 10–6 mol/L (P<0.05).
Effect of HO-1 Expression on the Antioxidant System
In an attempt to elucidate the mechanism by which D-4F attenuates endothelial sloughing and fragmentation and decreases O2–, we examined the effect of D-4F on vascular SOD. As shown in Figure 8, the level of EC-SOD was significantly reduced in diabetic rats compared with controls (P<0.003); however, treatment with D-4F significantly restored EC-SOD protein levels (P<0.001). Treatment with D-4F also increased the amount of EC-SOD in control rats (P<0.02 versus control, P<0.0001 versus diabetic). The levels of Cu/Zn-SOD and Mn-SOD proteins were not affected by D-4F in either control or diabetic rats.
Discussion
This study demonstrates that D-4F participates in the attenuation of diabetic vascular complications, presumably by increases in bilirubin (an antioxidant) and CO (which is cytoprotective) secondary to increased HO-1 and/or by a robust increase in EC-SOD. This conclusion is supported by 4 important key findings.
First, D-4F was shown to decrease the formation of reactive oxygen species, namely, superoxide (O2–). Prior study has indicated elevated O2– in the hyperglycemic state associated with diabetes, which was attenuated with upregulation of HO-1.21,36 A similar decrease in O2– was seen with administration of D-4F. This confirms the antioxidant nature of D-4F and supports earlier studies.1,3 Treatment of diabetic rats with D-4F induced increases of HO-1 and significantly increased EC-SOD but had no effect on the other SOD isoforms (Cu/Zn or Mn). We believe that the action of D-4F is largely or solely through the induction of HO-1; however, the present results, which indicate that D-4F dramatically induces HO-1 and EC-SOD, are novel, and together with our findings that D-4F dramatically reduced endothelial sloughing and improved vasoreactivity in diabetic rats, they constitute important new information about the actions of apolipoprotein A1 mimetic peptides. Although rats have a diminished level of EC-SOD compared with other species, there is evidence of EC-SOD in the vascular tissue.22 There is potentially an important role for EC-SOD in the attenuation of reactive oxygen species and peroxynitrite generation from inducible NO synthase– and endothelial NO synthase–derived NO. Peroxynitrite oxidizes and inactivates the NOS cofactor tetrahydrobiopterin into inactive molecules, such as dihydrobiopterin.37 This uncouples the enzyme, which then preferentially increases O2– production over NO production.37,38 Therefore, the HO-1 gene expression–mediated increase in EC-SOD may protect endothelial NO synthase from uncoupling. This is of particular importance in diabetes, in which hyperglycemia causes increased endothelial NO synthase uncoupling.39 The mechanism by which the HO-1 products CO and/or bilirubin may directly regulate this uncoupled state remains unclear.
Second, administration of D-4F increased both the level and activity of HO-1. These results confirmed our previous study,36 which established that diabetes results in a downregulation of HO-1 level and activity. Administration of D-4F to diabetic rats led to a reversal of this trend and a resultant increase in both the level and activity of HO-1, which strongly suggests a role for HO in the antioxidant effect of D-4F; however, D-4F–mediated HO-1 may be considered as a signaling mechanism in decreasing inducible NO synthase and O2– with an increase in EC-SOD. Gene transfer of antisense HO-1, as a specific inhibitor of HO-1, has been shown to decrease EC-SOD and increase circulating endothelial cells and inducible NO synthase.36
The third major finding indicated the vascular protective effect of D-4F administration by decreasing endothelial cell sloughing and debris. Many of the significant clinical problems associated with diabetes can be traced to vascular damage induced by hyperglycemia.20,27 Unfortunately, even patients with well-controlled blood glucose levels are not totally protected from these side effects.27,29 The increased vascular damage with resultant sloughing and fragmentation of endothelial cells typically found with diabetes was lessened in D-4F–treated animals. This demonstrates a possible clinical role for D-4F in protecting the vasculature from the damage associated with diabetes mellitus.
Finally, D-4F was shown to have positive effects on vascular reactivity to stimulation with acetylcholine. Reactivity of the blood vessels has been shown to be diminished in diabetes.35 Vascular reactivity in control, untreated diabetic, and D-4F–treated diabetic rats did not differ significantly in the presence of NONOate, which indicates a dependence on the bioavailability of NO.
There is an ever-increasing body of evidence that indicates that inflammation and oxidative stress a play major role in the formation of atherosclerotic disease. The oxidative-modification hypothesis indicates that only after oxidation does LDL become atherogenic.40–42 After incorporation into foam cells, oxidized LDL acts to recruit inflammatory cells, including T lymphocytes and monocytes, via O2–.43,44 There is also evidence that oxidative stress plays a role in the mechanisms for the documented risk factors of atherosclerosis, including hypertension, diabetes, and smoking.45,46 In individuals with diabetes, there is increased LDL oxidation even in the face of adequate glucose control.30 Prior study has shown that patients with documented atherosclerosis had LDL that was highly proinflammatory, independent of their total cholesterol or HDL levels.2,3 Superoxide, a marker for oxidative stress, has been implicated in many cardiovascular diseases, including atherosclerosis, and has been shown to rapidly inactivate endothelium-derived NO.47,48 Peroxynitrite, formed from the reaction of superoxide with NO, is known to promote lipid peroxidation, contributing to formation of atherosclerotic lesions.49 Superoxide-mediated impairment of NO may result in vascular smooth muscle cell proliferation and migration and the expression of proinflammatory molecules, which contribute to atherosclerosis.47,50 It is hypothesized that the elevated superoxide seen in the diabetic animals in the present study was vascular in origin, because EC-SOD is primarily vascular in nature. This hypothesis is supported by prior study showing that the apolipoprotein A1 mimetic L-4F can attenuate O2– production in cultured endothelial cells.4,5
In light of these recent discoveries, there is a growing potential for therapeutic agents that can attenuate oxidative stress. Mounting evidence demonstrates that D-4F can protect against oxidative injury, which makes it a potential therapeutic agent in the fight against atherosclerosis. With administration of D-4F, HDL becomes antiinflammatory, which increases HDL-mediated cholesterol efflux and reverses cholesterol transport from macrophages.1 In LDL-receptor null or apolipoprotein E null experimental mice fed Western diets, D-4F administration resulted in a 79% and 75% reduction in atherosclerotic lesions, respectively.3 This is in agreement with the potency of D-4F on induction of the HO-1–mediated increase in EC-SOD and subsequent decrease in O2–. The previously unknown mechanism for the antioxidant role of D-4F has now been shown to be due primarily to HO, a known antioxidant enzyme. In summation, the present results when added to the previous findings about D-4F indicate a possible clinical benefit in preventing the cardiovascular complications of diabetes.
Acknowledgments
This work was supported by National Institutes of Health grants HL55601 and the Phillip Morris Management Group (Dr Abraham). We thank Jennifer Brown for her excellent secretarial assistance.
Footnotes
Presented in part at the 77th Scientific Sessions of the American Heart Association, November 7–10, 2004, and published in abstract form (Circulation. 2004;110[suppl III]:III-129).
References
Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR, Wagner AC, Frank JS, Datta G, Garber D, Fogelman AM. Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice. Circulation. 2004; 109: 3215–3220.
Navab M, Ananthramaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fonarow GC, Vahabzadeh K, Hama S, Hough G, Kamranpour N, Berliner JA, Lusis AJ, Fogelman AM. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J Lipid Res. 2004; 45: 993–1007.
Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Hough G, Wagner A, Nakamura K, Garber DW, Datta G, Segrest JP, Hama S, Fogelman AM. Human apolipoprotein AI mimetic peptides for the treatment of atherosclerosis. Curr Opin Investig Drugs. 2003; 4: 1100–1104.
Ou Z, Ou J, Ackerman AW, Oldham KT, Pritchard KA Jr. L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-treated endothelial cells. Circulation. 2003; 107: 1520–1524.
Ou J, Ou Z, Jones DW, Holzhauer S, Hatoum OA, Ackerman AW, Weihrauch DW, Gutterman DD, Guice K, Oldham KT, Hillery CA, Pritchard KA Jr. L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation. 2003; 107: 2337–2341.
Motterlini R, Foresti R, Intaglietta M, Winslow RM. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol. 1996; 270: H107–H114.
Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Easton JW, Vercellotti GM. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem. 1992; 267: 18148–18153.
Hayashi S, Takamiya R, Yamaguchi T, Matsumoto K, Tojo SJ, Tamatani T, Kitajima M, Makino N, Ishimura Y, Suematsu M. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme. Circ Res. 1999; 85: 663–671.
Platt JL, Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med. 1998; 4: 1364–1365.
Nath KA, Haggard JJ, Croatt AJ, Grande JP, Poss KD, Alam J. The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am J Pathol. 2000; 156: 1527–1535.
Stocker R, McDonagh AF, Glazer AN, Ames BN. Antioxidant activities of bile pigments: biliverdin and bilirubin3. Methods Enzymol. 1990; 186: 301–309.
Wiesel P, Patel AP, DiFonzo N, Marria PB, Sim CU, Pellacani A, Maemura K, LeBlanc BW, Marino K, Doerschuk CM, Yet SF, Lee ME, Perrella MA. Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice. Circulation. 2000; 102: 3015–3022.
Wu P, Phillips MI, Bui J, Terilliger EF. Adeno-associated viral vector mediated transgene integration in nondividing cells. J Virol. 1998; 72: 5919–5926.
Otterbein LE, Bach FH, Alam J, Soares M, Tao LH, Wysk M, Davis RJ, Flavell RA, Choi AM. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med. 2000; 6: 422–428.
Leffler CW, Nasjletti A, Yu C, Johnson RA, Fedinec AL, Walker N. Carbon monoxide and cerebral microvascular tone in newborn pigs. Am J Physiol. 1999; 276 (pt 2): H1641–H1646.
Kushida T, LiVolti G, Goodman AI, Abraham NG. TNF-alpha-mediated cell death is attenuated by retrovirus delivery of human heme oxygenase-1 gene into human microvessel endothelial cells. Transplant Proc. 2002; 34: 2973–2978.
Ferris CD, Jaffrey SR, Sawa A, Takahashi M, Brady SD, Barrow RK, Tysoe SA, Wolosker H, Baranano DE, Dore S, Poss KD, Snyder SH. Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol. 1999; 1: 152–157.
Laniado-Schwartzman M, Conners MS, Dunn MW, Levere RD, Kappas A, Abraham NG. Heme oxygenase induction with attenuation of experimentally-induced corneal inflammation. Biochem Pharmacol. 1997; 53: 1069–1075.
Ishikawa K, Navab M, Leitinger N, Fogelman AM, Lusis AJ. Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. J Clin Invest. 1997; 100: 1209–1216.
Abraham NG, Kushida T, McClung J, Weiss M, Quan S, Lafaro R, Darzynkiewicz Z, Wolin M. Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells. Circ Res. 2003; 93: 507–514.
Quan S, Kaminski PM, Yang L, Morita T, Inaba M, Ikehara S, Goodman AI, Wolin MS, Abraham NG. Heme oxygenase-1 prevents superoxide anion associated endothelial cell sloughing in diabetic rats. Biochem Biophys Res Commun. 2004; 315: 509–516.
Faraci FM, Didion SP. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol. 2004; 24: 1367–1373.
Fattman CL, Schaefer LM, Oury TD. Extracellular superoxide dismutase in biology and medicine. Free Radic Biol Med. 2003; 35: 236–256.
Takatsu H, Tasaki H, Kim HN, Ueda S, Tsutsui M, Yamashita K, Toyokawa T, Morimoto Y, Nakashima Y, Adachi T. Overexpression of EC-SOD suppresses endothelial-cell-mediated LDL oxidation. Biochem Biophys Res Commun. 2001; 285: 84–91.
Quan S, Yang L, Shenouda S, Schwartzman ML, Nasjletti A, Goodman AI, Abraham NG. Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin II-mediated increase in oxidative injury. Kidney Int. 2004; 65: 1628–1639.
Da Ros R, Assaloni R, Ceriello A. Antioxidant therapy in diabetic complications: what is new; Curr Vasc Pharmacol. 2004; 2: 335–341.
Zou MH, Shi C, Cohen RA. High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H2 receptor-mediated apoptosis and adhesion molecular expression in cultured human aortic endothelial cells. Diabetes. 2002; 51: 198–203.
Ceriello A, dello Russo P, Amstad P, Cerutti P. High glucose induces antioxidant enzymes in human endothelial cells in culture: evidence linking hyperglycemia and oxidative stress. Diabetes. 1996; 45: 471–477.
Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991; 40: 405–412.
Scheffer PG, Henry RM, Wever EJ, van Rooij GJ, Bos G, Heine RJ, Dekker JM, Diamant M, Stehouwer CD, Nijpels G, Blankenstein MA, Teerlink T. LDL oxidative modifications in well- or moderately controlled type 2 diabetes. Diabetes Metab Res Rev. 2004; 20: 298–304.
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1–E7.
Challah M, Nadaud S, Philippe M, Battle T, Soubrier F, Corman B, Michel JB. Circulating and cellular markers of endothelial dysfunction with aging in rats. Am J Physiol. 1997; 273 (pt 2): H1941–H1948.
Woywodt A, Streiber F, de Groot K, Regelsberger H, Haller H, Haubitz M. Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis. Lancet. 2003; 361: 206–210.
Mohazzab H, Kaminski PM, Fayngersh RP, Wolin MS. Oxygen-elicited responses in calf coronary arteries: role of H2O2 production via NADH-derived superoxide. Am J Physiol. 1996; 270: H1044–H1053.
Reyes-Toso CF, Roson MI, Albornoz LE, Damiano PF, Linares LM, Cardinali DP. Vascular reactivity in diabetic rats: effect of melatonin. J Pineal Res. 2002; 33: 81–86.
Abraham NG, Rezzani R, Rodella L, Quan S, Kruger A, Li Volti G, Goodman AI, Kappas A. Overexpression of heme-oxygenase-1 attenuates endothelial cells sloughing in experimental diabetes. Am J Physiol Heart Circ Physiol. 2004; 287: H2468–H2477.
Milstien S, Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun. 1999; 263: 681–684.
Wever RM, Luscher TF, Cosentino F, Rabelink TJ. Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation. 1998; 97: 108–112.
Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004; 11: 89–97.
Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004; 84: 1381–1478.Review.
Reddy ST, Grijalva V, Ng C, Hassan K, Hama S, Mottahedeh R, Wadleigh DJ, Navab M, Fogelman AM. Identification of genes induced by oxidized phospholipids in human aortic endothelial cells. Vascul Pharmacol. 2002; 38: 211–218.
Briand O, Nizard FM, David-Dufilho M, Six I, Lestavel S, Brunet A, Fruchart JC, Torpier G, Bordet R, Clavey V, Duriez P. Human free apolipoprotein A-I and artificial pre-beta-high-density lipoprotein inhibit eNOS activity and NO release. Biochim Biophys Acta. 2004; 1683: 69–77.
Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991; 88: 2039–2046.
Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A. 1987; 84: 2995–2998.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Morrow JD, Frei B, Longmire AW, Gaziano JM, Lynch SM, Shyr Y, Strauss WE, Oates JA, Roberts LJ. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers: smoking as a cause of oxidative damage. N Engl J Med. 1995; 332: 1198–1203.
Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.
Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, Hamilton CA. Superoxide anion production is increased in a model of genetic hypertension: role of the endothelium. Hypertension. 1999; 33: 1353–1358.
White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A. 1994; 91: 1044–1048.
Dusting GJ, Fennessy P, Yin ZL, Gurevich V. Nitric oxide in atherosclerosis: vascular protector or villain; Clin Exp Pharmacol Physiol Suppl. 1998; 25: S34–S41.(Adam L. Kruger, DO; Steph)