AmericanJournal ofPathology, Vol. 136, No. 1, January 1990 Copyright© American Association ofPathologists
Immunohistochemical Localization of Heat Shock Protein-70 in Normal-Appearing and Atherosclerotic Specimens of Human Arteries
Paul A. Berberian,*4 Wendell Myers, Michael Tytell,* t Venkata Challa,t't and M. Gene Bond*4t From the Departments ofNeurobiology andAnatomy,* Pathology,j and The Stroke Research Center,j Bowman Gray School ofMedicine of Wake Forest University, Winston-Salem, North Carolina
Heat shock proteins (HSPs) are synthesized by cells under metabolic stress and are known to enhance a cell's ability to survive life-threatening stress. The authors have begun to examine HSPs in the context ofhuman atherosclerosis. This study demonstrated immunohistochemically the presence of HSP- 70 in human and rabbit arteries, and its distribution in relation to necrosis and lipid accumulation, as well as vascular smooth muscle cells and macrophages, in human atherosclerotic plaques. Advanced lesionsfrom 10 human carotid endarterectomy specimens were compared with 11 human aortic specimensfrom autopsy and 8 rabbit aortas. The immunostaining procedure used a mouse monoclonal antibody specific for the inducible form of HSP- 70. Normal rabbit aortas were tested for changes in HSP- 70 up to 24 hours after removal, and were used as controls for the human aortas. Representative plaques were examinedfor lipid content by osmium staining, and for smooth muscle cell and macrophage components using cell-specific monoclonal antibodies followed by immunostaining. The results indicated that HSP- 70 was present in human and rabbit arteries and remained unchanged in distribution or concentration up to 15 hours after death. HSP- 70 was present weakly throughout the media of normal-appearing arterial specimens. In contrast, HSP- 70 was concentrated in the central portions of more thickened atheromas around sites of necrosis and lipid accumulation. Macrophages were coincident with these areas and were observed to be lipid-loaded. In contrast, patches of smooth muscle cells were observed in very complicated plaques, but without consis-
tent association with necrosis or increased HSP- 70; plaque smooth muscle cells also were observed to contain lipid. Large, relatively avascular and collagenous areas of plaque also wera _ccasionally positivefor HSP- 70 staining. The results support the hypothesis that elevated HSPs indicate which plaque cells, particularly macrophages, are more stressed in the depth of atheroma, especially in association with necrosis, and shouldpromptfurther investigation of the significance of HSP accumulation to the evolution of atherosclerotic plaques. (AmJPathol 1990, 136:71-80)
The clinically important form of atherosclerosis is the advanced plaque, which may produce its effects either by lumen stenosis or by plaque rupture with subsequent thrombosis and embolization. These lafter effects are a consequence of one or more of the major components of advanced plaque, especially the necrotic center, which includes cell debris, cholesterol crystals, cholesterol esters, calcium,. and thrombolytic substances.1 Necrosis may be considered a complication that occurs in relation to fully developed lesions and is common in the central regions of plaques. This necrosis may be partially ischemic in origin because intimal thickening compromises the nutritional state of tissue that cannot be reached by vasa vasora;2 however, the specific cause of cell death in human atheroma is not well understood. Approximately ten proteins, described as heat shock proteins (HSPs) or stress proteins, are recognized to be present in virtually all organisms from Escherichia coli to humans, and have been shown to be induced by exposure to environmental stress, such as increased temperaPresented at the 72nd Annual Meeting of The American Association of Pathologists, Las Vegas, NV (FASEB J 1988, 2: A1597) and at the VlIlth International Symposium on Atherosclerosis, Rome, Italy (October 1988). Supported by NINCDS Grant NS-06655 and NIH Medical Student Short Research Training Grant DK-07400. Accepted for publication August 22, 1989. Address reprint requests to Paul A. Berberian, PhD, Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, 300 South Hawthorne Road, Winston-Salem, NC 27103.
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ture or anoxia;3 by chemicals such as amino acid analogues and heavy metal inhibitors of oxidative metabolism;4 by ischemia;5 and by infection.6 The HSPs are currently classified into four groups on the basis of size. Of these, the 70 kD HSPs (HSP-70) are regarded as most important because they are most often highly induced in stressed cells.7 HSPs have been the focus of increasing investigations during the last decade because the amino acid sequence of HSP-70 is very highly conserved across widely divergent species; thus, they are thought to represent a fundamental feature of a cell's ability to cope with unfavorable conditions. The underlying mechanism by which HSPs are protective may involve the stabilization of other proteins, particularly their tertiary structures.8'9 One study previously investigated the HSPs in arteries10; however, no one has documented the presence of these proteins in normal-appearing and atherosclerotic arteries of humans. Of relevance to the aim of the present study is the fact that the synthesis of HSPs is induced by oxygen deprivation, which may occur during ischemia at the site of advanced atheroma.1" An opposite extreme involving cell death caused by high concentrations of oxygen-free radicals during inflammation can be inhibited by heat shock and the subsequent production of HSPs.12 The possibility that viral infection causes arterial lipid accumulation and atheroma formation13 also may involve induced HSPs in monocytes and macrophages during inflammation.12 The objective of the present study was to determine the presence and distribution of HSP-70 in human arteries and its spatial relation with lipid accumulation and necrosis, as well as plaque smooth muscle cells and macrophages.
Materials and Methods
retinas known to contain HSP-7015 were used as positive controls. Retinal tissues were fixed in Carnoy's solution, prepared similarly to the arterial specimens, and then carried through the immunostaining procedure concurrently with each arterial specimen tested.
Time-After-Death Controls Eight normal male New Zealand white rabbits were killed and divided into four groups of two rabbits each for comparison of aortic HSP-70 after cold storage (4 C) of each rabbit cadaver for 0, 10, 15, and 24 hours, respectively. Following each period of storage, aortas were removed, prepared as intima-media, and immersion-fixed in 10% buffered formalin for histology and immunostaining of HSP-70. Two human autopsy aortic specimens obtained within 10 hours after death were each divided into six portions; two of the portions were immediately processed for study while the other four were stored at 4 C for periods equivalent to 15 and 24 hours after death, then formalin fixed and processed for study of HSP-70.
Histology Tissue specimens were fixed in 10% buffered formalin for a minimum of 48 hours, embedded in paraffin, sectioned at 5 to 7 ,u, and mounted on microscope slides. Representative sections were stained routinely with hematoxylin and eosin (H & E) whereas other sections underwent the immunostaining procedure employing a monoclonal antibody (MAb) specific for HSP-70. Controls for nonspecific staining consisted of tissue sections carried through the immunostaining procedure, but with the MAb omitted.
Source of Tissues Immunoperoxidase Staining of HSP-70 Ten human carotid endarterectomy specimens were studied. Routinely, less than one hour elapsed between surgical removal and analyses. Two human common carotid and 11 aortic specimens were obtained from autopsy. Previous findings14 support the use of autopsy tissue for analyzing arterial enzymes and cellular constituents. The autopsy specimens also contained normal-appearing areas from the same artery for controls. Eight male New Zealand white rabbits weighing 2 to 3 kg each were fed rabbit chow ad libitum and killed by intravenous injection of sodium pentobarbital. Entire aortas were removed, opened, and immersed in 10% buffered formalin for histologic preparation. The normal rabbit aortas were used as controls and for assessing postmortem alterations in HSP-70, similar to that which may have occurred in autopsy specimens as a result of autolysis. Normal rat
The fixed and paraffin-embedded tissues were mounted on slides, dried in a 47 C oven for at least 12 hours, and then deparaffinized with fresh xylene for 1 hour. Rehydration, beginning with 100% ethanol for 15 minutes, continued with 95%, 80%, and 70% ethanol solutions and ended with distilled water. Endogenous peroxidase activity was blocked by incubating the sections for 15 to 30 minutes in methanol containing 10% H202 between the 70% ethanol and distilled water steps during rehydration. To block nonspecific binding, the sections were covered with a solution of 2% powdered nonfat dry milk (Carnation, Los Angeles, CA) in 0.01 M phosphate-buffered saline (PBS), pH 7.2, for 30 minutes at room temperature in a humidified chamber. Excess milk solution was removed, and the sections were covered with a 1:5000 to 1: 10,000
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dilution of mouse monoclonal anti-HSP-70 antibody, C92F3-A (a gift of Dr. William J. Welch, University of California, School of Medicine, San Francisco, CA) in 2% powdered milk in PBS and incubated for two hours at room temperature. The sections were washed three times in PBS for five minutes each, and incubated for one hour with a 1:250 dilution of horse anti-mouse biotinylated antibody (Vector Laboratories, Burlingame, CA). The tissue sections were washed as before and incubated for 30 minutes to 1 hour with the avidin and biotinylated horseradish peroxidase contained in the Vectastain Peroxidase ABC Elite kit (Vector Laboratories) according to the manufacturer's recommendations. The sections were again rinsed twice for five minutes each in PBS followed by once in distilled water for five minutes. The reaction product was developed using diaminobenzidine (6 mg in 10 ml of 0.05M TRIS buffer, pH 7.6) plus 100 ,ul 3% H202 and counterstained with eosin. A brown-to-black precipitate was indicative of the presence of HSP-70.
Osmium Staining of Lipids Microscopic demonstration of lipids in the specimens was done according to a modification of the osmium tetroxide method described previously.17 Endarterectomy specimens were fixed in a mixture of glutaraldehyde and formaldehyde for one to two hours, followed by overnight fixation in 10% buffered formalin (0.01M phosphate buffer, pH 7.4). Thin slices were fixed for eight hours in a solution of 1% osmium tetroxide (Stevens Metallurgical Corp., New York, NY ) in 2.5% potassium dichromate, washed for 2 to 4 hours in running water and processed overnight in an automatic tissue processor for paraffin embedding. Six-micron sections were cut, deparaffinized, and counterstained with H & E. Using this method, lipids stain black and the background pink, with cell nuclei being blue to black.
Immunoperoxidase Staining of Smooth Muscle Versus Macrophage-Specific Antibody Binding Formalin-fixed, paraffin-embedded tissues were immunohistochemically tested for binding of cell-specific antibodies to vascular smooth muscle cells and to macrophages. All antibodies (ie, macrophage specific [MAC]; muscle specific actin [MSA]; and smooth muscle specific actin [SMSA]) were obtained from ENZO Biochemicals, New York, NY. Respective antibody dilutions applied were: 1: 500; 1:2000; and 1:50. Sections of normal human lymph node were used as positive tissue controls, inclusive of small arteries and arterioles for comparison of MSA and
SMSA specificity. Negative tissue controls (ie, the exact procedure less the cell-specific antibody) also were included to detect any false positive reactions. All samples were processed using an Autoprobe Detection System (Biomedia Corp., Foster City, CA) in combination with a Fisher Code-On Immunology System (Fisher, Pittsburgh, PA). The system involved a universal tissue conditioner to block nonspecific binding of the primary antibody followed by incubation of the tissue with the primary antibody. A peroxidase reagent containing a mixture of horseradish peroxidase conjugated antibodies to detect the primary antibodies was added next, followed by the application of a chromagen. The antigenic sites were visualized as a brownish-red precipitate on a clean tissue background when counterstained with a water-based hematoxylin.31
Results Normal-appearing specimens of human aortas from autopsy (Figure 1) and normal rabbit aortas demonstrated routinely a homogeneous distribution of HSP-70 throughout intima-media. As a control for postmortem changes in arterial HSP-70, cadavers of normal rabbits were kept at 4 C for up to 24 hours before their aortas were removed for HSP-70 analysis. There was no change in HSP-70 staining up to 15 hours after death, but by 24 hours, there was a dramatic decrease. Human aortic specimens from autopsy that were collected 8 to 10 hours after death also were stored at 4 C for periods up to 24 hours after death with similar results. Based on these findings, only autopsy aortic specimens up to 15 hours after death were included for study. Human atherosclerotic aortic specimens from autopsy demonstrated HSP-70 staining around areas of necrosis, typically in the central portions of atheroma (Figure 2). The same findings were observed for all ten human carotid endarterectomy specimens (Figure 3). The intensity of HSP-70 staining and its distribution appeared routinely to correlate with increased thickness of plaque (Figure 3), so that thinner regions often were without intense staining. HSP-70 staining in plaques appeared to be related more to a cellular than extracellular component around areas of necrosis and vacuolization partly due to lipid accumulation. To test the concurrence of HSP-70 intensity and increased lipid accumulation, separate sections of the same human carotid endarterectomy specimens were stained for lipid and for HSP-70 and compared. As illustrated in Figure 4, on the average, there was a general coincidence of plaque lipid with increased HSP-70 staining, most notably surrounding areas of necrosis. Rarely was there the occurrence of necrosis or lipid accumulation without intense HSP-70 staining. In contrast, how-
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Figure 1. Distribution of HSP-70 in normal-appearing human abdominal aorta less than ten hours postmortem. A and B: immunostainedfor HSP- 70; C and D: control section immunostained without HSP- 70 antibody. A and C:final magnification X 62 (M = media); B and D: final magnification, X 156 A weakly stained and relatively homogeneous distribution ofHSP- 70 was evidenced routinely. Small areas of increased staining were evidenced occasionally and usually proximal to lumen as seen in the enlarged view on right.
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Figure 2. Distribution of HSP-70 in human atherosclerotic abdominal aorta less than ten hours after death. Intimamedia preparation with necrosis is seen: A: immunostainedfor HSP- 70; B: immunostained without HSP-70 antibody. Final magnification, X62 (P = plaque). Intense HSP- 70 concentration was routinely present in the central areas of atherosclerotic plaques.
ever, there were plaque areas that did have intense HSP70 staining without notable necrosis and lipid. Examination of the cellular nature of the same arterial plaques that were studied for localization of HSP-70 revealed the presence of both smooth muscle cells and macrophages (Figure 5, 6). The distribution of these cell types in plaque and in relation to increased HSP-70 staining varied relative to the amount and complications of each plaque. Generally, however, macrophage-positive staining was viewed more often around areas of necrosis and coincident with increased HSP-70 staining. Additionally, patches of smooth muscle cells, when present in plaque, stained for HSP-70 equivalent in amount to that of the underlying medial cells and also comparable to vascular cells of small arteries when present in plaque (Figure 5C). It should be noted that lymphocytes and neutrophils also were observed in certain areas of complicated plaque, coincident with HSP-70 staining. In contrast, it also was evident that in complicated plaques especially, relatively acellular, collagenous areas stained lightly for HSP-70. Lastly, both macrophage- and smooth muscle
cell-positive staining were noted for lipid-filled cells when present and coincident with increased HSP-70 staining.
Discussion Based on examination of 23 individual human arterial specimens and 8 normal rabbit aortas, HSP-70 is present in human and rabbit arteries. These observations are consistent with a previous study in rat thoracic aortic cells.'0 The purpose of HSPs in normal arteries is unclear, but may relate to functions of HSPs in normal tissue. The results are consistent with observations by others indicating the presence of HSPs in a variety of normal cultured cell lines'8 and tissues.'9 There is, however, the possibility that a degree of unavoidable "stress" during procurement and preparation of the arterial samples added to the presence of HSP-70. This possibility has not been resolved, but if correct, appears minor because HSP-70 was present in normal arteries and did not vary significantly between different artery types (human carotid and aorta),
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B. Figure 3. Distribution ofHSP- 70 in human carotid endarterectomy specimen less than one hour after surgical removal. Advanced, complicated atherosclerotic plaque that contained lipid and necrosis. A: immunostained for HSP- 70; B: immunostained without HSP-70antibody. Final magniflcation, X15(n = necrosis; m = media). Endarterectomyspecimenscontained mostlyplaque circumscribed by a small rim of media; the size and complexity ofplaques varied between specimens. Intense HSP- 70 staining was found around areas of necrosis in contrast to other areas ofplaque that did not stain intensely.
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Figure 4. Comparison ofHSP- 70 and lipid localization in human carotid endarterectomy specimen less than one hour aftersurgical removal: A: osmium-stained lipids are evident as droplets; B: section ofsame specimen immunostainedfor HSP- 70. Original magnification, X200 (inset, X400). Generally, the more intense areas of lipid accumulation had greater concentrations of HSP-70. The reciprocal was not always true, suggesting that other factors in addition to lipid accumulations may be relevant to HSP levels in arteriosclerotic plaques (P = plaque; M = media; bars indicate areas ofinsets).
Heat Shock Protein and Atherosclerosis 77 AJPJanuary 1990, Vol. 136, No. 1
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Figure 5. Distribution ofsmooth muscle cell-specific antibody in human carotid endarterectomy specimens less than one hour after surgical removal: A and B include positive dark staining of vascular smooth muscle cells in media (M) in contrast to minimally stained areas of relatively uncomplicated plaque (P). Enlarged view of upper right also shows a mass of vascular smooth musqle cells at the leading edge ofplaque. Original magnifications, X 62 and X 156, respectively. C shows a portion of a complicated lesion. Arrow heads indicate small arteries representing neovasculazation ofplaque and positively stained for MSA antibody; the asterisk indicates an area of cholesterol clefts. Large positively stained dark areas, often seen as random whirls, were evident areas of vascular smooth muscle cellproliferation. Final magnification, X 62. C represents routine staining of small arteries andasarterioles (A) present in the human lymph node specimens included aspositive tissue control. Final magnification, X 156
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Figure 6. Distribution of macrophage-specific antibody in human carotid endarterectomy specimens less than one hour after surgical removal A represents a cross-section from media (M) through plaque (P) inclusive of a foci of necrosis (N) proximal to the luminal surface. The luminal halfofplaque containedpositive stainingfor macrophages, particularly at the borders of the necrosis. Final magnification, X 156 C evidences an enlarged view of necrosis (M) proximal to the luminal surface and dark stainedfor macrophages; the asterisk indicates an area of cholesterol clefts (N) also in association with the necrosis. Final magnification, X 156 D represents routine staining of macrophages (m) present in the human lymph node specimens included as positive tissue controls. Final magnification, X312.
different species (human and rabbit aorta), or different sources and times (rabbit surgical vs. autopsy aorta up to 15 hours after death). Because of the lack of differences in HSP-70 distribution and concentration in normal arteries, that level was taken as a relative baseline against which changes in HSP-70 in human atherosclerotic arteries could be contrasted. To examine HSP-70 in human atherosclerotic plaques, arrangements were made to obtain carotid endarterectomy specimens that typically contained advanced necrotic plaques immediately after surgery. One limitation to their use, however, was the minimal amount of more normal-appearing tissue included. As a comparison to the surgical carotid samples and as a source of more normalappearing artery for internal controls, aortic specimens from autopsy were examined. The use of autopsy aortic specimens for study of biochemical changes in human atherosclerosis for up to 24 hours after death has been validated previously.14 This validation was extended to
HSP-70 in the present study by comparing normal rabbit with human normal-appearing and atherosclerotic autopsy specimens kept in cold storage for up to 24 hours. Histologically, HSP-70 distribution and concentration did not differ significantly up to 15 hours postmortem. This finding suggests the potential use of HSPs as molecular markers of tissue viability as well as the quality of tissue preservation. Validation for the use of autopsy material also was implicated later, because the results of HSP-70 staining in atherosclerotic plaques were comparable regardless of the source or type artery. This study demonstrated that carotid arteries and autopsy aortas routinely had increased HSP-70 staining around areas of necrosis (Figure 2, 3). HSP-70 staining was most pronounced in the central portions of advanced atherosclerotic plaques (Figure 2, 3) and was often coincident with plaque lipid accumulations (Figure 4). The latter observation was meaningful because contiguous plaque areas did not always contain increased HSP-70 staining
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(Figure 3). The presence of HSP-70 was related more to the nature of the plaque, ie, lipid-filled and/or necrotic vs. fibrotic and cellular (smooth muscle cells) rather than to the size and thickness of the plaque per se (Figure 2). The observation that increased HSP-70 staining did not always coincide with lipid accumulation suggests that additional factors also may be influencing the metabolic state of plaque cells. Blood pressure-induced intramural stress2o possibly induces HSP responses depending on the arterial cell type, its intramural position, and degree of underlying disease. Although several potential arterial cell "stressors" may be involved, the present observations document that the central parts of advanced plaques that surround areas of necrosis and lipid accumulations were consistently most "stressed" based on the increased presence of HSP-70. To further resolve the significance of these observations, the coincidence of HSP-70 to either smooth muscle cells or macrophages in plaque was explored. Although the complicated plaque of human arteries provided excellent examples of necrotic foci, it was difficult to resolve any one major factor responsible for HSP-70 alterations; however, many aspects of the complicated arterial lesions appeared to relate to the distribution of plaque HSP-70. Importantly, macrophages were evidenced most routinely around areas of necrosis and coincident with increased HSP-70 staining; macrophages also were observed to be lipid-loaded. It remains unclear, however, whether the lipid-filled macrophages are stressed to the point of pending death to add to a necrotic foci or exist as scavengers in the process of cleaning up the extracellular debris. Relative to the extent and nature of the plaque, patches of smooth muscle cells also were observed to be positive for HSP-70. In contrast to macrophage-positive areas, necrosis was not consistently evident in association with the plaque smooth muscle cells; some plaque smooth muscle cells, however, also were observed to contain lipid but less routinely than macrophages. Additionally, large, relatively avascular and collagenous areas of plaque were sometimes positive for HSP-70 staining. The latter observation is consistent with the fact that HSPs can be secreted by cells2' and that at least one HSP is known to bind to collagen.22 It is clear that HSP-70 can be located both extra- and intracellularly in human plaque. Information on the deposition of the other HSPs in relation to atherosclerosis is also needed to obtain a more complete picture of how this molecular response of arterial cells relates to the disease. The implication that HSPs may have effects outside of the cells making them is especially noteworthy. The differences in stress-induced lipid accumulation between macrophages and arterial smooth muscle cells in early plaque evolution also warrants study. With respect to the latter, it may be possible to use HSP
production as a marker of arterial cells that are more stressed and possibly more vulnerable to die relative to their physiologic state in the plaque regardless of cell origin. Realizing the significance of these possibilities, it remains necessary to determine what relationship HSPs have to atherosclerosis, ie, are they protective, deleterious, or simply symptomatic? The relevance of HSPs to arterial lipid-filled foam cell genesis, as implicated by our results, deserves added consideration. A significant portion of arterial intracellular lipid accumulation occurs in lysosomes23 and lack of lysosomal integrity is a key factor in cell death.24 Theoretically, HSPs may have a protective effect on stressed arterial cells by preventing lysosomal breakdown. If so, and if lipids are present concurrently, the result may be intralysosomal lipid entrapment. This in turn would contribute to the genesis of arterial foam cells coincident with prevention of their death as part of additional plaque evolution. Lysosomes of lipid-filled foam cells are suspected to have increased membrane stability2526 and it is thought that one mechanism for HSP effects may include membrane stabilization.2728 It is also significant to note the relatively long period through which human plaques evolve,29 implicating that a number of lipid-filled cells remain intact and stable enough to survive long periods during plaque development. To explore these points, a biochemical examination of HSP-70 levels in normal versus atherosclerotic arteries relative to necrosis and lysosomal function is underway3' concomitant with an examination of the direct effects of purified HSP-70 on arterial cell survival and lysosomal function. The amounts and types of HSPs in different grades of atherosclerotic plaques as well as in different types of arterial cells enzymatically isolated and separated from atherosclerotic arteries are also under study in our laboratories. With regard to the latter, and of special significance to an understanding of which arterial cell types may be more prone to survive or die during atherogenesis, MAbs specific to macrophages or vascular smooth muscle cells are being applied to density-separated arterial cells as part of a future report.
References 1. Wissler RW: Principles of the Pathogenesis of Atherosclerosis, Heart Disease: A Textbook of Cardiovascular Medicine. Edited by E Braunwald. Philadelphia, WB Saunders, 1984, pp 1 183-1204 2. Kissane JM: Blood vessels and lymphatics, Anderson's Pathology, Volume 2. Edited by JM Kissane. St. Louis, CV Mosby, 1985, pp 687-696 3. Schlesinger MJ: Heat shock proteins: The search for functions. J Cell Biol 1986,103: 321-325
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4. Thomas GP, Welch WJ, Mathews MB, Feramisco JR: Molecular and cellular effects of heat shock and related treatments of mammalian tissue-culture cells. Cold Spring Harbor Sym Quant Biol 1982,12: 985-996 5. Dienel GA, Diessling M, Jaceqicz M, Pulsinnelli WA: Synthesis of heat shock proteins in rat brain cortex after transient ischemia. J Cereb Blood Flow Metab 1986,6: 505-510
6. Kennedy PG, LaThangue NB, Chan WL, Clements GB: Cultured human neural cells accumulate a heat shock protein during acute herpes simplex virus infection. Neurosci Left 1985,61:321-326 7. Craig EA: The heat shock response. CRC Crit Rev Biochem 1985,18:239-280 8. DeShaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R: A subfamily of stress proteins facilitates translocation of secretary and mitochondrial precursor polypeptides. Nature 1988, 332: 800-805 9. Chirico WJ, Waters MG, Blobel G: 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 1988, 332: 805-810 10. Hightower LE, White FP: Preferential synthesis of rat heatshock and glucose-regulated proteins in stressed cardiovascular cells, Heat Shock: From Bacteria to Man. Edited by M Schlesinger. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1982, pp 369-377 11. Bjornheden T, Bondjers G: Oxygen consumption in aortic tissue from rabbits with diet-induced atherosclerosis. Arteriosclerosis 1988,1: 238-247 12. Polla BS: A role for heat shock proteins in inflammation? Immunol Today 1988,9: 134-137 13. Hajjar DP, Fabricant CG, Minick CR, Fabricant J: Virus-induced atherosclerosis: Herpes virus infection alters aortic cholesterol metabolism and accumulation. Am J Pathol 1986,122:62-70 14. Berberian PA, Fowler S: The subcellular biochemistry of human arterial lesions: I. Biochemical constituents and marker enzymes in diseased and unaffected portions of human aortic specimens. Exp Mol Pathol 1979, 30: 27-40 15. Barbe MF, Tytell M, Gower DJ, Welch WJ: Hyperthermia protects against light damage in the rat retina. Science 1988, 241:1917-1820 16. Welch WJ, Suhan, JP: Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol 1986,103: 2035-2052 17. Luna LG: Osmium tetroxide method for fat (paraffin sections), Manual of Histologic Staining Methods. Edited by LG Luna. New York, McGraw-Hill, 1968, pp 143-144 18. Landry J, Bernier D, Chr6tien P, Nicole LM, Tanguay RM, Marceau N: Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res 1982, 42: 2457-2461 19. O'Malley K, Mauron A, Barchas JD, Kedes L: Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shocklike protein. Mol Cell Biol 1985, 5: 3476-3483 20. Thubrikar MJ, Baker JW, Nolan SP: Inhibition of atherosclerosis associated with reduction of arterial intramural stress in rabbits. Arteriosclerosis 1988,8: 410-420
21. Hightower LE, Guidon PT Jr: Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. J Cell Physiol 1989, 138: 257-266 22. Nagata K, Saga S, Yamada KM: Characterization of a novel transformation-sensitive heat-shock protein (HSP-47) that binds to collagen. Biochem Biophys Res Commun 1988, 153:428-434 23. Fowler S, Berberian PA, Haley NJ, Shio H: Lysosomes, vascular intracellular lipid accumulation and atherosclerosis, International Conference on Atherosclerosis. Edited by LA Carlson. New York, Raven Press, 1978, pp 19-27 24. Bowen ID: Techniques for demonstrating cell death, Cell Death in Biology and Pathology. Edited by ID Bowen and RA Lockshin. New York, Chapman and Hall, 1981, pp 379-444 25. Peters TJ, deDuve C: Lysosomes of the arterial wall: II. Subcellular fractionation of aortic cells from rabbits with experimental atheroma. Exp Mol Pathol 1974, 20: 228-256 26. Berberian PA, Jenison MW, Roddick V: Arterial prostaglandins and lysosomal function during atherogenesis: II. Isolated cells of diet-induced atherosclerotic aortas of rabbit. Exp Mol Pathol 1985, 43: 36-55 27. Minton KW, Karmin P, Hahn GM, Minton AP: Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: A model for the biological role of heat shock proteins. Proc Natl Acad Sci USA 1982, 79: 7107-7111 28. Lepock JR, Cheng KH, Al-Qysi H, Kruur J: Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: Relationship to hyperthermic cell killing. Can J Biochem Cell Biol 1983, 61: 421-427 29. Stary HC: Evolution and progression of atherosclerotic lesions in coronary arteries of children and young adults. Arteriosclerosis 1989, 9(suppl): 1-19-1-32 30. Berberian PA, Bond MG, Myers W, Tytell M, Challa V: Potential relevance of arterial heat shock proteins to lysosomal function and necrosis in human atheroma (abstr). FASEB J 1988,2: A1597 31. Brigat DJ, Budgeon LR, Unger ER, Koebler D, Cuomo C, Dennedy T, Perdomo JM: Immunocytochemistry is automated: Development of a robotic work-station based upon the capillary action principle. J Histotechnology 1988, 11: 165-183
Acknowledgments The authors thank Tonya Smith, Katrina Graham, and Carol R. Hollman for expert technical assistance; Linda Caudill and Sarah Graham for secretarial assistance; and Drs. W. Keith O'Steen and Judy K. Brunso-Bechtold, Department of Neurobiology and Anatomy, as well as Dr. Michael McWhorter, Department of Surgery and Dr. Larry Miller, the Immunohistochemistry Laboratory, Department of Pathology, for their cooperation and support of the study. HSP-70 antibody was provided by Dr. William J. Welch, Departments of Physiology and Medicine, University of California, School of Medicine, San Francisco, CA.
Immunohistochemical Localization of Heat Shock Protein-70 in Normal-Appearing and Atherosclerotic Specimens of Human Arteries
Paul A. Berberian,*4 Wendell Myers, Michael Tytell,* t Venkata Challa,t't and M. Gene Bond*4t From the Departments ofNeurobiology andAnatomy,* Pathology,j and The Stroke Research Center,j Bowman Gray School ofMedicine of Wake Forest University, Winston-Salem, North Carolina
Heat shock proteins (HSPs) are synthesized by cells under metabolic stress and are known to enhance a cell's ability to survive life-threatening stress. The authors have begun to examine HSPs in the context ofhuman atherosclerosis. This study demonstrated immunohistochemically the presence of HSP- 70 in human and rabbit arteries, and its distribution in relation to necrosis and lipid accumulation, as well as vascular smooth muscle cells and macrophages, in human atherosclerotic plaques. Advanced lesionsfrom 10 human carotid endarterectomy specimens were compared with 11 human aortic specimensfrom autopsy and 8 rabbit aortas. The immunostaining procedure used a mouse monoclonal antibody specific for the inducible form of HSP- 70. Normal rabbit aortas were tested for changes in HSP- 70 up to 24 hours after removal, and were used as controls for the human aortas. Representative plaques were examinedfor lipid content by osmium staining, and for smooth muscle cell and macrophage components using cell-specific monoclonal antibodies followed by immunostaining. The results indicated that HSP- 70 was present in human and rabbit arteries and remained unchanged in distribution or concentration up to 15 hours after death. HSP- 70 was present weakly throughout the media of normal-appearing arterial specimens. In contrast, HSP- 70 was concentrated in the central portions of more thickened atheromas around sites of necrosis and lipid accumulation. Macrophages were coincident with these areas and were observed to be lipid-loaded. In contrast, patches of smooth muscle cells were observed in very complicated plaques, but without consis-
tent association with necrosis or increased HSP- 70; plaque smooth muscle cells also were observed to contain lipid. Large, relatively avascular and collagenous areas of plaque also wera _ccasionally positivefor HSP- 70 staining. The results support the hypothesis that elevated HSPs indicate which plaque cells, particularly macrophages, are more stressed in the depth of atheroma, especially in association with necrosis, and shouldpromptfurther investigation of the significance of HSP accumulation to the evolution of atherosclerotic plaques. (AmJPathol 1990, 136:71-80)
The clinically important form of atherosclerosis is the advanced plaque, which may produce its effects either by lumen stenosis or by plaque rupture with subsequent thrombosis and embolization. These lafter effects are a consequence of one or more of the major components of advanced plaque, especially the necrotic center, which includes cell debris, cholesterol crystals, cholesterol esters, calcium,. and thrombolytic substances.1 Necrosis may be considered a complication that occurs in relation to fully developed lesions and is common in the central regions of plaques. This necrosis may be partially ischemic in origin because intimal thickening compromises the nutritional state of tissue that cannot be reached by vasa vasora;2 however, the specific cause of cell death in human atheroma is not well understood. Approximately ten proteins, described as heat shock proteins (HSPs) or stress proteins, are recognized to be present in virtually all organisms from Escherichia coli to humans, and have been shown to be induced by exposure to environmental stress, such as increased temperaPresented at the 72nd Annual Meeting of The American Association of Pathologists, Las Vegas, NV (FASEB J 1988, 2: A1597) and at the VlIlth International Symposium on Atherosclerosis, Rome, Italy (October 1988). Supported by NINCDS Grant NS-06655 and NIH Medical Student Short Research Training Grant DK-07400. Accepted for publication August 22, 1989. Address reprint requests to Paul A. Berberian, PhD, Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, 300 South Hawthorne Road, Winston-Salem, NC 27103.
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ture or anoxia;3 by chemicals such as amino acid analogues and heavy metal inhibitors of oxidative metabolism;4 by ischemia;5 and by infection.6 The HSPs are currently classified into four groups on the basis of size. Of these, the 70 kD HSPs (HSP-70) are regarded as most important because they are most often highly induced in stressed cells.7 HSPs have been the focus of increasing investigations during the last decade because the amino acid sequence of HSP-70 is very highly conserved across widely divergent species; thus, they are thought to represent a fundamental feature of a cell's ability to cope with unfavorable conditions. The underlying mechanism by which HSPs are protective may involve the stabilization of other proteins, particularly their tertiary structures.8'9 One study previously investigated the HSPs in arteries10; however, no one has documented the presence of these proteins in normal-appearing and atherosclerotic arteries of humans. Of relevance to the aim of the present study is the fact that the synthesis of HSPs is induced by oxygen deprivation, which may occur during ischemia at the site of advanced atheroma.1" An opposite extreme involving cell death caused by high concentrations of oxygen-free radicals during inflammation can be inhibited by heat shock and the subsequent production of HSPs.12 The possibility that viral infection causes arterial lipid accumulation and atheroma formation13 also may involve induced HSPs in monocytes and macrophages during inflammation.12 The objective of the present study was to determine the presence and distribution of HSP-70 in human arteries and its spatial relation with lipid accumulation and necrosis, as well as plaque smooth muscle cells and macrophages.
Materials and Methods
retinas known to contain HSP-7015 were used as positive controls. Retinal tissues were fixed in Carnoy's solution, prepared similarly to the arterial specimens, and then carried through the immunostaining procedure concurrently with each arterial specimen tested.
Time-After-Death Controls Eight normal male New Zealand white rabbits were killed and divided into four groups of two rabbits each for comparison of aortic HSP-70 after cold storage (4 C) of each rabbit cadaver for 0, 10, 15, and 24 hours, respectively. Following each period of storage, aortas were removed, prepared as intima-media, and immersion-fixed in 10% buffered formalin for histology and immunostaining of HSP-70. Two human autopsy aortic specimens obtained within 10 hours after death were each divided into six portions; two of the portions were immediately processed for study while the other four were stored at 4 C for periods equivalent to 15 and 24 hours after death, then formalin fixed and processed for study of HSP-70.
Histology Tissue specimens were fixed in 10% buffered formalin for a minimum of 48 hours, embedded in paraffin, sectioned at 5 to 7 ,u, and mounted on microscope slides. Representative sections were stained routinely with hematoxylin and eosin (H & E) whereas other sections underwent the immunostaining procedure employing a monoclonal antibody (MAb) specific for HSP-70. Controls for nonspecific staining consisted of tissue sections carried through the immunostaining procedure, but with the MAb omitted.
Source of Tissues Immunoperoxidase Staining of HSP-70 Ten human carotid endarterectomy specimens were studied. Routinely, less than one hour elapsed between surgical removal and analyses. Two human common carotid and 11 aortic specimens were obtained from autopsy. Previous findings14 support the use of autopsy tissue for analyzing arterial enzymes and cellular constituents. The autopsy specimens also contained normal-appearing areas from the same artery for controls. Eight male New Zealand white rabbits weighing 2 to 3 kg each were fed rabbit chow ad libitum and killed by intravenous injection of sodium pentobarbital. Entire aortas were removed, opened, and immersed in 10% buffered formalin for histologic preparation. The normal rabbit aortas were used as controls and for assessing postmortem alterations in HSP-70, similar to that which may have occurred in autopsy specimens as a result of autolysis. Normal rat
The fixed and paraffin-embedded tissues were mounted on slides, dried in a 47 C oven for at least 12 hours, and then deparaffinized with fresh xylene for 1 hour. Rehydration, beginning with 100% ethanol for 15 minutes, continued with 95%, 80%, and 70% ethanol solutions and ended with distilled water. Endogenous peroxidase activity was blocked by incubating the sections for 15 to 30 minutes in methanol containing 10% H202 between the 70% ethanol and distilled water steps during rehydration. To block nonspecific binding, the sections were covered with a solution of 2% powdered nonfat dry milk (Carnation, Los Angeles, CA) in 0.01 M phosphate-buffered saline (PBS), pH 7.2, for 30 minutes at room temperature in a humidified chamber. Excess milk solution was removed, and the sections were covered with a 1:5000 to 1: 10,000
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dilution of mouse monoclonal anti-HSP-70 antibody, C92F3-A (a gift of Dr. William J. Welch, University of California, School of Medicine, San Francisco, CA) in 2% powdered milk in PBS and incubated for two hours at room temperature. The sections were washed three times in PBS for five minutes each, and incubated for one hour with a 1:250 dilution of horse anti-mouse biotinylated antibody (Vector Laboratories, Burlingame, CA). The tissue sections were washed as before and incubated for 30 minutes to 1 hour with the avidin and biotinylated horseradish peroxidase contained in the Vectastain Peroxidase ABC Elite kit (Vector Laboratories) according to the manufacturer's recommendations. The sections were again rinsed twice for five minutes each in PBS followed by once in distilled water for five minutes. The reaction product was developed using diaminobenzidine (6 mg in 10 ml of 0.05M TRIS buffer, pH 7.6) plus 100 ,ul 3% H202 and counterstained with eosin. A brown-to-black precipitate was indicative of the presence of HSP-70.
Osmium Staining of Lipids Microscopic demonstration of lipids in the specimens was done according to a modification of the osmium tetroxide method described previously.17 Endarterectomy specimens were fixed in a mixture of glutaraldehyde and formaldehyde for one to two hours, followed by overnight fixation in 10% buffered formalin (0.01M phosphate buffer, pH 7.4). Thin slices were fixed for eight hours in a solution of 1% osmium tetroxide (Stevens Metallurgical Corp., New York, NY ) in 2.5% potassium dichromate, washed for 2 to 4 hours in running water and processed overnight in an automatic tissue processor for paraffin embedding. Six-micron sections were cut, deparaffinized, and counterstained with H & E. Using this method, lipids stain black and the background pink, with cell nuclei being blue to black.
Immunoperoxidase Staining of Smooth Muscle Versus Macrophage-Specific Antibody Binding Formalin-fixed, paraffin-embedded tissues were immunohistochemically tested for binding of cell-specific antibodies to vascular smooth muscle cells and to macrophages. All antibodies (ie, macrophage specific [MAC]; muscle specific actin [MSA]; and smooth muscle specific actin [SMSA]) were obtained from ENZO Biochemicals, New York, NY. Respective antibody dilutions applied were: 1: 500; 1:2000; and 1:50. Sections of normal human lymph node were used as positive tissue controls, inclusive of small arteries and arterioles for comparison of MSA and
SMSA specificity. Negative tissue controls (ie, the exact procedure less the cell-specific antibody) also were included to detect any false positive reactions. All samples were processed using an Autoprobe Detection System (Biomedia Corp., Foster City, CA) in combination with a Fisher Code-On Immunology System (Fisher, Pittsburgh, PA). The system involved a universal tissue conditioner to block nonspecific binding of the primary antibody followed by incubation of the tissue with the primary antibody. A peroxidase reagent containing a mixture of horseradish peroxidase conjugated antibodies to detect the primary antibodies was added next, followed by the application of a chromagen. The antigenic sites were visualized as a brownish-red precipitate on a clean tissue background when counterstained with a water-based hematoxylin.31
Results Normal-appearing specimens of human aortas from autopsy (Figure 1) and normal rabbit aortas demonstrated routinely a homogeneous distribution of HSP-70 throughout intima-media. As a control for postmortem changes in arterial HSP-70, cadavers of normal rabbits were kept at 4 C for up to 24 hours before their aortas were removed for HSP-70 analysis. There was no change in HSP-70 staining up to 15 hours after death, but by 24 hours, there was a dramatic decrease. Human aortic specimens from autopsy that were collected 8 to 10 hours after death also were stored at 4 C for periods up to 24 hours after death with similar results. Based on these findings, only autopsy aortic specimens up to 15 hours after death were included for study. Human atherosclerotic aortic specimens from autopsy demonstrated HSP-70 staining around areas of necrosis, typically in the central portions of atheroma (Figure 2). The same findings were observed for all ten human carotid endarterectomy specimens (Figure 3). The intensity of HSP-70 staining and its distribution appeared routinely to correlate with increased thickness of plaque (Figure 3), so that thinner regions often were without intense staining. HSP-70 staining in plaques appeared to be related more to a cellular than extracellular component around areas of necrosis and vacuolization partly due to lipid accumulation. To test the concurrence of HSP-70 intensity and increased lipid accumulation, separate sections of the same human carotid endarterectomy specimens were stained for lipid and for HSP-70 and compared. As illustrated in Figure 4, on the average, there was a general coincidence of plaque lipid with increased HSP-70 staining, most notably surrounding areas of necrosis. Rarely was there the occurrence of necrosis or lipid accumulation without intense HSP-70 staining. In contrast, how-
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Figure 1. Distribution of HSP-70 in normal-appearing human abdominal aorta less than ten hours postmortem. A and B: immunostainedfor HSP- 70; C and D: control section immunostained without HSP- 70 antibody. A and C:final magnification X 62 (M = media); B and D: final magnification, X 156 A weakly stained and relatively homogeneous distribution ofHSP- 70 was evidenced routinely. Small areas of increased staining were evidenced occasionally and usually proximal to lumen as seen in the enlarged view on right.
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Figure 2. Distribution of HSP-70 in human atherosclerotic abdominal aorta less than ten hours after death. Intimamedia preparation with necrosis is seen: A: immunostainedfor HSP- 70; B: immunostained without HSP-70 antibody. Final magnification, X62 (P = plaque). Intense HSP- 70 concentration was routinely present in the central areas of atherosclerotic plaques.
ever, there were plaque areas that did have intense HSP70 staining without notable necrosis and lipid. Examination of the cellular nature of the same arterial plaques that were studied for localization of HSP-70 revealed the presence of both smooth muscle cells and macrophages (Figure 5, 6). The distribution of these cell types in plaque and in relation to increased HSP-70 staining varied relative to the amount and complications of each plaque. Generally, however, macrophage-positive staining was viewed more often around areas of necrosis and coincident with increased HSP-70 staining. Additionally, patches of smooth muscle cells, when present in plaque, stained for HSP-70 equivalent in amount to that of the underlying medial cells and also comparable to vascular cells of small arteries when present in plaque (Figure 5C). It should be noted that lymphocytes and neutrophils also were observed in certain areas of complicated plaque, coincident with HSP-70 staining. In contrast, it also was evident that in complicated plaques especially, relatively acellular, collagenous areas stained lightly for HSP-70. Lastly, both macrophage- and smooth muscle
cell-positive staining were noted for lipid-filled cells when present and coincident with increased HSP-70 staining.
Discussion Based on examination of 23 individual human arterial specimens and 8 normal rabbit aortas, HSP-70 is present in human and rabbit arteries. These observations are consistent with a previous study in rat thoracic aortic cells.'0 The purpose of HSPs in normal arteries is unclear, but may relate to functions of HSPs in normal tissue. The results are consistent with observations by others indicating the presence of HSPs in a variety of normal cultured cell lines'8 and tissues.'9 There is, however, the possibility that a degree of unavoidable "stress" during procurement and preparation of the arterial samples added to the presence of HSP-70. This possibility has not been resolved, but if correct, appears minor because HSP-70 was present in normal arteries and did not vary significantly between different artery types (human carotid and aorta),
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B. Figure 3. Distribution ofHSP- 70 in human carotid endarterectomy specimen less than one hour after surgical removal. Advanced, complicated atherosclerotic plaque that contained lipid and necrosis. A: immunostained for HSP- 70; B: immunostained without HSP-70antibody. Final magniflcation, X15(n = necrosis; m = media). Endarterectomyspecimenscontained mostlyplaque circumscribed by a small rim of media; the size and complexity ofplaques varied between specimens. Intense HSP- 70 staining was found around areas of necrosis in contrast to other areas ofplaque that did not stain intensely.
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Figure 4. Comparison ofHSP- 70 and lipid localization in human carotid endarterectomy specimen less than one hour aftersurgical removal: A: osmium-stained lipids are evident as droplets; B: section ofsame specimen immunostainedfor HSP- 70. Original magnification, X200 (inset, X400). Generally, the more intense areas of lipid accumulation had greater concentrations of HSP-70. The reciprocal was not always true, suggesting that other factors in addition to lipid accumulations may be relevant to HSP levels in arteriosclerotic plaques (P = plaque; M = media; bars indicate areas ofinsets).
Heat Shock Protein and Atherosclerosis 77 AJPJanuary 1990, Vol. 136, No. 1
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Figure 5. Distribution ofsmooth muscle cell-specific antibody in human carotid endarterectomy specimens less than one hour after surgical removal: A and B include positive dark staining of vascular smooth muscle cells in media (M) in contrast to minimally stained areas of relatively uncomplicated plaque (P). Enlarged view of upper right also shows a mass of vascular smooth musqle cells at the leading edge ofplaque. Original magnifications, X 62 and X 156, respectively. C shows a portion of a complicated lesion. Arrow heads indicate small arteries representing neovasculazation ofplaque and positively stained for MSA antibody; the asterisk indicates an area of cholesterol clefts. Large positively stained dark areas, often seen as random whirls, were evident areas of vascular smooth muscle cellproliferation. Final magnification, X 62. C represents routine staining of small arteries andasarterioles (A) present in the human lymph node specimens included aspositive tissue control. Final magnification, X 156
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Figure 6. Distribution of macrophage-specific antibody in human carotid endarterectomy specimens less than one hour after surgical removal A represents a cross-section from media (M) through plaque (P) inclusive of a foci of necrosis (N) proximal to the luminal surface. The luminal halfofplaque containedpositive stainingfor macrophages, particularly at the borders of the necrosis. Final magnification, X 156 C evidences an enlarged view of necrosis (M) proximal to the luminal surface and dark stainedfor macrophages; the asterisk indicates an area of cholesterol clefts (N) also in association with the necrosis. Final magnification, X 156 D represents routine staining of macrophages (m) present in the human lymph node specimens included as positive tissue controls. Final magnification, X312.
different species (human and rabbit aorta), or different sources and times (rabbit surgical vs. autopsy aorta up to 15 hours after death). Because of the lack of differences in HSP-70 distribution and concentration in normal arteries, that level was taken as a relative baseline against which changes in HSP-70 in human atherosclerotic arteries could be contrasted. To examine HSP-70 in human atherosclerotic plaques, arrangements were made to obtain carotid endarterectomy specimens that typically contained advanced necrotic plaques immediately after surgery. One limitation to their use, however, was the minimal amount of more normal-appearing tissue included. As a comparison to the surgical carotid samples and as a source of more normalappearing artery for internal controls, aortic specimens from autopsy were examined. The use of autopsy aortic specimens for study of biochemical changes in human atherosclerosis for up to 24 hours after death has been validated previously.14 This validation was extended to
HSP-70 in the present study by comparing normal rabbit with human normal-appearing and atherosclerotic autopsy specimens kept in cold storage for up to 24 hours. Histologically, HSP-70 distribution and concentration did not differ significantly up to 15 hours postmortem. This finding suggests the potential use of HSPs as molecular markers of tissue viability as well as the quality of tissue preservation. Validation for the use of autopsy material also was implicated later, because the results of HSP-70 staining in atherosclerotic plaques were comparable regardless of the source or type artery. This study demonstrated that carotid arteries and autopsy aortas routinely had increased HSP-70 staining around areas of necrosis (Figure 2, 3). HSP-70 staining was most pronounced in the central portions of advanced atherosclerotic plaques (Figure 2, 3) and was often coincident with plaque lipid accumulations (Figure 4). The latter observation was meaningful because contiguous plaque areas did not always contain increased HSP-70 staining
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(Figure 3). The presence of HSP-70 was related more to the nature of the plaque, ie, lipid-filled and/or necrotic vs. fibrotic and cellular (smooth muscle cells) rather than to the size and thickness of the plaque per se (Figure 2). The observation that increased HSP-70 staining did not always coincide with lipid accumulation suggests that additional factors also may be influencing the metabolic state of plaque cells. Blood pressure-induced intramural stress2o possibly induces HSP responses depending on the arterial cell type, its intramural position, and degree of underlying disease. Although several potential arterial cell "stressors" may be involved, the present observations document that the central parts of advanced plaques that surround areas of necrosis and lipid accumulations were consistently most "stressed" based on the increased presence of HSP-70. To further resolve the significance of these observations, the coincidence of HSP-70 to either smooth muscle cells or macrophages in plaque was explored. Although the complicated plaque of human arteries provided excellent examples of necrotic foci, it was difficult to resolve any one major factor responsible for HSP-70 alterations; however, many aspects of the complicated arterial lesions appeared to relate to the distribution of plaque HSP-70. Importantly, macrophages were evidenced most routinely around areas of necrosis and coincident with increased HSP-70 staining; macrophages also were observed to be lipid-loaded. It remains unclear, however, whether the lipid-filled macrophages are stressed to the point of pending death to add to a necrotic foci or exist as scavengers in the process of cleaning up the extracellular debris. Relative to the extent and nature of the plaque, patches of smooth muscle cells also were observed to be positive for HSP-70. In contrast to macrophage-positive areas, necrosis was not consistently evident in association with the plaque smooth muscle cells; some plaque smooth muscle cells, however, also were observed to contain lipid but less routinely than macrophages. Additionally, large, relatively avascular and collagenous areas of plaque were sometimes positive for HSP-70 staining. The latter observation is consistent with the fact that HSPs can be secreted by cells2' and that at least one HSP is known to bind to collagen.22 It is clear that HSP-70 can be located both extra- and intracellularly in human plaque. Information on the deposition of the other HSPs in relation to atherosclerosis is also needed to obtain a more complete picture of how this molecular response of arterial cells relates to the disease. The implication that HSPs may have effects outside of the cells making them is especially noteworthy. The differences in stress-induced lipid accumulation between macrophages and arterial smooth muscle cells in early plaque evolution also warrants study. With respect to the latter, it may be possible to use HSP
production as a marker of arterial cells that are more stressed and possibly more vulnerable to die relative to their physiologic state in the plaque regardless of cell origin. Realizing the significance of these possibilities, it remains necessary to determine what relationship HSPs have to atherosclerosis, ie, are they protective, deleterious, or simply symptomatic? The relevance of HSPs to arterial lipid-filled foam cell genesis, as implicated by our results, deserves added consideration. A significant portion of arterial intracellular lipid accumulation occurs in lysosomes23 and lack of lysosomal integrity is a key factor in cell death.24 Theoretically, HSPs may have a protective effect on stressed arterial cells by preventing lysosomal breakdown. If so, and if lipids are present concurrently, the result may be intralysosomal lipid entrapment. This in turn would contribute to the genesis of arterial foam cells coincident with prevention of their death as part of additional plaque evolution. Lysosomes of lipid-filled foam cells are suspected to have increased membrane stability2526 and it is thought that one mechanism for HSP effects may include membrane stabilization.2728 It is also significant to note the relatively long period through which human plaques evolve,29 implicating that a number of lipid-filled cells remain intact and stable enough to survive long periods during plaque development. To explore these points, a biochemical examination of HSP-70 levels in normal versus atherosclerotic arteries relative to necrosis and lysosomal function is underway3' concomitant with an examination of the direct effects of purified HSP-70 on arterial cell survival and lysosomal function. The amounts and types of HSPs in different grades of atherosclerotic plaques as well as in different types of arterial cells enzymatically isolated and separated from atherosclerotic arteries are also under study in our laboratories. With regard to the latter, and of special significance to an understanding of which arterial cell types may be more prone to survive or die during atherogenesis, MAbs specific to macrophages or vascular smooth muscle cells are being applied to density-separated arterial cells as part of a future report.
References 1. Wissler RW: Principles of the Pathogenesis of Atherosclerosis, Heart Disease: A Textbook of Cardiovascular Medicine. Edited by E Braunwald. Philadelphia, WB Saunders, 1984, pp 1 183-1204 2. Kissane JM: Blood vessels and lymphatics, Anderson's Pathology, Volume 2. Edited by JM Kissane. St. Louis, CV Mosby, 1985, pp 687-696 3. Schlesinger MJ: Heat shock proteins: The search for functions. J Cell Biol 1986,103: 321-325
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4. Thomas GP, Welch WJ, Mathews MB, Feramisco JR: Molecular and cellular effects of heat shock and related treatments of mammalian tissue-culture cells. Cold Spring Harbor Sym Quant Biol 1982,12: 985-996 5. Dienel GA, Diessling M, Jaceqicz M, Pulsinnelli WA: Synthesis of heat shock proteins in rat brain cortex after transient ischemia. J Cereb Blood Flow Metab 1986,6: 505-510
6. Kennedy PG, LaThangue NB, Chan WL, Clements GB: Cultured human neural cells accumulate a heat shock protein during acute herpes simplex virus infection. Neurosci Left 1985,61:321-326 7. Craig EA: The heat shock response. CRC Crit Rev Biochem 1985,18:239-280 8. DeShaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R: A subfamily of stress proteins facilitates translocation of secretary and mitochondrial precursor polypeptides. Nature 1988, 332: 800-805 9. Chirico WJ, Waters MG, Blobel G: 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 1988, 332: 805-810 10. Hightower LE, White FP: Preferential synthesis of rat heatshock and glucose-regulated proteins in stressed cardiovascular cells, Heat Shock: From Bacteria to Man. Edited by M Schlesinger. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1982, pp 369-377 11. Bjornheden T, Bondjers G: Oxygen consumption in aortic tissue from rabbits with diet-induced atherosclerosis. Arteriosclerosis 1988,1: 238-247 12. Polla BS: A role for heat shock proteins in inflammation? Immunol Today 1988,9: 134-137 13. Hajjar DP, Fabricant CG, Minick CR, Fabricant J: Virus-induced atherosclerosis: Herpes virus infection alters aortic cholesterol metabolism and accumulation. Am J Pathol 1986,122:62-70 14. Berberian PA, Fowler S: The subcellular biochemistry of human arterial lesions: I. Biochemical constituents and marker enzymes in diseased and unaffected portions of human aortic specimens. Exp Mol Pathol 1979, 30: 27-40 15. Barbe MF, Tytell M, Gower DJ, Welch WJ: Hyperthermia protects against light damage in the rat retina. Science 1988, 241:1917-1820 16. Welch WJ, Suhan, JP: Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol 1986,103: 2035-2052 17. Luna LG: Osmium tetroxide method for fat (paraffin sections), Manual of Histologic Staining Methods. Edited by LG Luna. New York, McGraw-Hill, 1968, pp 143-144 18. Landry J, Bernier D, Chr6tien P, Nicole LM, Tanguay RM, Marceau N: Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res 1982, 42: 2457-2461 19. O'Malley K, Mauron A, Barchas JD, Kedes L: Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shocklike protein. Mol Cell Biol 1985, 5: 3476-3483 20. Thubrikar MJ, Baker JW, Nolan SP: Inhibition of atherosclerosis associated with reduction of arterial intramural stress in rabbits. Arteriosclerosis 1988,8: 410-420
21. Hightower LE, Guidon PT Jr: Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. J Cell Physiol 1989, 138: 257-266 22. Nagata K, Saga S, Yamada KM: Characterization of a novel transformation-sensitive heat-shock protein (HSP-47) that binds to collagen. Biochem Biophys Res Commun 1988, 153:428-434 23. Fowler S, Berberian PA, Haley NJ, Shio H: Lysosomes, vascular intracellular lipid accumulation and atherosclerosis, International Conference on Atherosclerosis. Edited by LA Carlson. New York, Raven Press, 1978, pp 19-27 24. Bowen ID: Techniques for demonstrating cell death, Cell Death in Biology and Pathology. Edited by ID Bowen and RA Lockshin. New York, Chapman and Hall, 1981, pp 379-444 25. Peters TJ, deDuve C: Lysosomes of the arterial wall: II. Subcellular fractionation of aortic cells from rabbits with experimental atheroma. Exp Mol Pathol 1974, 20: 228-256 26. Berberian PA, Jenison MW, Roddick V: Arterial prostaglandins and lysosomal function during atherogenesis: II. Isolated cells of diet-induced atherosclerotic aortas of rabbit. Exp Mol Pathol 1985, 43: 36-55 27. Minton KW, Karmin P, Hahn GM, Minton AP: Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: A model for the biological role of heat shock proteins. Proc Natl Acad Sci USA 1982, 79: 7107-7111 28. Lepock JR, Cheng KH, Al-Qysi H, Kruur J: Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: Relationship to hyperthermic cell killing. Can J Biochem Cell Biol 1983, 61: 421-427 29. Stary HC: Evolution and progression of atherosclerotic lesions in coronary arteries of children and young adults. Arteriosclerosis 1989, 9(suppl): 1-19-1-32 30. Berberian PA, Bond MG, Myers W, Tytell M, Challa V: Potential relevance of arterial heat shock proteins to lysosomal function and necrosis in human atheroma (abstr). FASEB J 1988,2: A1597 31. Brigat DJ, Budgeon LR, Unger ER, Koebler D, Cuomo C, Dennedy T, Perdomo JM: Immunocytochemistry is automated: Development of a robotic work-station based upon the capillary action principle. J Histotechnology 1988, 11: 165-183
Acknowledgments The authors thank Tonya Smith, Katrina Graham, and Carol R. Hollman for expert technical assistance; Linda Caudill and Sarah Graham for secretarial assistance; and Drs. W. Keith O'Steen and Judy K. Brunso-Bechtold, Department of Neurobiology and Anatomy, as well as Dr. Michael McWhorter, Department of Surgery and Dr. Larry Miller, the Immunohistochemistry Laboratory, Department of Pathology, for their cooperation and support of the study. HSP-70 antibody was provided by Dr. William J. Welch, Departments of Physiology and Medicine, University of California, School of Medicine, San Francisco, CA.
