| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 1998;98:2000-2003.)
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports |
Distribution of Inflammatory Cells in Atherosclerotic Plaques Relates to the Direction of Flow
From the Department of Cardiovascular Pathology, Academic Medical Center, University of Amsterdam, Netherlands.
Correspondence to Anton E. Becker, MD, Department of Cardiovascular Pathology, Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, Netherlands. E-mail m.i.schenker{at}amc.uva.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—The distribution of macrophages and smooth muscle cells (SMCs) within atherosclerotic plaques is highly variable. This is clinically relevant because these cell types have opposite effects on the stability of atherosclerotic plaques. The present study was designed to investigate whether local variations in arterial flow over the plaque surface could relate to differences in the distribution of SMCs and macrophages in plaques.
Methods and Results—Thirty-three entire carotid plaques were collected at autopsy and marked at their proximal (in relation to the direction of the blood flow) ends, and the cell composition of upstream parts (where high flow and high shear prevail) was compared with that of downstream parts (low flow and low shear stress). Seventy percent of plaques showed more SMCs in their downstream part, and 67% of plaques contained more macrophages in the upstream part. Immunostained macrophage areas were larger in upstream parts (P=0.011). Immunostained SMC areas were larger in downstream parts (P=0.031). Rupture sites of 6 of 9 ruptured plaques were in the upstream part.
Conclusions—Significant differences in cell composition between upstream and downstream parts of plaques indicate a role for arterial flow in the distribution of different cell types. The low-flow/low-shear downstream areas of plaques contain significantly more SMCs, which could provide the background for slowly progressive growth at distal ends of plaques. The significantly high number of macrophages in the upstream areas suggests a relationship between high flow/high shear and plaque instability.
Key Words: plaque • stress • carotid arteries • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Atherosclerotic plaques show marked variability with respect to the distribution of inflammatory cells, not only from one lesion to the other but also within one and the same plaque.1 2 3 This phenomenon is significant because plaque inflammation is widely considered to play a role in plaque destabilization and, eventually, plaque erosion and rupture.1 4 5 6 7 8 By the same token, plaques dominated by smooth muscle cells (SMCs) are considered stable. Indeed, coronary atherectomy specimens obtained from patients with chronic stable angina contain SMCs as the dominant cellular component, but in those obtained from patients with unstable angina or acute myocardial infarction, inflammation prevails.4 9 10
In view of these considerations, it is important to know which mechanisms could be responsible for these major variations in the cellular composition of atherosclerotic plaques. Hemodynamic factors, such as shear stress, are considered to play a role in plaque growth,11 12 but thus far the effect of these factors on the cellular composition of atherosclerotic plaques has not been studied. This may well be an interesting enterprise, because the geometry of a bulging plaque dictates differences in the impact of blood flow in relation to the direction of flow. In fact, the luminal endothelial lining on the upstream (proximal) sites of a plaque is under high shear stress, whereas at downstream (distal) sites, low shear stress prevails.13 14 We speculated that these differences may also have implications for local variation in the cellular composition of plaques.
To verify this hypothesis, we investigated the relationship between blood flow direction and the cellular composition of carotid plaques by quantitatively comparing SMC and macrophage contents of the upstream shoulder part of plaques with those of the downstream shoulder parts. The common carotid artery with its bifurcation site was chosen, because previous studies by Zarins et al12 and Ku et al15 have shown that these arterial sites may serve as a good model to investigate the relationship between fluid dynamics and atherosclerosis and also because the carotid bifurcation is a predilection site for plaque formation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Tissue Sampling
Sixty-three left and right carotid artery bifurcations were studied in a consecutive autopsy series of 45 patients (mean age, 69 years; range, 26 to 89 years). In all cases, the postmortem interval was <24 hours. Excluded from the study were immunocompromised patients and patients who had died under septic conditions. Left and right carotid arteries were carefully dissected from the surrounding tissues, and a segment was removed that contained 10 mm of the common carotid artery at the upstream end, the carotid bifurcation, and 10 mm of the internal and the external carotid arteries at the downstream ends. The arterial segment was opened longitudinally along its ventral side, fixed in 4% buffered formalin, and, if necessary, decalcified in EDTA for 4 days. Arteries with total luminal occlusion were excluded; none of the arteries showed recanalization. From the remaining samples, a longitudinal transmural tissue block of
20x4 mm was removed
from both the internal and external carotid arteries. These tissue
blocks were marked immediately with india ink at the upstream
(proximal) site to ensure their topographic relation with the flow
direction. They were then routinely processed for paraffin embedding
and microscopic sectioning (Figure 1
).
|
Light Microscopy
Serial sections 6 µm thick were cut parallel to the long
axis of the arterial segment, and 2 sections were stained
with hematoxylin-eosin and an elastic–van Gieson stain for screening.
Arterial segments that appeared to contain diffuse
atherosclerosis or fatty streaks, as well as
plaque-free segments, were all excluded from the study. Ruptured or
eroded plaques were excluded from morphometric evaluation; these
plaques were studied to determine at which site of the plaque (upstream
or downstream) rupture or erosion had occurred. The remaining segments,
containing raised plaques with intact upstream and downstream shoulders
and cap parts, were used for further investigation (Figure 1
).
Immunohistochemistry
Adjacent serial sections were stained for SMCs with an
anti–
-actin antibody (SMA, clone 1A4, DAKO, dilution 1:200).
Macrophages were stained with an anti-CD68 antibody (clone
PG-M1, DAKO, dilution 1:100). A 3-step indirect streptavidin-biotin
technique with peroxidase was used, in which final visualization of the
peroxidase activity was performed with diaminobenzidine as chromogen.
Nuclei were faintly counterstained with hematoxylin. In negative
controls, the primary antibody was replaced by an irrelevant mouse
monoclonal antibody of the same subclass.
Morphometry
Surface areas of the upstream (proximal) shoulder and the
downstream (distal) shoulder of plaques were planimetrically quantified
in tissue sections with image-analysis software running on a PC
connected with a video-mounted microscope. The shoulder parts were
defined as the plaque area reaching from the adjacent normal intima
(upstream or downstream) to the outer sides of the lipid core
(atheroma) at both ends (Figure 1). These areas were
outlined manually, and the percentage of immunostained
surface was measured automatically with gray-scale detection. In this
way, we calculated the anti-CD68 (macrophages) and
anti–-actin (SMC) immunopositive areas as a percentage of the total
area of each shoulder part in square millimeters. The ratios of
macrophage-positive areas and SMC-positive areas were
calculated for each plaque individually. Results were recorded
as mean±SD.
Statistical Analysis
For comparison of morphometric data between different plaque
areas, which were not compatible with a normal frequency distribution,
a paired Student's t test with the logarithmic
transformation of individual values was used (±SD). Values of
P<0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Six carotid arteries (10%) were occluded. Histopathological analysis of the carotid artery samples resulted in 41 plaque-free segments (36%), 31 segments containing diffuse atherosclerosis (27%), and 9 segments showing rupture or erosion (8%). The site of rupture or erosion was upstream in 6 of the 9 plaques. Each of the remaining 33 carotid artery segments (29%) contained an entire atherosclerotic plaque, and these were used for further study (Figure 2
).
Immunohistochemistry revealed large variations in the numbers of SMCs
and macrophages among plaques of different patients but also
within one and the same plaque. Some plaques contained only SMCs,
whereas others were almost totally infiltrated by macrophages.
On average, however, SMC areas were larger than macrophage
areas.
|
The results of area quantification of SMCs and
macrophage areas in the immunostained sections are
shown in the Table. In 23 of the
33 sections (70%) stained with anti– -actin, the SMC areas of
the downstream (distal) shoulder were larger than those of the upstream
shoulder part, with an upstream/downstream ratio [ln(U/D) ratio] of
-0.41. In the downstream (distal) shoulder, the SMC areas were
significantly larger than in the upstream (proximal) shoulder
(P=0.031) (Figure 2). In 22 of 33 cases (67%),
anti-CD68–stained sections showed larger macrophage areas in
the upstream (proximal) shoulder part of the plaque, with a U/D ratio
[ln(U/D)] of 0.74. Macrophage areas were significantly larger
in the upstream shoulder areas of plaques (P=0.011) (Figure 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Several risk factors for atherosclerosis, including hemodynamic factors, may be implicated in determining the cellular composition of plaques. To the best of our knowledge, this is the first study that shows marked topographic variation in cellular composition within atherosclerotic plaques related to the direction of the blood stream. Planimetric quantification of the macrophage contents in carotid plaques showed statistically significantly larger macrophage-rich areas in the upstream shoulder than in the downstream shoulder of the same atherosclerotic lesion. This phenomenon may render the upstream part of an atherosclerotic plaque more vulnerable to erosion or rupture than the downstream part. Indeed, 6 of 9 rupture sites were upstream. Differences in fluid mechanics at the luminal site of different regions of the plaque could be responsible for differences in plaque architecture.
The carotid artery has been used by several investigators to study the relationship between flow dynamics and plaque formation. Zarins et al12 and Ku et al15 showed that the upstream sites of plaques are preferentially under high flow/high shear stress, whereas downstream parts are under low flow/low shear stress. Plaque growth, moreover, has been shown to occur predominantly in regions of low shear stress.11 12 15 16 17 18 A recent angiographic study of femoral arteries also revealed that plaque growth in the downstream direction occurs significantly more frequently than in the upstream direction.19
It is known from in vitro studies of endothelial cells under shear stress conditions that high shear stress induces increased expression of endothelial adhesion molecules, such as ICAM and VCAM, resulting in enhanced leukocyte adherence, including monocytes and lymphocytes.20 21 Conversely, other investigators found a relationship between low shear and macrophage infiltration due to prolonged and intimate contacts between mononuclear cells and the endothelial lining.11 12 However, in the in vivo situation of human arteries, shear stress may not be the only rheological factor interfering with leukocyte adherence and influx. Recently, Tropea et al22 studied the differences in monocyte binding upstream and downstream to artificial coarctations in lipid-fed New Zealand White rabbits. They demonstrated that monocyte adhesion and VCAM-1 expression were increased upstream of the stenosis, which resulted in enhanced intimal thickening and accumulation of macrophages at these sites.
Because the common carotid artery conveys blood with high flow and high kinetic energies, one may assume that at sites of atherosclerotic plaques, similar mechanisms are involved, as described by Tropea et al.22 This, then, could account for the large macrophage-rich areas in the upstream shoulder of the lesions. Nevertheless, the dominant overall cell type in most plaques appeared to be the SMC. In individual plaques, however, the downstream shoulders showed (on average) larger SMC-rich areas than their upstream counterparts. This is of interest because an increase of shear stress activates endothelium-derived nitric oxide (NO) synthase and NO production,23 24 and a chronic increase in NO has an inhibitory effect on SMC protein synthesis and SMC proliferation.23 24 In areas with low shear stress, such as the downstream parts, there is no step-up in NO production. In fact, it has been shown that low shear stress increases endothelin production, which acts as a stimulating factor for the production of extracellular matrix components by SMCs and for SMC proliferation.25 Platelet adherence in low-flow areas, with release of platelet-derived growth factor and basic fibroblast growth factor, could provide another stimulus for SMC growth.2 26 SMC growth with connective tissue production is generally considered to be the mechanism responsible for a gradually progressive growth of atherosclerotic plaques.2 These phenomena, therefore, could provide an explanation for the differences in SMC content between the upstream and the downstream parts of plaques and the slowly progressive growth downstream of the main lesion.
Obviously, shear stress cannot be the only factor involved. It is likely that a balance between local hemodynamic variables, such as pulse pressure, wall stress, and turbulence, all play a role in the eventual component makeup of an atherosclerotic plaque.11 12 15 16 17 27 And because this study is based on specimens obtained at autopsy, without much clinical information, a variety of intrinsic and environmental risk factors for plaque development and growth could have been involved, which could be reflected in variability in macrophage and SMC contents in plaques of different patients. Conversely, one may anticipate that such factors affect the overall composition of plaques rather than inducing local changes. However, it appears from this study that the overall effect of the variables involved in human plaque formation results in increased macrophage infiltration at the upstream site and increased SMC growth at the downstream sites.
Our observations in human carotid arteries are of clinical relevance. Plaque instability leading to plaque rupture is considered to be a disbalance between reparative activities (SMC growth and collagen synthesis) and degrading activities induced by macrophages.1 7 8 Therefore, the large amounts of macrophages in the upstream parts of plaques could indicate a relationship between high flow/high shear stress and plaque instability.
|
Acknowledgments |
|---|
The authors thank Dr ir J. Oosting for assistance with the statistical analyses. Technical and secretarial assistance was provided by Hanneke Ploegmakers and Marsha Schenker, respectively.
Received March 3, 1998; revision received June 24, 1998; accepted July 1, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Davies MJ, Richardson PD, Woolf N, Katz DR, Mann J. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage, and smooth muscle cell content. Br Heart J. 1993;69:377–381.
2. Gown AM, Tsukada T, Ross R. Human atherosclerosis, II: immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191–207.[Abstract]
3. van der Wal AC, Becker AE, van der Loos CM, Tigges AJ, Das PK. Fibrous and lipid-rich plaques are part of interchangeable morphologies related to inflammation: a concept. Coron Artery Dis. 1994;5:463–469.[Medline] [Order article via Infotrieve]
4.
Moreno PR, Falk E, Palacios JF, Newell JB, Fuster V,
Fallon JT. Macrophage infiltration in acute coronary
syndromes: implications for plaque rupture. Circulation. 1994;90:775–778.
5.
van der Wal AC, Becker AE, van der Loos CM, Das PK.
Site of intimal rupture or erosion of thrombosed coronary
atherosclerotic plaques is characterized by an inflammatory process
irrespective of the dominant plaque morphology. Circulation. 1994;89:36–44.
6. Richardson PD, Davies MJ, Born GVR. Influence of plaque configuration and stress distribution on fissuring of coronary atherosclerotic plaques. Lancet. 1989;2:941–944.[Medline] [Order article via Infotrieve]
7. Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques. Circulation. 1995;92:1565–1569.
8. Lendon CL, Davies MJ, Born GVR, Richardson PD. Atherosclerotic plaque caps are locally weakened when macrophages density is increased. Atherosclerosis. 1991;87:87–90.[Medline] [Order article via Infotrieve]
9. Davies MJ. A macro and microview of coronary vascular insult in ischaemic heart disease. Circulation. 1990;82(suppl II):II-38–II-46.
10.
van der Wal AC, Becker AE, Koch KT, Piek JJ, Teeling P,
van der Loos CM, David GK. Clinically stable angina pectoris is not
necessarily associated with histologically stable
atherosclerotic plaques. Heart. 1996;76:312–316.
11. Glagov S, Zarins C, Giddens DP, Ku DN. Hemodynamics and atherosclerosis: insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med. 1988;112:1018–1031.[Medline] [Order article via Infotrieve]
12.
Zarins CK, Giddens DP, Bharadvaj BK, Sottiurai VS,
Mabon RF, Glagov S. Carotid bifurcation
atherosclerosis: quantitative correlation of plaque
localization with flow velocity profiles and wall shear stress.
Circ Res. 1983;53:502–514.
13. Zarins CK, Bomberger RA, Glagov S. Local effects of stenoses: increased flow velocity inhibits atherogenesis. Circulation. 1981;64(suppl II):II-221–II-227.
14.
Levesque MJ, Liepsch D, Moravec S, Nerem RM.
Correlation of endothelial cell shape and wall shear
stress in a stenosed dog aorta.
Arteriosclerosis. 1986;6:220–229.
15.
Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow
and atherosclerosis in the human carotid bifurcation:
positive correlation between plaque location and low and oscillating
shear stress. Arteriosclerosis. 1985;5:293–302.
16. Moore JE Jr, Xu C, Glagov S, Zarins CA, Ku DN. Fluid wall shear stress measurements in a model of the human abdominal aorta: oscillatory behavior and relationship to atherosclerosis. Atherosclerosis. 1994;110:225–240.[Medline] [Order article via Infotrieve]
17.
Asakura T, Karino T. Flow patterns and spatial
distribution of atherosclerotic lesions in human coronary
arteries. Circ Res. 1990;66:1045–1066.
18.
Gibson CM, Diaz L, Kandarpa K, Sacks FM, Pasternak RC,
Sandor T, Feldman C, Stone PH. Relation of vessel wall shear stress to
atherosclerosis progression in human coronary
arteries. Arterioscler Thromb. 1993;13:310–315.
19.
Smedby O. Do plaques grow upstream or downstream? An
angiographic study in the femoral artery. Arterioscler Thromb
Vasc Biol. 1997;17:912–918.
20. Gimbrone MA Jr, Cybulsky MI, Kume N, Collins T, Resnick N. Vascular endothelium: an integrator of pathophysiological stimuli in atherogenesis. Ann N Y Acad Sci. 1995;748:122–132.[Medline] [Order article via Infotrieve]
21.
Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL.
Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries
exposed to altered shear stress. Arterioscler Thromb Vasc
Biol. 1995;15:2–10. (Published erratum appears in
Arterioscler Thromb Vasc Biol. 1995;15:429.)
22. Tropea BI, Huie P, Cooke JP, Tsao PS, Sibley RK, Zarins CK. Hypertension-enhanced monocyte adhesion in experimental atherosclerosis. J Vasc Surg. 1996;23:596–605.[Medline] [Order article via Infotrieve]
23.
Kolpakov V, Gordon D, Kulik TJ. Nitric
oxide–generating compounds inhibit total protein and collagen
synthesis in cultured vascular smooth muscle cells. Circ
Res. 1995;76:305–309.
24. Garg UC, Hassid A. Nitric oxide-generating vasodilatators and 8-bromo-cyclic GMP inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774–1777.
25. Yoshizumi M, Kurihara H, Sugiyama T, Takaku F, Yanagisawa M, Masaki T, Yazaki Y. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biomech Biophys Res Commun. 1989;161:859–864.
26. Sterpetti AV, Cucina A, Fragale A, Lepidi S, Cavallaro A, Santoro-D'Angelo L. Shear stress influences the release of platelet derived growth factor and basic fibroblast growth factor by arterial smooth muscle cells. Eur J Vasc Surg. 1994;8:138–142.[Medline] [Order article via Infotrieve]
27.
Davies PF, Remuzzi A, Gordon EJ, Dewey CF, Gimbrone MA.
Turbulent fluid shear stress induces vascular
endothelial cell turnover in vitro. Proc Natl
Acad Sci U S A. 1986;83:2114–2117.
This article has been cited by other articles:
 |
|
 |
|
  N. Gonzalo, H. M. Garcia-Garcia, E. Regar, P. Barlis, J. Wentzel, Y. Onuma, J. Ligthart, and P. W. Serruys In Vivo Assessment of High-Risk Coronary Plaques at Bifurcations With Combined Intravascular Ultrasound and Optical Coherence Tomography J. Am. Coll. Cardiol. Img., April 1, 2009; 2(4): 473 - 482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. J. H. Gijsen, J. J. Wentzel, A. Thury, F. Mastik, J. A. Schaar, J. C. H. Schuurbiers, C. J. Slager, W. J. van der Giessen, P. J. de Feyter, A. F. W. van der Steen, et al. Strain distribution over plaques in human coronary arteries relates to shear stress Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1608 - H1614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fukumoto, T. Hiro, T. Fujii, G. Hashimoto, T. Fujimura, J. Yamada, T. Okamura, and M. Matsuzaki Localized elevation of shear stress is related to coronary plaque rupture: a 3-dimensional intravascular ultrasound study with in-vivo color mapping of shear stress distribution. J. Am. Coll. Cardiol., February 12, 2008; 51(6): 645 - 650. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Segers, F. Helderman, C. Cheng, L. C.A. van Damme, D. Tempel, E. Boersma, P. W. Serruys, R. de Crom, A. F.W. van der Steen, P. Holvoet, et al. Gelatinolytic Activity in Atherosclerotic Plaques Is Highly Localized and Is Associated With Both Macrophages and Smooth Muscle Cells In Vivo Circulation, February 6, 2007; 115(5): 609 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Krams, S. Verheye, L. C.A. van Damme, D. Tempel, B. M. Gourabi, E. Boersma, M. M. Kockx, M. W.M. Knaapen, C. Strijder, G. van Langenhove, et al. In vivo temperature heterogeneity is associated with plaque regions of increased MMP-9 activity Eur. Heart J., October 2, 2005; 26(20): 2200 - 2205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fisher, A. Paganini-Hill, A. Martin, M. Cosgrove, J. F. Toole, H. J.M. Barnett, and J. Norris Carotid Plaque Pathology: Thrombosis, Ulceration, and Stroke Pathogenesis Stroke, February 1, 2005; 36(2): 253 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kinlay, J. Grewal, D. Manuelin, J. C. Fang, A. P. Selwyn, J. A. Bittl, and P. Ganz Coronary Flow Velocity and Disturbed Flow Predict Adverse Clinical Outcome After Coronary Angioplasty Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1334 - 1340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ichikawa, H. Kajiwara, Y. Noishiki, I. Yamazaki, K. Yamamoto, T. Kosuge, S. Sato, and Y. Takanashi Flow dynamics in internal thoracic artery grafts 10 years after coronary artery bypass grafting Ann. Thorac. Surg., January 1, 2002; 73(1): 131 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sander, C. Kukla, J. Klingelhofer, K. Winbeck, and B. Conrad Relationship Between Circadian Blood Pressure Patterns and Progression of Early Carotid Atherosclerosis : A 3-Year Follow-Up Study Circulation, September 26, 2000; 102(13): 1536 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Ward, G. Pasterkamp, A. C. Yeung, and C. Borst Arterial Remodeling : Mechanisms and Clinical Implications Circulation, September 5, 2000; 102(10): 1186 - 1191. [Full Text] [PDF]
|
|
|
|
|
|
P. K. Witting, K. Pettersson, J. Letters, and R. Stocker Site-Specific Antiatherogenic Effect of Probucol in Apolipoprotein E-Deficient Mice Arterioscler Thromb Vasc Biol, August 1, 2000; 20 (8): e26 - e33. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||