Ultrastructural Localization of Secretory Type II Phospholipase A₂ in Atherosclerotic and Nonatherosclerotic Regions of Human Arteries

From the Wallenberg Laboratory (M.R., E.R., E.H.-C.) and the Department of Pathology (S.B.), Göteborgs University, Göteborg 41 345, Sweden.

	Abstract

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Abstract—We recently reported on the immunolocalization of type II secretory nonpancreatic phospholipase A₂ (snpPLA₂) in human atherosclerotic lesions. In the present study, we present data on the distribution and ultrastructural localization of snpPLA₂ in adjacent nonatherosclerotic and atherosclerotic regions of human arteries. Electron microscopy (EM) of immunogold labeling techniques with a monoclonal antibody was used to analyze arterial tissue. The human specimens analyzed were obtained from autopsy and surgery cases. The results with EM showed a stronger snpPLA₂ immunoreactivity in regions of arteries with atherosclerotic lesions than in regions without lesions from the same individual. snpPLA₂ immunoreactivity was stronger in the arterial intima of atherosclerotic than of nonatherosclerotic tissue. EM-immunogold examination revealed that the majority of snpPLA₂ was localized along the extracellular matrix, associated with collagen fibers and other extracellular matrix structures. Intracellular snpPLA₂ was observed in electron-dense vesicles in intimal cells. snpPLA₂ was also found in contact with large, extracellular lipid droplets. These results support the hypothesis that extracellular snpPLA₂ is localized at sites where it may hydrolyze phospholipids from lipoproteins and lipid aggregates retained in the extracellular matrix of the arterial wall. This may be a mechanism for in situ release of proinflammatory lipids, free fatty acids, and lysophosphatidylcholine in regions of apolipoprotein B accumulation, which are abundant in atherosclerotic lesions.

Key Words: atherosclerosis • inflammation • phospholipase A₂ • matrix

	Introduction

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Type II secretory nonpancreatic phospholipase A₂ is a 14-kD, calcium-dependent enzyme that hydrolyzes phospholipids at the sn-2 position, yielding nonesterified free fatty acids (NEFAs) and lysophospholipids.¹ These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators.^{2 3 4 5 6} Additionally, polyunsaturated free fatty acids are susceptible to free radical–mediated oxidation.⁷ Lysophosphatidylcholine (lyso-PC), on the other hand, has recently been documented as a mediator of a broad range of cellular processes that can be defined as proinflammatory.^{8 9 10 11 12 13 14 15 16 17 18 19 20} A correlation exists between elevated levels of extracellular snpPLA₂ and several inflammatory diseases.^{21 22 23} However, the mechanisms for increased snpPLA₂ during the inflammatory response are not yet clearly understood. The levels of extracellular snpPLA₂ appear to be regulated at the secretion step of the already synthesized enzyme and by modulation of gene expression. One hypothesis is that the elevation of circulating snpPLA₂ may be a consequence of increased synthesis of snpPLA₂ in vascular SMCs, saturation of its binding sites in the vascular wall, and release of snpPLA₂ into the circulation.^{23 24}

Atherosclerosis shares many characteristics of an inflammatory process.²⁵ Furthermore, a high incidence of atherosclerosis and high mortality from cardiovascular accidents is common in patients with chronic inflammatory diseases who have prolonged periods of high extracellular snpPLA₂ activity.^{26 27} The concentration of lyso-PC in the atherosclerotic aorta has been reported to be higher and the PC to lyso-PC ratio lower than in comparable control tissue in rabbits.²⁸ Previous immunolocalization studies from our laboratory showed the presence of snpPLA₂ associated with SMCs in normal human arterial media and in the intima-media of atherosclerotic arteries.²⁹ These results agree with those of other groups, indicating that SMCs are an important source of snpPLA₂.^{30 31} However, these studies were performed by light microscopy and provided no data about the ultrastructural localization of snpPLA₂ in human arterial tissue. Knowledge about the extracellular and intracellular distribution of snpPLA₂ within atherosclerotic regions compared with that in nonatherosclerotic regions in arteries may give further insights into the possible role of snpPLA₂ in the atherosclerotic process. In the present study, we used high-resolution EM with the immunogold technique to study the extracellular and intracellular distribution of snpPLA₂ in nonatherosclerotic and atherosclerotic regions in tissue samples from the same arteries.

	Methods

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Chemicals
LR White resin was purchased from London Resin Co Ltd. Protein G–gold (20-nm) complexes were purchased from Sigma Chemical Co. Formvar/carbon-coated copper grids of 300 mesh and polytetrafluoroethylene (Teflon) molds for flat embedding were purchased from Ted Pella Inc. Formaldehyde (methanol-free) 16%, ultrapure EM grade, was purchased from Polysciences Inc. All other chemicals were of analytical grade and were obtained from Merck or Sigma.

Antibodies
Mouse mAb (subclass IgG1k) against human sperm snpPLA₂ from Upstate Biotechnology Inc was used for immunogold detection with EM. This antibody reacts against both snpPLA₂ isolated from human arteries and human recombinant snpPLA₂.^{29 32 33} As a control antibody, we used mouse mAb subclass IgG2a, a negative control from Dako.

Human Tissue Specimens
The specimens were 1x2-mm sectors of human coronary arterial or abdominal aortic wall, with atherosclerotic lesions ranging from fatty streaks (type II lesions) to fibroatheroma (type IV lesions) or without lesions (normal controls). The human tissue material used in the present work was obtained in accordance with local rules for collection of samples from human autopsy tissue. The latter two were taken adjacent to the lesions. The macroscopic designation of the samples (nonatherosclerotic and atherosclerotic tissue) were fully verified microscopically. The tissue samples studied were from 1 female and 3 male autopsy cases, with age the age at ranging from 57 to 80 years. Four to eight tissue samples from each case were obtained and fixed between 33 and 54 hours after death. The tissue specimens were fixed in 4% formaldehyde in 0.1 mol/L cacodylate buffer, pH 7.3, overnight at 4°C. To quench any free aldehydes, the formaldehyde-fixed tissue samples were washed with 3x 0.1 mol/L glycine buffer, pH 7.4, for 35 minutes. The tissue samples were dehydrated in 70% ethanol twice for 30 minutes each, followed by immersion in 1:1 (vol/vol) LR white/ethanol and then 2:1 (vol/vol) LR white/ethanol for 30 minutes, each at 4°C. The tissue blocks were infiltrated at 4°C with LR white for 1 hour, followed by overnight infiltration in fresh LR White/ethanol, and then a final change of resin the following morning for 1 hour before being embedded in Teflon molds for 24 hours at 50°C. Ultrathin sections of the embedded tissues were cut on an LKB Bromma 2088 Ultrotome (Pharmacia-LKB) ultramicrotome with a diamond knife and picked up on copper grids for immunogold labeling; thick sections of the same material were placed on Superfrost glass slides (Menzel-Glaser) for immunofluorescence labeling as described below.

Immunogold EM
Thin sections of embedded tissues were labeled at room temperature by floating the grids sections upside down on droplets placed on wax sheets under three different conditions as follows: (1) blocking of nonspecific binding, achieved by incubation in 3% BSA in PBS containing 0.1% Tween 20 (buffer A) for 15 minutes; (2) binding of primary antibody by placing the grids on drops with the primary mAb diluted 1:20 in buffer A for 1 hour, followed by eight 2-minute washes in buffer A. Optimal dilution of the primary mAb was selected on the basis of maximal specificity and minimal background staining; and (3) gold labeling of the grids by floating them on drops containing gold-labeled protein G, diluted 1:50 in buffer A for 1 hour, followed by eight 2-minutes washes in buffer A. The grids were rinsed with distilled water and dried by gently touching them to filter paper after each step. Labeled grids were stained with saturated aqueous uranyl acetate for 4 minutes at 60°C. The grids were then rinsed with distilled water, dried, stained with lead citrate (Reynolds) for 4 minutes at room temperature, rinsed again with distilled water, dried, and examined in a Philips C12 EM at 60 kV.

	Results

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Morphological Characterization of Human Nonatherosclerotic and Atherosclerotic Tissue
Morphological characterization of human arterial specimens used in this study was done by following the definitions established by the committee on vascular lesions of the American Heart Association.³⁴ Fig 1 shows images of toluidine blue–stained thick sections from a nonatherosclerotic (A) and an adjacent atherosclerotic (B) region of a coronary artery. Nonatherosclerotic specimens were characterized by an intimal thickening made of layers of ECM and SMCs (Fig 1A). Extracellular or intracellular lipid accumulation was not observed. Atherosclerotic specimens corresponded to the definition of type III to type IV atheromatous lesions. These lesions, as shown in Fig 1B, were characterized by the presence of extracellular lipid droplets and foam cells among the layers of SMCs and the ECM of a colocalized intimal thickening. Fig 1 shows that the extent of intimal disruption and thickening was greater in the atherosclerotic tissue (B) than in the nonatherosclerotic region (A). The intima-media thickness in nonatherosclerotic tissue was between 425 and 625 µm, compared with 800 µm in atherosclerotic tissue. Cholesterol crystal clefts and necrotic acellular regions in the deep intima were also found in the atherosclerotic specimens.

View larger version (68K): [in this window] [in a new window]   Figure 1. Overview photomicrograph at light-microscopic level showing the nature of the lesions analyzed in this study. A and B show the morphology of toluidine blue–stained sections from nonatherosclerotic and atherosclerotic human coronary artery tissue, respectively. Solid arrow indicates cholesterol clefts. Open arrow indicates foam cells in the deep intima of atherosclerotic tissue. Arrowheads indicate the limit between media and adventitia. Original magnification, x200.

Ultrastructural Detection of snpPLA₂ in Nonatherosclerotic and Atherosclerotic Tissue
EM immunochemistry is the method of choice to study the ultrastructural location of specific antigens. However, one of the major problems with EM immunocytochemistry is the reduction of antigenicity caused by the fixation and embedding routines.³⁵ In particular, fixation often adversely affects the antigenicity of the proteins, resulting in a markedly reduced labeling intensity.³⁶ Therefore, the fixation procedure of choice is always a compromise between good morphological preservation and retention of antigenicity. Thus, in the present study before doing the EM analysis of human arterial specimens, we first characterized the immunoreactivity of several antibodies obtained after different fixation procedures. Glutaraldehyde is a common reagent used for fixation and preservation of tissue for further EM analysis. However, snpPLA₂ was found to be extremely sensitive to cross-linking by glutaraldehyde at any concentration used, thereby completely eliminating its immunodetection by a panel of mAbs (Upstate Biotechnology; mAb 187³⁷; and Boehringer Mannheim) and the polyclonal rabbit antibody produced in our laboratory. Therefore, glutaraldehyde was omitted entirely from the tissue fixation protocol. The best immunogold labeling intensity was obtained by fixing arterial tissues with 4% formaldehyde (methanol-free) in 0.1 mol/L cacodylate buffer, pH 7.3, at 4°C.

The ultrastructural pattern of immunogold labeling of nonatherosclerotic and atherosclerotic tissue with anti-snpPLA₂ mAb was compared in areas of the arterial intima. In atherosclerotic tissue, snpPLA₂ was mainly located extracellularly and associated with ECM components. Immunogold particles were found extracellularly and associated with collagen fibers (Figs 2, 3A, and 3B). Fig 3B shows at higher magnification (x95 000) the colocalization of snpPLA₂ with collagen fibers in atherosclerotic tissue. Fig 4 shows a typical section in which immunogold particles were also detected and found to be associated with other ECM components localized close to the cells.³⁸ In lipid-rich regions, snpPLA₂ was detected in areas containing large, extracellular lipid droplets as shown in Fig 5. Intracellularly, immunogold particles were found in the cytoplasm of SMCs and on electron-dense vesicles (Figs 2 and 3A). In nonatherosclerotic tissues, we observed a weak pattern of extracellular gold particles in the ECM (Fig 6). Immunogold labeling of sections with a control, nonspecific mAb resulted in an image of the ECM and the cells that was almost free of gold particles (Fig 7).

View larger version (183K): [in this window] [in a new window]   Figure 2. Electron photomicrograph of atherosclerotic tissue taken from the same coronary artery as the nonatherosclerotic tissue shown in Fig 6. Ultrastructural immunogold labeling pattern was obtained with an mAb against snpPLA2. Immunogold particles (20 nm) are found all over the extracellular matrix (ecm) associated with collagen fibers. Immunogold particles are also found intracellularly in SMCs (smc). Original magnification, x35 000; scale bar=286 nm.

View larger version (142K): [in this window] [in a new window]   Figure 3. Electron photomicrograph of human atherosclerotic tissue showing ultrastructural immunogold labeling pattern obtained with an mAb against snpPLA2. A, Immunogold particles can be observed distributed over the ECM (ecm) associated with collagen fibers and intracellularly in an electron-dense vesicle indicated by an arrow. Original magnification, x46 000; scale bar=217 nm. B, A higher-magnification view of an ECM region showing immunogold particles associated with collagen fibers. Original magnification, x95 000; scale bar=105 nm.

View larger version (173K): [in this window] [in a new window]   Figure 4. Electron photomicrograph of human atherosclerotic tissue from a coronary artery showing the ultrastructural immunogold labeling pattern obtained with an mAb against snpPLA2. Immunogold particles are found all over the ECM (ecm) associated with collagen fibers and with proteoglycans indicated by arrows. Immunogold particles are also found intracellularly in SMCs (encircled). Original magnification, x35 000; scale bar=286 nm.

View larger version (167K): [in this window] [in a new window]   Figure 5. Electron photomicrograph of human atherosclerotic tissue from a coronary artery showing ultrastructural labeling pattern obtained with an mAb against snpPLA2. Immunogold particles are found associated with the ECM (ecm) and in close proximity to lipid droplets (li). Original magnification, x35 000; scale bar=286 nm.

View larger version (192K): [in this window] [in a new window]   Figure 6. Electron photomicrograph of human nonatherosclerotic tissue taken from the same coronary artery as the one shown in Fig 2. Ultrastructural immunogold labeling pattern was obtained with an mAb against snpPLA2. Some immunogold particles (20 nm) are found over the ECM (ecm). No immunogold particles were observed associated with SMCs (smc). Original magnification, x35 000; scale bar=286 nm.

View larger version (198K): [in this window] [in a new window]   Figure 7. Electron photomicrographs of human atherosclerotic tissue as in Fig 2. Ultrastructural immunogold labeling pattern was obtained with a primary nonspecific mAb (negative control mAb). ECM (ecm) and SMCs (smc) are almost free of gold particles. Original magnification, x35 000; scale bar=286 nm.

	Discussion

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A key event for the development of atherosclerosis is presently believed to be accumulation of apoB-100–containing lipoproteins in the arterial intima.³⁹ The interaction of apoB lipoproteins with ECM components has been studied extensively and is considered to be one of the main reasons for apoB lipoproteins retention in the arterial wall.⁴⁰ Earlier EM and EM–immunogold labeling studies by other groups have reported the presence of apoB lipoproteins in the ECM, colocalized with chondroitin sulfate proteoglycans and along collagen fibers in the arterial intima.^{41 42 43 44} This entrapment of apoB lipoproteins in the ECM may provide the opportunity for further structural, hydrolytic, and oxidative modifications of the lipoproteins, with consequences for the cells in the arterial wall.⁴⁵ There is indirect evidence that one modification that occurs rapidly, once apoB-100 lipoproteins are in the arterial intima, is the hydrolysis of PC by PLA₂-like activity^{46 47 48} Lyso-P, a product of hydrolysis of PC by PLA₂, is found in atherosclerotic lesions, and the PC to lyso-PC ratio in these lesions is lower than that in comparable control tissue.^{28 49} Lyso-PC triggers a series of cellular processes that are associated with an inflammatory response.^{8 9 10 11 12 13 14 15 16 17 18 19 20} Another effect of lyso-PC and free fatty acids is their "detergent" action, which can alter cell membrane permeability.¹⁵

The cDNA for snpPLA₂ encodes a 144–amino acid protein including a 20–amino acid signal peptide, indicating that snpPLA₂ is a secretory enzyme. snpPLA₂ has seven disulfide bridges, which make it very stable. In addition, snpPLA₂ is a very basic protein (pI, 10.5) containing 23 arginine and lysine residues but only 8 glutamic and aspartic acid residues.⁵⁰ One consequence of its net positive charges is that snpPLA₂ binds to sulfated GAGs.^{33 51} For its activity, snpPLA₂ requires millimolar concentrations of calcium. Some of these characteristics indicate that snpPLA₂ can be active extracellularly. The results obtained in this work, with both immunofluorescent and EM techniques, clearly show stronger immunolabeling of snpPLA₂ in atherosclerotic regions than in adjacent nonatherosclerotic regions from the same human artery. EM-immunogold examination reveled that the majority of snpPLA₂ was found extracellularly and colocalized with collagen fibers. In regions of atherosclerotic tissue containing extracellular lipids, snpPLA₂ was detected close to large lipid droplets. snpPLA₂ was also found colocalized with a finely woven meshwork of proteoglycan structures situated in the ECM close to the cells. These types of proteoglycan mainly contain chondroitin sulfate and dermatan sulfate GAG chains.³⁸

We reported previously the capacity of snpPLA₂ to interact with the GAG moiety of proteoglycans synthesized by human arterial SMCs.33 It will be interesting to further investigate whether snpPLA₂ around collagen fibers binds directly to collagen or through the chondroitin sulfate or dermatan sulfate GAG chains of small proteoglycans, like decorin or biglycan, which are known to be part of the collagen network organization in the arterial wall.^{52 53 54} The interaction between decorin or biglycan and collagen type I has been studied extensively in vitro and in vivo.^{55 56 57 58} Both proteoglycans colocalize with collagen types I and III in atherosclerotic plaques.⁵⁹ Furthermore, decorin and biglycan have chondroitin/dermatan sulfate GAG chains that can potentially bind both LDL and snpPLA₂. Therefore, decorin and biglycan may link both LDL and snpPLA₂ with collagen fibers in the arterial ECM. This colocalization through the binding to GAGs may consequently facilitate the hydrolysis of LDL phospholipids by snpPLA₂.

snpPLA₂ was also present intracellularly in electron-dense vesicles localized near the cell membrane of arterial cells. To our knowledge, this has not been reported before. Such accumulation of snpPLA₂ in intracellular vesicles may be part of a transport and secretory function. In mast cells, studies with EM-immunogold technique have reported the presence of snpPLA₂ in "proteoheparin" secretory granules in mast cells.^{60 61} The composition and function(s) of the snpPLA-positive, electron-dense vesicles in vascular cells need to be characterized in detail.

EM data also show that atherosclerotic regions contain more snpPLA₂ than do adjacent nonatherosclerotic regions from the same human artery. A number of reports have suggested a correlation between elevated levels of snpPLA₂ and several inflammatory diseases.^{21 22 23 62} However, the mechanisms for the increased snpPLA₂ in the inflammatory response are not clearly understood. The levels of extracellular snpPLA₂ appear to be regulated through both secretion of already synthesized enzyme and modulation of its gene expression. Atherosclerosis has characteristics of an inflammatory process,²⁵ a phenomenon that may be related to the high incidence of atherosclerosis and mortality from cardiovascular diseases in patients with chronic inflammation and prolonged periods of high extracellular snpPLA₂ activity.²⁶ Inflammatory cytokines, including interleukin-1ß, interferon-, and tumor necrosis factor-, can regulate genes in vascular wall cells and macrophages, which are involved in atherogenesis.²⁵ Several in vitro studies indicate that cytokines, such as interleukin-1ß, interleukin-6, and tumor necrosis factor-, can stimulate several animal^{63 64} and human^{65 66} cell types to release snpPLA₂. T lymphocytes are present in human atherosclerotic lesions.⁶⁷ Furthermore, extracellular snpPLA₂ increases T-lymphocyte response.¹⁰ Therefore, activation of T lymphocytes in the arterial wall may be part of a positive-feedback mechanism sustaining chronic inflammation through cytokine production and increased snpPLA₂ expression by arterial SMCs.

Our previous findings showed that snpPLA₂ isolated from human arteries can hydrolyze LDL phospholipids.²⁹ The present study shows that snpPLA₂ is located mainly extracellularly in atherosclerotic lesions at places similar to where apoB lipoproteins and lipid droplets have been reported trapped in the arterial ECM. Taken together, these findings support the hypothesis that snpPLA₂ may contribute to atherogenesis by modifying apoB lipoproteins and producing inflammatory lipid factors locally in the arterial wall.⁶⁸

	Selected Abbreviations and Acronyms

 

ECM = extracellular matrix EM = electron microscopy GAG = glycosaminoglycan mAb = monoclonal antibody PC = phosphatidylcholine SMC = smooth muscle cell snpPLA2 = type II secretory nonpancreatic phospholipase A2

	Acknowledgments

 
This work was supported by grants from the Heart and Lung Foundation of Sweden (project No. 51014, 51013, and 61538), the Swedish Medical Research Council (MFR, project No. 4531), and the Sahlgrenska University Hospital fund for research on heart diseases.

	Footnotes

 
Reprint requests to Eva Hurt-Camejo, Wallenberg Laboratory, Fack 16, Sahlgrenska University Hospital, Göteborg 41 345, Sweden.

Received July 18, 1997; accepted September 5, 1997.

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