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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:27-36.)
© 1995 American Heart Association, Inc.

Articles

Differential Distribution of 70-kD Heat Shock Protein in Atherosclerosis

Its Potential Role in Arterial SMC Survival

A. Daniel Johnson; Paul A. Berberian; Michael Tytell; M. Gene Bond

From the Department of Neurobiology and Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC, and the Department of Atherosclerosis and Vascular Biology (P.A.B.), Sandoz Research Institute, East Hanover, NJ.

Correspondence to Dr A. Daniel Johnson, Texas Heart Institute, Vascular Cell Biology Laboratory, PO Box 20345, Mail Code 2-255, Houston, TX 77225.

	Abstract

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Abstract Smooth muscle cell death may contribute to necrotic plaque rupture and subsequent thromboembolus. Stress-induced synthesis of heat-shock proteins (HSPs) normally protects cells from death, but vascular HSPs may become insufficient as cytotoxicity increases in advanced plaques. To determine whether vascular HSP content is altered near necrosis, we compared 70-kD HSP (HSP70) distribution between fibrotic and necrotic plaques in immunostained carotid endarterectomy specimens. Average levels of HSP70 immunoreactivity were compared by video densitometry between fibrotic and necrotic plaques or between their underlying media. Both necrotic plaques and their underlying media contained significantly more HSP70 staining than did fibrotic tissues. To test whether cellular HSP70 correlated with resistance to toxicity in vitro, aortic smooth muscle cells (aSMCs) were heat shocked to induce endogenous HSPs or given 2 to 50 µg/mL purified HSP70. Cells were then serum deprived or exposed to 12 to 96 µmol/L cholestanetriol (C3ol) or 25-hydroxycholesterol, and survival was determined. Cellular HSP70 content was assayed by immunoblotting, and protein synthesis was monitored by ³⁵S radiolabeling. Serum deprivation inhibited general protein synthesis but induced HSP70; C3ol exposure inhibited both overall protein and HSP70 synthesis, including post–heat shock. Induction of endogenous HSPs or 10 µg/mL exogenous HSP70 improved viability of serum-deprived cells (P<.05 and P<.01, respectively), while only exogenous HSP70 protected against C3ol (P<.002). The results suggest that insufficient HSP70 accumulates in aSMCs residing near necrosis to protect against plaque toxicity; aSMC death might then occur, allowing resident macrophages to degrade and destabilize the matrix, leading to rupture.

Key Words: plaque necrosis • cytoprotection • HSP70 • stress response • smooth muscle cells

	Introduction

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During atherosclerotic progression, two major subtypes of advanced plaques appear, with differing paths to vascular occlusion.¹ Fibrotic plaques are characterized by extensive proliferation of arterial smooth muscle cells (aSMCs) with extracellular matrix deposition; they may also arise through organization of a lipid-rich plaque by SMCs, as observed during plaque regression.^{2 3} Generally, fibrotic primary plaques slowly grow to occlude blood flow^{4 5 6} and minimal necrosis is present.^{1 7} Necrotic plaques differ in that cell death leads to a disorganized, relatively acellular central core that can subsequently rupture and cause sudden thromboembolic occlusion.^{8 9} Both plaque subtypes can coexist within a single vessel under similar physiological conditions (References 1 and 7 and A.D. Johnson, unpublished data, 1992).

The specific causes for development of plaque necrosis have not yet been identified, but it may arise from restricted availability of oxygen and nutrients^{10 11 12 13} ; loss of essential growth factors¹⁴ ; free radical injury¹⁵ ; toxic cytokines generated by both monocyte/macrophages and SMCs^{16 17} ; oxidation of lipids, lipoproteins, and cholesterol derivatives^{18 19 20 21} ; or alterations in calcium regulation.²² Plaque destabilization and rupture may also result from hydrolysis of the extracellular matrix around the necrotic core by macrophage-derived proteases.^{23 24 25} We have adopted a hypothetical working model in which there is a dynamic equilibrium between organization and fibrosis by aSMCs^{8 9} and destabilization and matrix proteolysis by macrophages. In such a model, loss of aSMCs due to plaque toxicity would make rupture more likely; therefore, our research has focused in part on endogenous mechanisms to prevent aSMC death, such as synthesis of heat-shock proteins (HSPs).

HSPs are a family of cytoprotective proteins that are rapidly and preferentially synthesized by cells in response to many potentially lethal stimuli.^{26 27} HSPs are induced by several plaque-associated factors, including oxygen radicals,^{23 24} toxic cytokines,^{28 29 30} ischemia,^{31 32} serum deprivation,³³ or mechanical trauma.³⁴ HSPs are normally present in vessels,^{35 36 37 38} and induction of HSPs has been correlated with enhanced stress resistance in endothelial cells^{35 38} and vascular SMCs.^{36 39 40} A change in the distribution of 70-kD molecular weight HSP (HSP70) correlates with increasing plaque size in human carotid endarterectomy specimens⁴¹ and human autopsy or macaque diet-induced atherosclerotic aortas.⁴² While those results suggest that HSP70 is induced during plaque evolution, no comparison has yet been made of the levels of HSP70 expression in advanced plaques with differing amounts of cell death, ie, between necrotic and fibrotic plaques. It is also possible that increased HSP70 expression in plaques is only a marker of increased stress rather than being actively involved in the preservation of plaque cells. To address these questions, we used video analysis to compare HSP70 accumulation in fibrotic and necrotic plaques obtained from human carotid endarterectomy surgical specimens. We also exposed cultured aSMCs to serum deprivation or oxysterols (oxidized cholesterol derivatives) as two models of cytotoxic conditions believed to be present in atherosclerotic plaques^{11 21 43} to determine the relation between HSP70 expression and stress-induced cell death. Furthermore, we examined whether elevating endogenous HSP levels by heat shock^{26 27} or by the addition of exogenous HSP70⁴⁴ could protect aSMCs from plaque-associated cytotoxicity. The results showed a significant correlation between HSP70 accumulation and the presence of necrosis in carotid vessels. We also found that elevated HSP70 levels protected stressed aSMCs in vitro but that protection depended on the severity of the stress applied.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Nomenclature for 70-kD HSPs
In this study, HSC73 refers to the 73-kD HSP isoform produced constitutively by mammalian cells and induced by stress in primate-derived cells (including human cells).²⁶ HSP72 refers to the highly stress-inducible 72-kD HSP isoform. HSP70 refers generically to either isoform, while purified bovine brain HSP70 or exogenous HSP70 refers specifically to a mixture of 95% HSC73 and 5% HSP72 (a gift from Dr David Gower, Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Winston-Salem, NC).

Tissue Retrieval and Histology
Carotid endarterectomy specimens, 20 to 30 mm total length, were obtained from 18 patients immediately after surgery and placed in saline. Specimens removed from the common and internal carotid arteries consisted of intimal plaques with subjacent portions of underlying media. Specimens were cut into four to six 5-mm-thick cross-sectional segments. Each segment was fixed for 24 hours in Carnoy's fixative before being embedded in separate paraffin blocks.

Serial 8-µm cross sections were cut from the proximal ends of each of the four to six segments from each patient's specimen and stained with hematoxylin/eosin (H&E) or Verhoeff/van Gieson's (VVG) elastin stains. Lesions were separated into two groups based on the criteria of Stary¹ : atheromatous (types IV to V) plaques, in which necrotic cores were consistently present, or fibrotic (type VIII) plaques, in which necrosis was absent from all segments of the specimen.

Immunohistochemistry and Video Analysis
Additional serial sections matching the H&E-stained sections from the proximal end of each segment were incubated overnight with N27F3-4, a monoclonal antibody against both HSC73 and HSP72 (StressGen), diluted 1:5000 in 1% normal horse serum/Dulbecco's phosphate-buffered saline (PBS). Matching control sections were stained by switching the monoclonal antibody to nonimmune murine immunoglobulin (Ig) G (Sigma). Immunoreactivity was then visualized by using avidin/biotin/diaminobenzidine/H₂O₂ (Vector Laboratories).⁴²

For video analysis, images were collected at x10 magnification with an Olympus BH-2 microscope fitted with a DAGE-MTI CCD72 video camera using National Institutes of Health (NIH) IMAGE (version 1.32) on a Macintosh IIfx computer. During each work session, intensity measurements were initially made by using a piece of exposed color negative film mounted on a glass slide; this was used to set the light source to a consistent output level. Next, immunostained cross sections of each of the four to six segments from seven necrotic and seven fibrotic plaque specimens were examined; the four remaining specimens were excluded due to insufficient underlying media for analysis. A schematic of the sampling procedure is shown in Fig 1. Each image spanned approximately one third of the circumference of the specimen. To determine HSP70 immunostaining density, each image was subdivided into five to seven regions, with one region representing a 1-mm-wide wedge of the circumference of the original plaque. VVG-stained sections were used to delineate the intima from media in each region, and a boundary was drawn around the entire media or intima within each of the five to seven subdivisions. Next, the mean gray level (reflecting the density of immunostaining) within each intimal or medial subdivision was measured separately and expressed on a linear scale of 0 (white) to 255 (black). The process was then repeated twice more to include all of one cross section. The 15 to 20 individual data points were averaged to give the mean HSP70 immunostaining for a single plaque segment cross section, and then averaged again for the four to six cross sections examined from each patient's specimen. Finally, the mean HSP70 immunoreactivity was statistically compared between the seven necrotic and seven fibrotic intimal plaques or between media underlying those plaques by using Student's t test. To determine cell density, nuclei per unit area of tissue were counted on matching H&E-stained sections using the x20 objective and the same sampling protocol. In necrotic plaques, intimal HSP70 and nuclear counts were made within organized tissue around the necrotic core only; area analysis did not include the mostly acellular lipid gruel filling the core itself.

View larger version (25K): [in this window] [in a new window]   Figure 1. Schematic of the sampling procedure for 70-kD heat-shock protein determination in endarterectomy specimens. An image spanning approximately one third the total original segment in cross section was divided into five or six 1-mm-wide wedge-shaped regions. Boundaries were separately drawn around the intima and media within each region. Average gray level was determined within each intimal or medial region and then averaged for all cross sections taken from a single patient's specimen. Statistical comparisons of the means and SDs were made between data from fibrotic and necrotic plaques.

To ensure that all slides to be compared were immunostained as identically as possible, control serial sections from an atherosclerotic plaque of a human aorta and of rat retinas were included in each staining run. Equivalent results were obtained when multiple control sections processed within a single batch or sections processed on different days (data not shown) were compared. Other studies have demonstrated linearity of the immunoreactivity of HSP70 by Western blots of serial dilutions of purified HSP70⁴⁵ or homogenates of arterial tissue (data not shown). Prior studies using various whole-body stress animal models have also shown that arterial HSP70 immunoreactivity by immunostaining of tissues increases or declines in patterns consistent with total HSP70 determined by Western blotting or HSP70 mRNA on Northern or in situ hybridization analyses.^{31 32 34 37 46 47} As additional controls, sections were immunostained for -actin or fibronectin (which are both altered in atherosclerosis^{48 49} ) and analyzed. Patterns of -actin or fibronectin immunostaining were similar to prior reports; the video methods described successfully detected a significant difference in distribution for both markers (data not shown).

Macaque and Rabbit Cell Lines
Two groups of macaque aSMCs were used. First, aSMCs from six adult male cynomolgus macaques obtained from Charles River Laboratories were isolated as described.⁴⁴ These primary isolates were used immediately without subculturing. Second, a passaged macaque smooth muscle (PMSM) cell line was obtained from Dr Richard St. Clair, Department of Comparative Medicine, Bowman Gray School of Medicine. These cells were derived from an explant of thoracic aorta of a fetal male cynomolgus macaque and subsequently grown in T-75 flasks in minimal essential medium/10% fetal calf serum (FCS). They were subcultured at a 1:3 ratio with 0.25% trypsin/PBS as necessary. PMSM cells were used at passages 15 through 25 for these studies.

Adult male New Zealand White rabbits were obtained from Franklin's Rabbitry and were maintained in accordance with institutional and Public Health Service Animal Welfare Assurance guidelines. Aortic SMCs were isolated by collagenase/elastase digestion⁴⁴ and plated into T-75 flasks and cultured for 10 to 12 days in Dulbecco's modified Eagle's medium (DMEM)/10% FCS until confluent. Log phase cells at passages 1 through 4 were used for all studies.

Viability of Serum/Amino Acid–Deprived Cells Over Time
Macaque aSMCs were tested for loss of viability over time in response to serum/amino acid deprivation, referred to here simply as serum deprivation. We used serum deprivation as a model for the nutrient and serum-derived growth factor deprivation described in advanced plaques.^{10 11 12 13} Glucose deprivation has been used as a model for tissue ischemia,^{26 50 51} and the effects of endogenous HSP induction on survival of glucose-deprived cells were qualitatively similar to the results reported here for serum deprivation. However, glucose deprivation does not mimic limited protein availability to aSMCs, which may affect their ability to maintain the extracellular matrix essential for plaque integrity. Therefore, we chose to use serum rather than glucose deprivation.

Samples of 2.0x10⁵ cells suspended in 200 µL serum/amino acid–free Hank's balanced salt solution (SL-HBSS plus 0.9 mmol/L Mg²⁺ and 0.09 mmol/L Ca²⁺) were incubated at 37°C for 0 to 20 hours in the trials using primary cell isolates or 0 to 40 hours for studies using PMSM cells. To determine if prior induction of HSP70 improved cell viability, T-75 flasks containing PMSM cells near confluence were heat shocked in a circulating water bath for 30 minutes at 43°C and then returned to 37°C for 6 hours. Shocked cells were dissociated from the flasks by using 0.25% trypsin/PBS and then serum deprived as described. To test for effects of exogenous HSP70 on aSMC survival,⁴⁴ primary isolates or PMSM cells were pretreated by adding 2 to 100 µg/mL purified bovine brain HSP70 to the SL-HBSS stress media at time zero and then incubating at 37°C for 0 to 20 or 0 to 40 hours as before. Nonspecific effects due to exogenous proteins were determined by substituting lactate dehydrogenase, histone H2a, parathyroid hormone, or bovine serum albumin (BSA) (all from Sigma) at 10 µg/mL in SL-HBSS instead of exogenous HSP70. Viability of control, shocked, or exogenous HSP70-treated cells was determined at 5-hour intervals by 0.4% trypan blue dye exclusion. Triplicate viability determinations were made for each of at least three replicates per treatment. Results were analyzed by ANOVA, and individual means were compared by post hoc protected t tests.

Effects of Serum Deprivation on HSP70 Accumulation and Total Protein Synthesis
HSP70 induction in PMSM cells by serum deprivation was determined by treating cells 0 to 35 hours as described above. Cell suspensions were triturated to lyse nonviable cells; cells were centrifuged for collection and then resuspended and washed twice with PBS. To ensure that only viable cells were collected by this method, suspensions were checked for >95% trypan blue dye exclusion. Viable aSMCs were collected by centrifugation and lysed with 150 µL/10⁶ cells BUST (2% ß-mercaptoethanol, 8 mol/L urea, 1% sodium dodecyl sulfate (SDS), 100 mmol/L Tris, pH 6.8, and 0.01% phenol red), after which total protein was determined by using a BioRad assay. Lysates containing equal cellular protein were resolved on 10% polyacrylamide gels run under reducing conditions and using purified bovine brain HSP70 as a standard. Proteins were electroblotted to nitrocellulose and then probed with one of two murine monoclonal antibodies against HSP70 (both from StressGen) dissolved in 5% dry milk/PBS: antibody N27F3-4 (against both HSC73 and HSP72) diluted 1:5000 or C92F3A-5 (against HSP72 only) diluted 1:1000. Two different antibodies were required because primate-derived cells express both HSC73 and HSP72 during a stress response, whereas rabbit cells express only HSP72.²⁶ HSP70 immunoreactive bands were visualized by using horseradish peroxidase–conjugated rabbit anti-murine IgG secondary antibody at 1:1000 in PBS (Sigma) and diaminobenzidine/H₂O₂ (Vector Labs). Blot band densities were quantified by using NIH IMAGE (version 1.32).

To analyze total protein synthesis, 35-mm dishes containing 10⁶ PMSM cells were serum deprived in SL-HBSS for 30 minutes or 5 hours at 37°C, after which newly synthesized proteins were labeled for an additional 2 hours with 100 µCi/dish Tran³⁵S-Label (a mixture of ³⁵S-labeled methionine and cysteine; ICN). Cells were lysed in BUST, and samples containing either an equal amount of cellular protein or equal trichoroacetic acid–precipitable counts per minute were resolved by SDS–polyacrylamide gel electrophoresis, and the gels were processed for fluorography.

Oxysterol Toxicity to aSMCs and Protection by Heat Shock or Exogenous HSP70
Oxysterols are highly toxic to aSMCs that are actively proliferating, whereas quiescent aSMCs are essentially oxysterol insensitive.^{21 52 53} Since the primary isolates from macaques consisted mainly of quiescent cells, oxysterol toxicity experiments were conducted initially with the PMSM cell line, and then repeated using rabbit aSMCs grown for 3 days as subconfluent cultures in DMEM/10% FCS.⁵¹ Cells were plated at 2.5x10⁵/well in 24-well plastic plates 24 hours before assays. Identical, parallel sets of cultures were used for the heat-shocked and unshocked conditions. For each test group, cells were either left untreated (control), heat shocked to induce endogenous HSPs (30 minutes at 43°C followed by 6 hours of recovery at 37°C), or treated with 2 to 50 µg/mL exogenous bovine brain HSP70 in complete media. Cells were then given 12, 48, or 96 µmol/L cholestane-3ß,5,6ß-triol (C3ol) or 25-hydroxycholesterol (25HC) in 4 µL ethanol vehicle or 4 µL ethanol vehicle alone and returned to 37°C for 24 hours. Cell survival was assayed by vigorously washing cells five times with Ca²⁺/Mg²⁺-free PBS to remove nonviable cells and quantifying the number of cells still attached by using crystal violet staining.^{54 55} In our hands, the crystal violet method gives quantitatively equivalent results as trypan blue (data not shown) without requiring direct visual counting by investigators. However, cells must be attached for the assay, making it unsuitable for use in the serum deprivation studies. The number of cells before and after oxysterol treatment was compared by using ANOVA and protected t tests.

Effect of Oxysterols on HSP70 Accumulation and Total Protein Synthesis in aSMCs
Rabbit aSMCs were preplated at 1.5x10⁶ cells/60-mm dish and grown to 95% confluency in DMEM/10% FCS. Identically plated dishes of cells were either heat shocked at 43°C for 30 minutes before oxysterol or vehicle addition or kept at 37°C (control). Triplicate dishes of heat-shocked and control cells were then treated with 12, 48, or 96 µmol/L C3ol or 25HC (delivered in 4 µL ethanol vehicle) in complete media; control dishes were given an equivalent volume of vehicle alone. After 30 minutes or 6 hours of exposure at 37°C, the medium was removed, the cells were washed as described for crystal violet viability assays, lysed in BUST, and cellular HSP72 in an equivalent amount of cellular protein was determined by Western blot using C92F3A-5 monoclonal antibody. Effects of oxysterols on overall cellular protein synthesis were determined by using ³⁵S radioisotopic labeling of proteins as described above.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Video Analysis of Plaque-Associated HSP70 Immunoreactivity
Representative HSP70-immunostained necrotic and fibrotic plaques and matching VVG elastin–stained sections used to delineate intima from underlying media are shown in Fig 2. HSP70 immunoreactivity appeared higher overall both in necrotic plaques and in the media underlying necrotic plaques compared with either fibrotic plaques or their underlying media (Fig 2B versus 2D). Video analyses of the immunoreaction product in the media underlying carotid plaques are summarized in Table 1. Average HSP70 staining intensity in media underlying necrosis was significantly higher (P<.02), whereas nuclear counts revealed no significant difference in cell density of media underlying necrotic versus fibrotic plaques. Data analyses for intimal plaques are summarized in Table 2. In parallel with the observation seen for the underlying medial tissue, necrotic intimal plaques had higher average HSP70 immunoreactivity than did fibrotic intimal plaques (P<.02). However, nuclear counts were 2.5-fold greater per unit area in necrotic than in fibrotic intimal plaques (P<.0005).

View larger version (121K): [in this window] [in a new window]   Figure 2. Photomicrographs showing representative necrotic and fibrotic plaques from human carotid endarterectomy specimens, stained for elastin with Verhoeff/van Gieson's stain (VVG) or with N27F3-4 monoclonal antibody against 70-kD heat-shock protein (HSP70). A, Necrotic plaque, VVG. B, Necrotic plaque, HSP70; gray scale range of 93 to 177 linear scale units. C, Fibrotic plaque, VVG. D, Fibrotic plaque, HSP70; gray scale range of 77 to 114 linear scale units (A through D, original magnification x13).

View this table: [in this window] [in a new window]   Table 1. Relative Density of HSP70 Immunostaining of Media Underlying Necrotic and Fibrotic Carotid Endarterectomy Specimens

View this table: [in this window] [in a new window]   Table 2. Relative Density of HSP70 Immunostaining of Intimal Plaques From Necrotic and Fibrotic Carotid Endarterectomy Specimens

Heat Shock and Exogenous HSP70 Protect aSMCs From Serum Deprivation–Induced Cell Death
Macaque aSMC primary isolates were serum deprived for 0 to 20 hours; viability was determined by trypan blue dye exclusion, while HSP70 content of surviving cells was monitored by Western blotting using N27F3-4 antibody. Fig 3 shows the cellular HSP70 content of still viable control cells; endogenous HSP70 declined over time, coincident with a significant loss (P<.02) in overall cell viability. Exogenous HSP70 (10 µg/mL) added at time zero significantly improved cell survival after 10 to 20 hours of deprivation stress over time-matched controls (P<.01). Four other test proteins failed to improve cell viability significantly at 20 hours compared with serum-deprived controls (Table 3), indicating that the effect was specific to HSP70.

View larger version (19K): [in this window] [in a new window]   Figure 3. Graphs showing Western blot band densities for 70-kD heat-shock protein (HSP70) content using N27F3-4 antibody () of macaque arterial smooth muscle cell primary isolates and viability by trypan blue dye exclusion (bars; mean±SEM, n=6 replicates) vs time of serum deprivation. *P<.01, decrease in viability of control cells compared with time zero. ++P<.01, viability between cells treated with 10 µg/mL HSP70 at time zero (solid bars) vehicle-only controls (open bars).

View this table: [in this window] [in a new window]   Table 3. Change in Viability of Macaque aSMC Primary Isolates After 20 Hours of Serum Deprivation

The results for serum-deprived PMSM cells over 40 hours (Fig 4) were similar to those for primary cell isolates. Densitometric analysis of bands detected on Western blots by N27F3-4 or C92F3A-5 antibody of serum-deprived PMSM cells demonstrated a transient increase in HSP70 accumulation after 5 hours (Fig 4A and 4B); in contrast, ³⁵S radiolabeling of proteins synthesized by serum-deprived PMSM cells showed that the overall protein synthesis rate decreased by 5 hours (Fig 4C), suggesting that activation of HSP70 synthesis was selective. Viability of PMSM cells (Fig 4B) fell significantly after 10 hours of serum deprivation (P<.01), which was paralleled by a decrease in cellular HSP70 (Fig 4A and 4B); both parameters decreased still further through 40 hours.

View larger version (39K): [in this window] [in a new window]   Figure 4. A, Western blot showing a decrease in N27F3-4 immunoreactive 70-kD heat-shock protein (HSP70) content of non–heat-shocked, serum-deprived passaged macaque smooth muscle (PMSM) cells over time. Lanes 1 and 10, 250 ng HSP70 and molecular weight standards (S); lane 2, time zero; lanes 3-9, 5, 10, 15, 20, 25, 30, and 35 hours of serum deprivation, respectively. An incompletely resolved, immunoreactive band of unknown origin seen just below HSP70 became more prominent with increasing deprivation, but was not an HSP70 degradation product, since it was also recognized by nonimmune mouse immunoglobulin G (not shown). B, Graphs showing HSP70 band densities using C92F3A-5 (; n=1) or N27F3-4 (; n=2) antibodies and viability (bars; mean±SEM, n=6 replicates) vs time of serum-deprived PMSM cells. A rise in cellular HSP70 was detected with both antibodies after 5 hours of initiating serum deprivation, which then declined along with cell viability until 15 hours. After 15 hours, no further decline in immunoreactive HSP70 could be detected using N27F3-4. HSP70 was no longer detectable beyond 15 hours of deprivation using the C92F3A-5 antibody against HSP72. Viability first declined significantly by 10 hours compared with time zero and declined continuously thereafter. *P<.01, decline in cell viability vs time zero. C, Fluorogram of [35S]methionine/cysteine radiolabeled proteins synthesized by PMSM cells during serum deprivation. Lanes were loaded with equal amounts of protein from arterial smooth muscle cell lysed after 30 minutes or 5 hours of serum deprivation and then labeled with [35S]methionine/cysteine. Overall protein synthesis declined dramatically by 5 hours, while the relative level of HSP70 synthesis rose as determined by video densitometry.

Heat-shock pretreatment of PMSM cells increased cellular HSP70 levels through 20 hours and slightly elevated HSP70 at the remaining times examined (Fig 5A versus 4A; Fig 5B); 10 µg/mL exogenous HSP70 had no effect on the endogenous HSP70 synthesis rate compared with BSA-exposed controls (Fig 5C), indicating that any improvement in survival was a specific effect of the exogenous HSP70 rather than via induction of endogenous HSPs. After 15 hours of serum deprivation, both induction of endogenous HSPs by heat shock or the addition of 5 or 10 µg/mL exogenous HSP70 at time zero improved survival compared with unprotected controls (Fig 5B). By 30 hours, heat shock–induced protection had disappeared, while exogenous HSP70 protected cells through 35 hours.

View larger version (41K): [in this window] [in a new window]   Figure 5. A, Western blot of 70-kD heat-shock protein (HSP70) content (using N27F3-4 antibody) of heat-shocked, serum-deprived passaged macaque smooth muscle (PMSM) cells over time. Cells were shocked 30 minutes at 43°C and allowed to recover for 6 hours at 37°C before serum deprivation was initiated. Markers are as noted in Fig 4A. Lanes 1 and 10, 250 ng HSP70 and molecular weight standards (S); lane 2, time zero; lanes 3-9, 5, 10, 15, 20, 25, 30, and 35 hours of serum deprivation, respectively. Heat shock dramatically raised cellular HSP70 accumulation compared with the unshocked PMSM cells shown in Fig 4A. B, HSP70 band densities using N27F3-4 antibody (triangles; mean of n=2) and viability (bars; mean±SEM, n=6) vs time for serum-deprived PMSM cells after heat shock or addition of exogenous HSP70. Cellular HSP70 was induced as described above. HSP70 was elevated in heat-shocked PMSM cells () compared with unshocked cells () through 20 hours of deprivation. Viability of heat-shocked PMSM cells (diagonally hatched bars) was higher than serum-deprived, unshocked controls (open bars) at 15-20 hours (P<.05). Exogenous HSP70 at 2 or 10 µg/mL (shaded and black bars, respectively) improved cell viability over controls at 15-35 hours (*P<.01). C, Fluorogram of sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel containing equal trichoroacetic acid–precipitable counts per minute from PMSM cells labeled with [35S]methionine/cysteine after 30 minutes or 5 hours of serum deprivation. B indicates serum deprivation in the presence of 10 µg/mL bovine serum albumin (BSA); H, serum deprivation in the presence of 10 µg/mL HSP70. Labeled HSP70 content was not significantly higher in exogenous HSP70 versus BSA-treated cells.

Exogenous HSP70 but Not Heat Shock Protects Against Oxysterol Toxicity
Both C3ol and 25HC decreased the viability of PMSM cells in a dose-dependent manner (Fig 6A and 6B). Heat-shock pretreatment did not significantly protect cells from C3ol (Fig 6A); in cells exposed to 25HC (Fig 6B), heat shock improved cell survival over controls only at the highest dose of 25HC tested (P<.05). Addition of exogenous HSP70 to C3ol-treated aSMCs (Fig 6A) increased viability 15% above controls but only at the highest dose of HSP70 and lowest dose of C3ol tested (P<.002). Exogenous HSP70 (Fig 6B) increased aSMC survival in the presence of 25HC by 9% to 31% above controls (P<.0002) depending on the concentration of 25HC used.

View larger version (38K): [in this window] [in a new window]   Figure 6. Bar graphs. A, Viability (mean±SEM; n=6) of passaged macaque smooth muscle (PMSM) cells vs dose of cholestane-3ß,5,6ß-triol (C3ol) after 24 hours. Heat shock (diagonally hatched bars) failed to improve viability of cells at any dose of C3ol compared with EtOH (ethanol only) controls (open bars); 10 µg/mL exogenous 70-kD heat-shock protein (HSP70) (cross-hatched and solid bars) improved viability only at the lowest dose (hatched bars) of C3ol tested (**P<.002). B, Viability (mean±SEM; n=6) of PMSM cells vs dose of 25-hydroxycholesterol (25HC) after 24 hours. Compared with controls (open bars), heat shock (diagonally hatched bars) and 2 µg/mL exogenous HSP70 (cross-hatched bars) improved viability of cells at the highest dose of 25HC (P<.05 and *P<.0001, respectively) Exogenous HSP70 (10 µg/mL; solid bars) significantly improved viability at all doses of 25HC (**P<.0002).

Rabbit aSMCs were less sensitive than PMSM cells to either oxysterol, with only 96 µmol/L C3ol causing significantly decreased viability; results are summarized in Fig 7. C3ol killed approximately 45% of rabbit aSMCs, but cotreatment with 2 or 10 µg/mL exogenous HSP70 at time zero significantly increased survival at 24 hours (P<.05 and 0.01, respectively). Neither heat shock nor 20 or 50 µg/mL exogenous HSP70 improved viability.

View larger version (54K): [in this window] [in a new window]   Figure 7. Bar graph showing viability (mean±SEM; n=6 replicates) of rabbit arterial smooth muscle cells exposed to 96 µmol/L cholestane-3ß,5,6ß-triol for 24 hours. Heat shock or 20 or 50 µg/mL exogenous 70-kD heat-shock protein (HSP70) did not improve viability over untreated controls; 2 or 10 µg/mL HSP70 improved viability (*P<.05 and .01, respectively).

Oxysterols Limit the Inducibility of HSP70 in aSMCs
Induction of HSP72 by oxysterols alone and the effects of oxysterol on heat-shock induction of HSP72 in rabbit aSMCs were examined by using Western blot analyses and C92F3A-5 antibody (Fig 8). C3ol (Fig 8A) or 25HC (not shown) induced little or no HSP72 after 6 hours' exposure compared with heat-shocked positive control cells (Fig 8B or 8C, vehicle controls). Moreover, HSP72 induction was not observed on repetition of the same experiment (data not shown). In cells that were heat shocked after addition of only the ethanol vehicle, little inducible protein was observed at 30 minutes postshock, but by 6 hours postshock a strong HSP72 band was present (Fig 8B and 8C, lane 2 versus 1). In contrast, HSP72 production after heat shock was significantly reduced by C3ol exposure (Fig 8B, 96 µmol/L versus vehicle control at 6 hours), whereas 25HC did not appear to alter HSP70 synthesis (Fig 8C). Analysis of ³⁵S radiolabeling showed that C3ol profoundly inhibited overall protein synthesis, while 25HC did not (Table 4), correlating well with the relative effects of the two oxysterols on heat inducibility of HSP72.

View larger version (59K): [in this window] [in a new window]   Figure 8. Western blots using C92F3A-5 antibody for 70-kD heat-shock protein (HSP70) induction by oxysterols in rabbit arterial smooth muscle cells (aSMCs). Cells were treated with (A) 12, 48, or 96 µmol/L cholestane-3ß,5,6ß-triol (C3ol) or 25-hydroxycholesterol (25HC) (not shown), (B) heat shock plus C3ol, or (C) heat shock plus 25HC. Veh. indicates ethanol vehicle–only control; 30', 30 minutes' exposure to oxysterol or vehicle; 6h, 6 hours of exposure. Dose of oxysterol is given under each pair of bands. C3ol and 25HC (not shown) without heat shock were poor inducers of HSP70. In heat-shocked cells, bands in 96 µmol/L oxysterol vs vehicle control showed that C3ol but not 25HC significantly inhibited HSP70 accumulation by aSMCs (B and C, respectively).

View this table: [in this window] [in a new window]   Table 4. 35S Radiolabeling of Total Protein Synthesis in aSMCs Treated With Ethanol or Oxysterols

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Human and diet-induced animal models of atherosclerosis have shown that distribution of HSPs in arteries changes very early in the development of plaques and becomes increasingly more heterogeneous with disease progression.^{42 56 57} Furthermore, lipid-loaded macrophages near areas of necrosis accumulate high levels of immunoreactive HSP70, while HSP70 staining of aSMCs is lower and is seen less consistently.⁴¹ Such observations suggest that sites of increased HSP expression during plaque evolution represent areas within the arterial wall experiencing greater cytotoxic stress. Comparison of HSP70 expression in fibrotic and necrotic carotid plaques by this study confirmed that significantly more immunoreactive HSP70 was present in specimens containing necrotic plaques. It may be argued that the greater HSP70 immunoreactivity within necrotic intimas resulted from the presence of more cells compared with fibrotic plaques (Table 2), which constitutively expressed HSP70, or that phenotypic differences in the cell types comprising the two forms of plaque accounted for this observation. However, neither argument can explain why HSP70 immunostaining was elevated in medial tissue underlying areas of necrosis, where cell density was equivalent to that in subfibrotic media, and aSMCs comprised the overwhelming majority of cells. We therefore believe our immunohistochemical observations support the hypothesis that cells in or near necrotic plaques experience greater cytotoxicity (of still incompletely defined etiology) than do cells within fibrotic plaques of approximately the same size and anatomic location.

We have adopted a working paradigm of advanced plaques in which aSMCs initially resist local cytotoxicity but later succumb to chronic stress. To test whether the observed increase in HSP70 in necrotic plaques is important for protection during initial stress resistance, we used serum deprivation or oxysterol exposure as in vitro models of known plaque-associated stresses. Western blot analyses and radiolabeling of newly synthesized proteins in serum-deprived aSMCs (Fig 4) indicated a transient increase in HSP70 synthesis after 5 hours of deprivation, while overall protein synthesis declined. After 10 hours of deprivation, cellular HSP70 content also declined, in step with a significant decrease in cell viability. C3ol-treated cells were similar to serum-deprived cells in that increasing levels of C3ol caused overall protein synthesis to decrease profoundly; however, no inhibition of protein synthesis was seen when the 25HC dose was increased (Table 4). The difference in the effect of C3ol versus 25HC was not unexpected; other studies have shown differences in their relative cytotoxicity, although both are present in human plaques at concentrations similar to those used here.²⁰ In contrast to serum deprivation, neither C3ol nor 25HC consistently induced significant endogenous HSP70 accumulation (Fig 8). This result was not merely a consequence of the time of sampling, since cells collected at earlier or later times after oxysterol addition also showed no increased accumulation of HSP70 (data not shown).

When aSMC-associated HSPs were increased by heat shock prior to serum deprivation, viability was significantly improved compared with unshocked controls (Fig 5C). However, C3ol inhibited HSP70 synthesis after heat shock, similar to its effect on overall protein synthesis; therefore, heat-shock pretreatment failed to protect either PMSM cells or rabbit aSMCs from exposure to C3ol (Fig 6A and Fig 7). We also found that the effects of inducing endogenous HSP70 on cell viability could be mimicked or surpassed by using exogenous HSP70. We used exogenous HSP70 to increase cell-associated HSPs because prior studies have demonstrated that HSPs are present in the extracellular milieu,⁴² that HSP70 can be transported between cells,⁵⁸ and that exogenous HSP70 can inhibit overall cellular protein synthesis to a similar extent as occurs during classic heat-shock responses.⁴⁵ In both serum-deprived and oxysterol-treated cells exogenous HSP70 protected against stress-induced cell death (Figs 5 and 6); this is especially notable since heat shock alone did not protect cells against C3ol toxicity. Furthermore, the in vitro data indicated that exogenous HSP70 protected aSMCs against moderate levels of plaque-associated stress sufficient to potentially cause 30% to 50% loss of viability in both models but that exogenous HSP70 was ineffective when stress was severe enough to cause greater than 50% loss of viability in vitro. The cumulative data indicate that aSMC viability in these models was highly correlated with cell-associated HSP70 regardless of the source of HSP70. However, HSP70 did not protect aSMCs indefinitely against continued toxicity, as seen during extended serum deprivation (30 hours; Fig 5C) or with high doses of C3ol (Fig 6A). These observations support our hypothesis that acute cytoprotective mechanisms such as the heat-shock response are gradually insufficient to protect aSMCs against rising toxicity in plaques, eventually allowing cell death to occur.

We also found that exogenous HSP70 protected aSMCs even after heat shock–induced protection subsided (Fig 5B), suggesting there are normal regulators or even inhibitors (such as C3ol) of the heat-shock response in vivo that limit endogenous HSP accumulation and thus protection. Since exogenous HSP70 was not subject to downregulation, more was available to protect cells, and greater survival occurred. However, the precise effector mechanisms for exogenous HSP70 are still incompletely defined. We know that exogenous HSP70 does not enter cells and augment intracellular HSP pools but acts via the plasmalemma, apparently without using a receptor-mediated pathway.⁴⁵ Therefore, different sites of action for the two sources of HSP70 might account for the extended protection rather than differences in availability. It is also possible that HSP70 enhances the activity of other cell-signaling molecules, which in turn have cytoprotective effects of their own. One possibility is fibroblast growth factor (FGF), a peptide mitogen that is secreted in response to heat shock.⁵⁹ FGF protects during ischemia of brain cells,⁶⁰ and a similar function may occur in aSMCs during advanced atherosclerosis; FGF-induced proliferation and matrix production may counteract destabilization by macrophages, with HSPs acting to chaperone FGF to the extracellular space.

Our results showing that 2 or 10 µg/mL exogenous HSP70 protected rabbit aSMCs against C3ol toxicity, while 20 or 50 µg/mL HSP70 did not (Fig 7) also need to be addressed further. The lack of effect at the higher doses may have arisen because 20 and 50 µg/mL HSP70 injured cells directly and negated any beneficial effects seen at lower concentrations. Indeed, data show⁶¹ that HSP70 at those concentrations can induce ion-conductive pores in unilamellar lipid vesicles, suggesting that such doses are toxic to aSMCs. More data are needed to clarify whether exogenous HSP70 is an appropriate model for studying actions of endogenous HSPs during plaque progression. Beyond this caveat, however, we are still actively exploring whether exogenous HSP70 could be used to directly treat necrotic plaques and thereby delay their rupture.

Initially, the 15% to 20% increase in cell survival observed during exogenous HSP70 treatment of cells in the serum-deprivation or C3ol studies may not appear very substantial. However, if a prerupture plaque exists in a dynamic equilibrium as we believe, then a small shift toward additional viable aSMCs could be sufficient to delay or prevent rupture. A small number of aSMCs might achieve this by increasing structural stabilization events such as collagen deposition or extracellular matrix repair after macrophage invasion. Additional studies of matrix integrity in aSMC/macrophage coculture after addition of HSP70 or heat-shock preinduction are planned to test how a relatively small increase in viable aSMCs may affect overall vascular wall structural stability. We must also address a potential problem raised by our data, ie, to distinguish between insufficient HSP production to protect against plaque stress and an inability of any amount of expressed or added HSP to protect against injury. Advancing atherosclerosis in vivo may cause sufficient injury to aSMCs that no amount of cytoprotective proteins are beneficial. If so, then formation of some sites of necrosis may be inevitable. However, preventing expansion of those necrotic foci to a point of rupture would remain a potential application for exogenous HSP70 or a function of endogenous HSPs requiring more study.

There is one major aspect of the hypothesized dynamic equilibrium between macrophages and SMCs prior to necrosis that was not addressed by this study: are macrophages indeed resistant, while aSMCs are killed by plaque-associated toxicity? There is currently no direct evidence that this occurs; however, greater stress resistance by macrophages is implicit if they actively destabilize plaques prior to rupture.³ There is also circumstantial evidence that resident macrophages are already more stress resistant. Resident macrophages and foam cells in atherosclerotic plaques and elsewhere typically maintain high constitutive levels of multiple HSP isoforms,^{41 56 62} which has been hypothesized to protect them against their own cytolytic activities. HSP60 in particular has also been strongly implicated as a presented antigen that helps recruit T lymphocytes and additional macrophages into atherosclerotic plaques, thus enhancing the local inflammatory response.^{56 57} Such evidence suggests that macrophages are more innately stress resistant than aSMCs because of higher baseline HSP expression or differences in the HSP subtypes accumulated. However, additional studies will be necessary to determine whether altered HSP levels in macrophages can be directly correlated with greater stress resistance, matrix proteolysis, or structural destabilization of advanced necrotic plaques.

	Acknowledgments

 
This study was supported by North Carolina Heart Association grant NC91G5, awarded to A.D. Johnson, and the Bowman Gray Stroke Center. The authors would like to thank the following people at Bowman Gray School of Medicine: Dr Venkata Challa, Department of Pathology, and the staff of the Surgical Pathology laboratory, North Carolina Baptist Hospital, for help in obtaining surgical specimens; Dr Richard St. Clair, Department of Comparative Medicine, for supplying the initial stock of PMSM cells; Dr David J. Gower, Department of Neurobiology and Anatomy, for supplying purified HSP70; Ruyou Xiao and Carol R. Hollman for technical assistance during video analysis; and Dr Greg Evans for consultations on the statistical analyses. Additional thanks go to Dr Timothy Scott-Burden (Texas Heart Institute, Houston, Tex) for helpful comments on the manuscript.

Received February 16, 1994; accepted October 4, 1994.

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