Atherosclerosis accounts for half of the morbidity and

Transcription

1 Brief Rapid Communications Reduced Endothelial Nitric Oxide Synthase Expression and Production in Human Atherosclerosis Barry S. Oemar, MD; Marcel R. Tschudi, PhD; Nelson Godoy, MD; Victor Brovkovich, PhD; Tadeusz Malinski, PhD; Thomas F. Lüscher, MD Background NO regulates vascular tone and structure, platelets, and monocytes. NO is synthesized by endothelial NO synthase (enos). Endothelial dysfunction occurs in atherosclerosis. Methods and Results With a porphyrinic microsensor, NO release was measured in atherosclerotic human carotid arteries and normal mammary arteries obtained during surgery. enos protein expression was analyzed by immunohistochemistry. In normal arteries, the initial rate of NO release after stimulation with calcium ionophore A23187 (10 mol/l) was ( mol/l)/s (n 10). In contrast, the initial rate of NO release was markedly reduced in atherosclerotic segments, to ( mol/l)/s (n 10, P ). NO peak concentration in normal arteries was mol/l (n 10) and in atherosclerotic segments, mol/l (n 10, P ). Reduced NO release in atherosclerotic segments was accompanied by marked reduction of immunoreactive enos in luminal endothelial cells, although specific endothelial cell markers (CD31) were present (n 13). Endothelial cells of vasa vasorum of atherosclerotic segments, however, remained positive for enos, as was the endothelium of normal arteries. Conclusions In clinically relevant human atherosclerosis, enos protein expression and NO release are markedly reduced. This may be involved in the progression of atherosclerosis. (Circulation. 1998;97: ) Key Words: atherosclerosis arteries endothelium nitric oxide stroke Atherosclerosis accounts for half of the morbidity and mortality in Western countries. Its pathogenesis, however, is not clear. Risk factors of atherosclerosis, ie, LDL levels, diabetes mellitus, hypertension, and smoking, are associated with vascular dysfunction, including monocyte adhesion and invasion, smooth muscle proliferation and migration, platelet activation, and extracellular matrix formation. 1,2 In animal models, the endothelial NO pathway appears to be involved in atherosclerosis. 3 6 However, little is known about this in human atherosclerosis. 7 In endothelial cells, NO is formed from L-arginine 8,9 by enos. 10 enos is constitutively expressed and activated by cell surface receptors or mechanical forces such as shear stress and stretch. 1,11 NO relaxes vascular smooth muscle, inhibits platelet activation, and modulates migration and growth of vascular smooth muscle. Furthermore, NO regulates genes that lead to the expression of adhesion molecules for monocytes Indirect evidence suggests that alterations in the NO pathway might be involved in endothelial dysfunction and atherosclerosis. In hypercholesterolemia and atherosclerosis, endothelium-dependent relaxation is reduced. 3 5 Chronic administration of L-arginine to hypercholesterolemic animals improves endothelium-dependent relaxation. 6 In patients with coronary artery disease, basal NO release seems to be impaired, as suggested by a blunted response to L-NMMA. 7 However, in human atherosclerosis, no direct measurement of NO release or NOS protein has been reported. This study presents the first direct evidence that NO release and enos expression are markedly reduced in clinically manifest human atherosclerosis. Methods Blood Vessels Thirty-five arteries of patients undergoing carotid atherectomy or coronary bypass surgery were collected. For NO measurement, carotid arteries from 10 patients (mean age, 66 years; range, 53 to 78 years) and IMAs (normal segments) from 10 age-matched control subjects (mean age, 66 years; range, 54 to 75 years; P NS) were used. The presence or absence of atherosclerosis was confirmed visually, microscopically, and by histology. The absence of atherosclerosis in IMAs is in line with previous studies. 15 Immunohistochemistry for enos and CD31 was performed in 13 carotid arteries and 2 IMAs. Porphyrinic NO Microsensor Measurements of NO were carried out with a porphyrinic microsensor as described 16,17 immediately after surgical removal of segments ( 30 minutes). The amperometric signal was recorded with a chart recorder (Recom Electronic AG), and NO concentration was determined from the measured current by means of a calibration curve Received March 3, 1998; revision received April 21, 1998; accepted April 28, From Cardiovascular Research, Institute of Physiology, University of Zürich (B.S.O., M.R.T.); Cardiology, University Hospital Zürich (T.F.L.); and Neurosurgery, University Hospital Bern (N.G.), Switzerland; and the Department of Chemistry, Institute of Biotechnology, Oakland University, Rochester, Minn (V.B., T.M.). Correspondence to Thomas F. Lüscher, MD, Professor and Head of Cardiology, University Hospital Zürich, CH-8091 Zürich, Switzerland. karluh@usz.unizh.ch 1998 American Heart Association, Inc. 2494

2 Oemar et al June 30, Selected Abbreviations and Acronyms enos endothelial NO synthase IMA internal mammary artery L-NMMA N G -monomethyl-l-arginine NOS NO synthase with NO standards. Standard NO solutions (1.8 mmol/l) were prepared from aqueous solution saturated with pure gaseous NO (Garbagas). Experimental Protocols Isolated vascular segments were cut longitudinally and pinned on the bottom of organ chambers filled with fresh HBSS buffer. The active tip of the L-shaped NO microsensor was placed on the endothelial surface of the IMA or carotid strips. A precision stereo zoom microscope (PZM) and a micromanipulator (M3301) were used for positioning. Ten microliters of a 10 mol/l calcium ionophore A23187 solution was injected on the luminal side of the vascular strips with a pneumatic picoinjector (PV820) positioned with a micromanipulator (PZM, PV820, and M3301 are all from World Precision Instruments). In each tissue sample, NO release was measured at 3 different anatomic sites. Immunohistochemistry Serial paraformaldehyde-fixed paraffin sections were mounted onto silane-coated slides, dewaxed and rehydrated, washed in PBS, and incubated in 1 g/ml anti-enos monoclonal antibodies (Transduction Laboratories; 2 hours at room temperature or overnight at 4 C). Adjacent sections were incubated in a 1:100 dilution of primary monoclonal antibodies for CD-31 (JC/70A; Dako) or preimmune control serum for 2 hours at room temperature, washed in PBS for 10 minutes, incubated in biotinylated secondary antibody for 30 minutes at room temperature, and visualized by use of the avidin-biotinperoxidase labeling system (ABC-elite kit; Vector Laboratories). Sections were counterstained with hematoxylin and mounted with Kaiser s solution (Merck). Calculations and Statistical Analysis For statistical analysis, the initial rate of NO release [slope of NO peak, ( mol/l)/s] and the maximal concentration of NO produced (NO peak; mol/l) were measured. In each tissue sample, NO release was measured at 3 different anatomic sites, and the mean value was calculated for n 1. In each set of experiments, n is the number of blood vessels studied. Data are given as mean SEM. Statistical analysis was performed with unpaired Student s t test. Values of P 0.05 were considered statistically significant. Results NO Release Representative amperometric curves of NO release are shown in Figure 1A. In normal segments, a rapid release of NO was observed after injection of 10 mol/l calcium ionophore A23187 solution (Figure 1A, top). In contrast, the initial rate of NO release and NO peak concentration were significantly reduced in atherosclerotic carotid arteries (Figure 1A, bottom). The initial rate of NO release after calcium ionophore administration was ( mol/l)/s in normal arteries but only ( mol/l)/s in atherosclerotic carotid arteries (Figure 1B, left; n 10, P ). NO peak concentration was mol/l in normal arteries but was reduced to mol/l in atherosclerotic arteries (Figure 1B, right; n 10, P ). Figure 1. A, Amperograms of NO release from normal IMA (top) and atherosclerotic carotid artery (bottom) to calcium ionophore A23187 (10 mol/l) measured on endothelial surface with a porphyrinic microsensor. B, Initial rate of NO release (left) and maximal concentration of NO (right) to calcium ionophore A23187 (10 mol/l) of isolated IMAs (solid bars) and carotid arteries (open bars). Values are mean SEM (P , n 10). Expression of enos Labeling of normal arterial segments with monoclonal antibody for enos revealed high-level expression of enos in luminal endothelial cells (Figure 2A). In contrast, immunoreactive enos was undetectable in luminal endothelial cells of carotid segments with advanced atherosclerosis (Figure 3A). However, endothelial cells of the vasa vasorum inside the atherosclerotic plaques were strongly positive for enos staining (Figure 4A and 4C), indicating that the negative staining in luminal endothelial cells was not due to technical failure but rather reflects downregulation of the enzyme. Smooth muscle cells of normal segments and of atherosclerotic carotid arteries as well as foam cells and macrophages in the plaque were negative for enos. Expression of Endothelial Cell Marker Anti-CD31 staining, a specific marker for endothelial cells, was positive in all instances, indicating the presence of endothelial cells (Figures 2C, 3C, and 4D) and demonstrating that both enos-expressing cells in normal arterial segments and enos-negative cells in atherosclerotic carotid arteries were indeed endothelial cells. The negative staining with control serum demonstrated the specificity of both anti-enos and anti-cd31 antibodies (Figures 2B, 3B, and 4B).

3 2496 enos and Human Atherosclerosis Figure 2. Immunohistology of normal IMA. Serial sections were labeled with anti-enos antibody (A), control nonimmune serum (B), or monoclonal anti-cd31 antibody (C). Positive staining is indicated by dark purple color of luminal endothelial cells. Positive staining for enos (A) demonstrated normal expression of enos protein in CD31-positive (C) endothelial cells. Arrows indicate position of internal elastic lamina. Sections were counterstained with hematoxylin. Magnification 400. LU indicates lumen. Discussion This study provides the first direct evidence that NOS protein expression and NO release are diminished in advanced human atherosclerosis. Initial and peak NO release as determined by direct in situ measurement with a porphyrinic sensor were markedly reduced in atherosclerotic carotid arteries. The impaired NO release was accompanied by a substantial reduction of immunoreactive enos protein in luminal endothelial cells of atherosclerotic plaques but not in vasa vasorum. Figure 3. Immunohistology of atherosclerotic carotid artery. Serial sections were labeled with anti-enos antibody (A), control nonimmune serum (B), or monoclonal anti-cd31 antibody (C). Positive staining is indicated by dark purple color of luminal endothelial cells. Negative staining for enos (A) demonstrated diminished expression of enos protein in CD31-positive (C) endothelial cells. Arrows indicate intact endothelial cell layer in these areas. Sections were counterstained with hematoxylin. Magnification 400. LU indicates lumen. Endothelial NO plays an important role in vascular homeostasis, adhesion of white blood cells, and platelet function. 10 There are at least 3 major mechanisms in the NO pathway that may lead to endothelial dysfunction and consequently atherogenesis: (1) functional abnormalities of NOS due to substrate or cofactor deficiency 10,18 ; (2) increased breakdown of NO 3,5 ; or (3) reduced expression of enos. Until now, even with invasive in vivo studies in humans, no clear distinction between these possibilities could be made. 7 Moreover, studies of the NO pathway in rabbit models of

4 Oemar et al June 30, Figure 4. Immunohistology of atherosclerotic carotid artery. Serial sections were labeled with anti-enos antibody (A and C), control nonimmune serum (B), or monoclonal anti-cd31 antibody (D). Positive staining is indicated by dark purple color. Endothelial cells of vasa vasorum inside plaque are positive for enos. Positive labeling for CD31 confirms endothelial cells. Sections were counterstained with hematoxylin. Magnification 400. atherosclerosis gave unexpected results, ie, both enos protein and mrna are increased in the atherosclerotic aorta of these animals, despite impaired endothelium-dependent vascular relaxation (most likely due to inactivation of NO by superoxide 4 ). In contrast, our results provide the first direct evidence that enos protein expression is markedly diminished in advanced human atherosclerosis and NO release is reduced. These findings are supported by studies in explanted vein grafts in which enos expression is reduced only in diseased segments. 19 The differences in NO release observed in normal and atherosclerotic segments were mirrored by marked differences in NOS expression and are in line with previous work showing potent endothelium-dependent relaxations in normal mammary arteries 20 and reduced responses in atherosclerotic human coronary arteries. 21 We confirmed the presence of endothelial cells in both normal and atherosclerotic segments by light microscopy and by endothelial cell marker (CD31). A heterogeneity in vascular beds is an unlikely explanation for the observed differences, because both mammary and carotid arteries are branches of the subclavian artery and brachiocephalic trunk. Differences in NO production due to anatomic heterogeneity in receptor expression could be excluded by use of the receptor-independent agonist calcium ionophore. Moreover, the amount of NO released from normal arteries was comparable to that of other normal vessels. 17 Finally, it should be noted that IMA samples were obtained from age-matched patients with documented atherosclerosis in the coronary circulation and angiographically normal mammary artery. The discrepancies between our findings in human atherosclerosis and animal models might be explained by the duration of the atherosclerotic process, ie, months in Watanabe rabbits versus decades in patients. Furthermore, the severity of clinically relevant lesions and species differences may play a role. Indeed, vascular lesions of hypercholesterolemic rabbits mimic human plaques only in part. Nevertheless, it is conceivable that enos protein expression and NO release may be normal or increased in early human atherosclerosis, whereas at later stages NOS expression and NO release are reduced. Hence, abnormal endothelium-dependent responses during early atherosclerosis might be due to reduced substrate and/or cofactor concentrations or increased superoxide formation. This could explain why the administration of L-arginine 22 or the cofactor tetrahydrobiopterin 18 improves endothelial function only in early stages of atherosclerosis and in the microcirculation, where no plaques develop, but not in epicardial coronary arteries of patients with clinically relevant coronary disease. 22 Thus, at later stages of the atherosclerotic process, when enos expression and NO production are decreased, these therapies are no longer effective.

5 2498 enos and Human Atherosclerosis Acknowledgments This work was supported by Swiss National Science Foundation grants , , and /1; the Swiss Cardiology Foundation; and in part by grant HL from the US Public Health Service. The help of Drs D.S. Hartman, K. Hischikawa, and M. Pech is appreciated. References 1. Lüscher TF, Noll G. The endothelium in coronary vascular control. Heart Disease. 1995;Update 3: Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362: Harrison DG, Freiman PC, Armstrong ML, Marcus ML, Heistad DD. Alterations of vascular reactivity in atherosclerosis. Circ Res. 1987;61: Kanazawa K, Kawashima S, Mikami S, Miwa Y, Hirata K, Suematsu M, Hayashi Y, Itoh H, Yokoyama M. Endothelial constitutive nitric oxide synthase protein and mrna increased in rabbit atherosclerotic aorta despite impaired endothelium-dependent vascular relaxation. Am J Pathol. 1996;148: Minor R Jr, Myers PR, Guerra R Jr, Bates JN, Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest. 1990;86: Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90: Quyyumi AA, Dakak N, Mulcahy D, Andrews NP, Husain S, Panza JA, Cannon RO. Nitric oxide activity in the atherosclerotic human coronary circulation. J Am Coll Cardiol. 1997;29: Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987; 327: Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;333: Cooke JP, Dzau VJ. Nitric oxide synthase: role in the genesis of vascular disease. Annu Rev Med. 1997;48: Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996;93: Khan BV, Harrison DG, Olbrych MT, Alexander RW, Medford RM. Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells. Proc Natl Acad Sci U S A. 1996;93: Takahashi M, Ikeda U, Masuyama JI, Funayama H, Kano S, Shimada K. Nitric oxide attenuates adhesion molecule expression in human endothelial cells. Cytokine. 1996;8: Tsao PS, Buitrago R, Chan JR, Cooke JP. Fluid flow inhibits endothelial adhesiveness: nitric oxide and transcriptional regulation of VCAM-1. Circulation. 1996;94: Sims FH. The pathology of the internal thoracic artery and its contribution to the study of atherosclerosis. In: Green GE, Singh RN, Sosa JA, eds. Surgical Revascularization of the Heart: The Internal Thoracic Arteries. New York, NY: Igaku-Shoin; 1991; Malinski T, Taha Z. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature. 1992;358: Tschudi MR, Barton M, Bersinger NA, Moreau P, Cosentino F, Noll G, Malinski T, Lüscher TF. Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary artery. J Clin Invest. 1996;98: Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Lüscher T, Rabelink T. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest. 1997;99: Buttery LD, Chester AH, Springall DR, Borland JA, Michel T, Yacoub MH, Polak JM. Explanted vein grafts with an intact endothelium demonstrate reduced focal expression of endothelial nitric oxide synthase specific to atherosclerotic sites. J Pathol. 1996;179: Lüscher TF, Diederich D, Siebenmann R, Lehmann K, Stulz P, von Segesser L, Yang ZH, Turina M, Grädel E, Weber E, Bühler FR. Difference between endothelium-dependent relaxation in arterial and in venous coronary bypass grafts. N Engl J Med. 1988;319: Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5 -monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest. 1987;79: Drexler H, Zeiher AM, Meinzer K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolaemic patients by L-arginine. Lancet. 1991;338: