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Abstract
We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ERβ)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE−/− mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P≤0.001) in atherosclerotic lesion area in apoE−/−HSP27o/e mice compared to apoE−/− mice, but primarily in females. Serum HSP27 levels were >10-fold higher in female apoE−/−HSP27o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r2=0.78, P≤0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)—an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A−/− mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1β and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.
Studies from our laboratory as well as others reveal that estrogen receptor β (ERβ) may play a key role in vascular homeostasis.1 For example, ERβ given that its mRNA and protein expression are upregulated after vascular injury in male arteries.2,3 Moreover, ERβ is the predominant receptor expressed in the intima, media, and adventitia in male arteries, and unlike ERα, its expression correlates with the degree of calcification—a marker of severe atherosclerosis.4,5 Recently we discovered that heat shock protein 27 (HSP27) interacts with estrogen receptor β (ERβ) to reduce estrogen-mediated transcriptional signaling, as reflected by the results of an estrogen response element (ERE) activity assay. Moreover, we showed that HSP27 expression diminishes with the progression of atherosclerosis.1 Hence, this loss of HSP27 acting as a corepressor on estrogen signaling may result in the untoward expression of genes associated with atherogenesis.
Heat shock proteins are involved in a wide variety of processes, both physiological and pathological.6,7 Heat shock protein 27 (HSP27) is a member of the small (15 to 30 kDa) heat shock protein family. Principally described as an intracellular chaperone, HSP27 is capable of binding and stabilizing the actin cytoskeleton in response to stress (see review by Ciocca et al8). In addition, HSP27 can bind cytochrome c and prevent downstream caspase activation, thereby making it a potent antiapoptotic protein.9,10 Hence, given the interaction between HSP27 and ERβ, we sought to further elucidate the vascular role of HSP27 and the role of estrogens in this process. Our laboratory as well as others recently proposed that HSP27 is a biomarker for atherosclerosis, with vascular HSP27 expression diminishing with the progression of disease.1,11,12 Indeed, we demonstrated that HSP27 tissue expression diminishes in human coronary arteries of young individuals (mean age: 27 years) as atherosclerosis stage increases.1 Separate proteomic studies discovered decreased HSP27 levels secreted from human carotid endarterectomy plaques versus normal (disease-free) arteries11 and preserved HSP27 expression in cardiac biopsy specimens (primarily blood vessels) correlated with freedom from cardiac allograft vasculopathy.13,14 Moreover, HSP27 is present in the serum of individuals free of atherosclerosis11,12 and presumably acts in a yet to be defined “atheroprotective” manner. In other tissues (eg, nerve, gastromucosal and myocardium) HSP27 is protective against the sequelae of acute injury/stress, perhaps via antiapoptotic mechanisms.15–19
Hence, the central postulate of this study is that HSP27 is protective against atherogenesis, and to address this claim we determined whether overexpression of human HSP27 in atherosclerosis-prone apoE−/− mice was protective against development of disease. We now report for the first time how extracellular HSP27 may have a potent atheroprotective effect that is modulated by estrogens and high-fat diet, and involves interaction with the scavenger receptor A with antiinflammatory effects.
Materials and Methods
Briefly, our studies involve 3 major components: (1) a mouse model of atherogenesis that uses apoE−/− mice that are cross bred to overexpress HSP27 (apoE−/−HSP27o/e), fed a high-fat diet for 4 weeks before aortic lesion analysis is performed; (2) the characterization of the signals for the release of HSP27 from cells, including secretion of HSP27 in vitro in human macrophages in response to both acLDL and estrogen; and (3) the novel interaction of extracellular HSP27 and scavenger receptor-A, demonstrated using immunolabeling, immunoprecipitation, and competitive binding studies. For a detailed account of the methodologies used in this manuscript, please refer to http://circres.ahajournals.org.
Results
HSP27 Overexpression Protects Against Atherogenesis in Female Mice
Using a mouse model of inflammatory atherosclerosis, we sought to determine whether overexpression of human HSP27 (HSP27o/e) is protective against atherogenesis in apoE−/− mice that are prone to atherosclerosis when fed a diet supplemented with cholesterol.19 apoE−/−HSP27o/e and apoE−/− mice were placed on a high-cholesterol diet for 4 weeks and euthanized at 10 weeks of age. Mean body weight and length, as well as total serum cholesterol levels, were similar for these 2 groups of mice (see supplemental Figure I). The percentage aortic lesion area, measured by quantitative histomorphology of oil red O–stained en face specimens was 35% reduced in apoE−/−HSP27o/e versus apoE−/− female mice (n=9/group; P<0.001; Figure 1). The male apoE−/− HSP27o/e and apoE−/− mice (n=6/group) had similar percentage aortic lesion areas (12.9±1.2% versus 13.5±1.0% lesion area compared to total arch area, respectively; P=0.69). Serum HSP27 levels measured using an ELISA were low in all apoE−/−HSP27o/e mice fed a normal chow diet (Figure 2A). However, after 4 weeks of a high-fat diet, female apoE−/−HSP27o/e mice had more than 10-fold higher circulating levels of HSP27 compared to male mice (P≤0.05). This difference in serum HSP27 levels was not apparent until after 2 weeks of ingestion of a cholesterol-enriched diet (eg, serum HSP27 levels after 2 weeks in females: 536±308 pg/mL versus males: 123±81 pg/mL; P=0.456). When circulating HSP27 levels were compared to total en face lesion area, there was a remarkable inverse correlation between serum HSP27 levels and atherosclerotic lesion area (r2=0.78; P<0.001, Figure 2B) in both males and females. When only females were used for correlation analysis, the relationship between circulating HSP27 and atherosclerotic lesion area was even stronger (r2=0.9; P<0.001).
Figure 1. HSP27 overexpression reduces atherosclerotic lesion size in female mice. Total aortic en face atherosclerotic lesion area was analyzed in mice overexpressing human HSP27 (apoE−/−HSP27o/e) (A, C) and compared to their apoE−/− littermates (B, D); pictured here are female mice. Quantification of lesion area/total aortic arch area (E) demonstrated a 35% reduction in lesion burden in HSP27o/e apoE−/− compared to apoE−/− female mice (*P<0.001). No difference was observed in the male mice.
Figure 2. A, HSP27 serum levels increase in response to an atherogenic diet. Serum HSP27 levels from mice fed a normal chow diet (left columns) and from the same mice 4 weeks after ingesting a high-fat atherogenic diet (right columns) (*P≤0.001). B, Regression analysis comparing HSP27 serum levels with aortic en face lesion area in male and female mice HSP27 overexpressing mice.
HSP27 is Secreted In Vitro in Response to Estrogen and acLDL
As serum HSP27 levels inversely correlate with aortic lesion area in mice fed a cholesterol-enriched diet, and is >10-fold higher in females than in males, we determined in vitro whether HSP27 is released on stimulation with estradiol (E2) or atherogenic acetylated low-density lipoprotein (acLDL). Human macrophages (U937) were plated in replicates at a density of 1×106 per well and treated with estradiol or acLDL. Conditioned media was collected and before analysis for secreted HSP27, overall cell viability was measured using an LDH-release assay and revealed no difference in cell viability or membrane integrity between any of the treatments (data not shown). Treatment of macrophages with estrogen (E2) for 24 hours caused a dose-dependent increase in HSP27 release into the media compared to controls (Figure 3A).20,21 Estrogen-induced HSP27 release also increased over time, with maximum secretion after 24 hours (Figure 3B). Macrophages were subjected to increasing concentrations of acLDL (1 to 100 μg/mL) for 24 hours. HSP27 protein was detected by Western blot in conditioned media from cells treated with 100 μg/mL acLDL (Figure 3C). The addition of acLDL to the media containing estrogen caused a further increase in HSP27 secretion when compared to estrogen or acLDL treatment alone, indicating that these 2 mechanisms of secretion may act synergistically (Figure 3D). On treatment with estrogen, examination of intracellular protein levels revealed that HSP27 protein levels increased slightly in response to estrogen treatment, suggestive of an intracellular pool of HSP27 that is secreted without necessitating de novo protein synthesis (Figure 3E). On treatment with acLDL, there was a concomitant dose-dependent increase in intracellular HSP27 expression, then an apparent decrease corresponding to increased HSP27 protein release into the media (Figure 3F).
Figure 3. HSP27 is secreted from macrophages in response to estrogen and acLDL. A, Human U937 macrophages in culture were treated with 10% charcoal-stripped FBS with or without increasing concentrations of 17β-estradiol (E2) for 24 hours, and conditioned media was subjected to Western blotting using an antibody against HSP27. HSP27 secretion into the media increased in a dose-dependent manner with E2. *P≤0.05 compared to control. B, U937 cells were treated with or without 100 nmol/L E2 for 1, 3, and 24 hours, and conditioned media showed increasing HSP27 released into the extracellular space over time. *P≤0.05 compared to control; #P≤0.05 compared to all treatment groups. C, U937 cells were treated in culture with increasing concentrations of acLDL (0 to 100 μg/mL). HSP27 was detected in the conditioned media on treatment with 100 μg/mL acLDL (top panel). *P≤0.05 compared to control. D, U937 cells were treated with 100 nmol/L E2 or 100 μg/mL acLDL, or both together for 24 hours and conditioned media was analyzed by Western blot. HSP27 in the conditioned media increased with treatment of E2 and further increased with treatment of acLDL. An equal number of cells and volume of conditioned media was analyzed for each treatment condition for all conditioned media experiments. *P≤0.05 compared to control; #P≤0.05 compared to all treatment groups. E, Intracellular protein from U937 cells treated with increasing concentrations of E2 and probed using an antibody to HSP27. Equal protein loading was confirmed using Ponceau S staining and α-actin blotting (see supplemental Figure III). F, Intracellular protein from U937 cells treated with increasing concentrations of acLDL, and probed using an antibody to HSP27. Equal protein loading was confirmed using Ponceau S staining and α-actin blotting (see supplemental Figure III).
To examine the pathway of HSP27 secretion, we used 2 independent experiments: the first used human U937 macrophages treated with E2 and immunolabeled for HSP27 and a marker of the lysosomal membrane (LAMP1); second, mouse J774 macrophages were transfected with a fluorescently tagged HSP27 (HSP27-ECFP), and treated with acLDL in the presence of Lysotracker, which labels acidic organelles (ie, lysosomes) in live cells. Using both methods, we localized intracellular HSP27 in macrophage lysosomes after treatment with E2 or acLDL. Specifically, human macrophages treated with 100 nmol/L E2 overnight displayed colocalization of HSP27 (red) and LAMP1 (green; Figure 4A). Similarly, mouse macrophages transfected with fluorescently-tagged HSP27 (green) treated with 100 μg/mL acLDL and incubated in the presence of Lysotracker (red) displayed HSP27 colocalization within lysosomes after treatment for 1 hour and 24 hours (merged image, Figure 4B). Without estrogen treatment, HSP27 showed minimal colocalization with the lysosome (Figure 4A and 4B, bottom row). Cells transfected with empty ECFP alone with or without acLDL treatment did not show colocalization with the lysosome, indicating that HSP27-ECFP was not simply degraded and targeted to the lysosome (data not shown). These results indicate that HSP27 is found within secretory lysosomal-like vesicles in macrophages under conditions which stimulate its secretion (eg, on treatment with E2 or acLDL).
Figure 4. A, U937 cells were treated with 100 nmol/L E2 overnight and immunolabeled using antibodies to LAMP1 (green, a marker for the lysosomal membrane) and HSP27 (red). Merged image (yellow) shows colocalization of HSP27 in the lysosome (magnification, 100×). Images were obtained with sequential scanning to avoid bleed through, and arrows indicate locations which are positive for LAMP1 in the green channel and where no staining is observed in the red channel. B, J774 cells transfected with HSP27-ECFP (green) were treated with 100 μg/mL acLDL 24 hours in the presence of Lysotracker red. Colocalization is seen as a yellow color (magnification, 100×). Images were obtained with sequential scanning to avoid bleed through, and arrows indicate locations which are positive for HSP27-ECFP in the green channel and where no staining is observed in the red channel. Experiments were performed in triplicate for all treatment conditions. See supplemental Figure IV for additional controls.
Extracellular HSP27 Binds the Scavenger Receptor-A and Prevents acLDL Uptake
Given that HSP27 is secreted not only in vitro by atherogenic lipids, but in vivo in response to high-fat diet, the next step was to determine whether extracellular HSP27 is involved in cholesterol trafficking and thus potentially the progression of atherosclerosis. Other groups have shown that members of the heat shock protein family (eg, HSP70) bind a variety of cell surface receptors, including toll-like receptors and scavenger receptors.22–25 We investigated whether extracellular HSP27 binds the SR-A—an important receptor for the uptake of atherogenic lipids and the progression of atherosclerosis.26,27 Immunolabeling studies reveal that recombinant extracellular HSP27 is capable of binding the surface of macrophages, and colocalizes with SR-A (Figure 5A). In the presence of fucoidan, a specific competitor for SR-A, HSP27 binding to SR-A was reduced, indicating that this interaction is specific.28 Furthermore, in macrophages from SR-A−/− mice, HSP27 binding was absent (Figure 5B).
Figure 5. Extracellular HSP27 colocalizes with the SR-A on the surface of macrophages. A, Recombinant HSP27 (5 μg/mL) was administered to J774 macrophages for 2 hours at 4°C. Immunolabeling of HSP27 (green) and SR-A (red) was visualized using confocal microscopy (100×). Cells were also treated with an SR-A specific competitive ligand (fucoidan, 10 μg/mL) before administration of HSP27 (bottom panel). Colocalization is seen as a yellow color. B, Macrophages from SR-A+/+ (see supplemental Figure VB) and SR-A−/− mice were harvested and incubated with recombinant HSP27 (5 μg/mL) for 2 hours at 4°C. Immunolabeling was performed as described above. C, HSP27-ECFP (or empty ECFP control) secreted in response to acLDL was applied to naïve macrophages at 4°C for 2 hours (see supplemental Figure VC). Cells were crosslinked with increasing concentrations of DTSSP (0.1 to 2 mmol/L) and immunoprecipitated using an antibody to ECFP. Lane containing nonimmunoprecipitated cell lysate represents 25% to 30% of input (see supplemental Figure VD). Experiments were performed in triplicate for all treatment conditions.
Next, we explored whether endogenous HSP27 secreted in response to acLDL is capable of binding SR-A. Conditioned media from macrophages transfected with HSP27-ECFP (or empty ECFP) and treated with 100 μg/mL acLDL was applied to naïve, untreated macrophages at 4°C for 2 hours to allow HSP27-ECFP to bind the cell surface. Cells were then treated with DTSSP, a reversible, membrane-impermeable cross-linking agent, to cross-link HSP27 to the cell surface. Immunoprecipitation was carried out using antibodies to the fluorescent tag (anti-ECFP) and cross-linked proteins were reduced to reverse cross-linking, then separated on an SDS-PAGE gel and subjected to immunoblotting. Using antibodies to SR-A, we demonstrated that HSP27-ECFP is secreted from cells treated with acLDL and binds the SR-A under various concentrations of DTSSP (Figure 5C). Macrophage whole cell lysates were also probed and displayed a ≈80 kDa band corresponding to SR-A in these cells.
As foam cell formation is a hallmark of atherogenesis, and prevention of lipid uptake by macrophages may serve to reduce lesion development and vessel wall inflammation, we hypothesized that HSP27 binds the SR-A to prevent uptake of atherogenic lipids such as acLDL to attenuate foam cell formation. Mouse macrophages were cultured in vitro in the presence of fluorescently labeled acLDL for 6 hours, and acLDL uptake was measured using a fluorometer before being normalized for total cell number (n=10 wells per treatment; experiments performed in triplicate). When extracellular HSP27 was added to the culture media, there was a 41% reduction in specific acLDL uptake by macrophages (P≤0.05; Figure 6; normalized to nonspecific uptake in the presence of the SR-A competitor fucoidan). To investigate whether a reduction in overall lipid uptake occurs in vivo in the presence of HSP27, peritoneal macrophages from HSP27o/eapoE−/− and apoE−/− mice were harvested and stained with Oil Red O, which stains intracellular lipids red. Semiquantitative analysis was performed and the total number of lipid-laden cells were counted and expressed as a percentage of total cell number by 2 independent blind reviewers. Only 3% of macrophages from HSP27o/eapoE−/− were considered lipid-laden, whereas 21% of cells from apoE−/− mice were considered lipid-laden (Figure 6B and supplemental Table I). Moreover, using Western blotting we noted that the reduction in macrophage acLDL uptake by HSP27 attenuated acLDL-induced release of IL-1β, a potent proinflammatory cytokine (Figure 7A). Extracellular HSP27 also increased the released of the antiinflammatory cytokine IL-10 (Figure 7B). Previous reports involving human monocytes suggest that HSP27 can induce the release of IL-10 but not TNF-α, and hence agree with our observations that extracellular HSP27 is primarily an antiinflammatory signaling protein.29 These results suggest that extracellular HSP27 is capable of reducing foam cell formation and the accompanying inflammation.
Figure 6. A, Extracellular HSP27 inhibits uptake of acLDL by macrophages via the SR-A. Fluorescently labeled acLDL (5 μg/mL) was added to macrophages in the presence (right column) or absence (left column) of recombinant HSP27 (5 μg/mL). Cells were also treated with an SR-A specific competitor (fucoidan, 10 μg/mL) before administration of acLDL or HSP27. Cells were harvested, and fluorescent acLDL uptake was measured using a fluorometer and normalized to cell number. Nonspecific uptake in the presence of fucoidan was subtracted to define specific acLDL uptake; at least 10 samples were analyzed per group and comparisons were made using a 1-way ANOVA; *P=0.005 compared to acLDL alone. B, Peritoneal macrophages from apoE−/− and HSP27o/eapoE−/− mice were plated, fixed, and stained for Oil Red O. Cells were counted and those that were “lipid-laden” (deep red color) were expressed as a % of total cell number. HSP27o/eapoE−/− macrophages contained only 3% lipid-laden cells compared to apoE−/− macrophages which contained 21% lipid-laden cells (see supplemental Table I).
Figure 7. Extracellular HSP27 results in release of cytokines involved in the inflammatory response. Fluorescently labeled acLDL (5 μg/mL) was added to J774 macrophages in the presence or absence of recombinant HSP27 (5 μg/mL). Conditioned media was collected after 30 minutes and 4 hours, and 40 μL was subjected to Western blotting for IL-1β (A) and IL-10 (B). Addition of HSP27 caused a decrease in the acLDL-induced secretion of IL-1β into the extracellular space. An equal number of cells and volume of conditioned media was analyzed for each treatment condition for all conditioned media experiments. Experiments were performed in triplicate for all treatment conditions.
HSP27 Overexpression Reduces Cell Adhesion and Migration
To further investigate how HSP27 might be protective against the development of atherosclerosis, peritoneal macrophages were harvested from apoE−/−HSP27o/e and apoE−/− mice after a high-fat diet. Cells were plated in culture on a collagen matrix and allowed to adhere for 2 hours. There was a 53% reduction in cell adhesion in apoE−/−HSP27o/e macrophages compared to apoE−/− (P≤0.001; Figure 8A and 8B). Macrophages were also maintained overnight in a transwell migration chamber and allowed to migrate toward a 10% FBS gradient. There was a 42% reduction in cell migration in apoE−/−HSP27o/e macrophages compared to apoE−/− (P≤0.01; Figure 8C). No cell migration was detectable in the absence of FBS (data not shown). Taken together, macrophages that overexpress HSP27 display reduced adherence and migration in vitro, suggesting that in vivo these cells are less likely to incorporate into vascular lesions and exacerbate disease.
Figure 8. HSP27 overexpression results in decreased macrophage adhesion and migration. A, Peritoneal macrophages harvested from apoE−/−HSP27o/e and apoE−/− mice were plated on type I collagen. After 2 hours incubation, the cells were washed and fixed. B, Cell nuclei were stained with Hoechst 33258 and the number of cells per high power field (HPF) was manually counted. C, Peritoneal macrophages as in A were subject to CytoSelect migration assay for 24 hours and quantified as total number of cells migrated toward 10% FBS. Experiments were performed in triplicate for all treatment conditions.
Discussion
The present study is an extension of previous work by our group and others that demonstrate reduced tissue and serum levels of HSP27 with the development of atherosclerosis.1,11,12 However, in this article we bring forth important new mechanistic information regarding the atheroprotective effects of HSP27. Specifically, we asked whether overexpression of HSP27 is protective against the development of atherosclerotic lesions in apoE−/− mice fed a cholesterol-enriched diet. Overexpression of HSP27 in apoE−/− mice resulted in a 35% decrease in aortic lesion area relative to apoE−/− mice—but only in females (P≤0.001). With the commencement of an atherosclerotic diet serum HSP27 levels remained low in male mice, yet showed a dramatic increase in females. Interestingly, there was a remarkable inverse correlation between serum HSP27 levels and lesion area in both male and female mice (r2=0.78; P<0.001); when females were examined independently of males, this relationship was even stronger (r2=0.90; P<0.001). We confirmed these in vivo observations by showing in vitro that both acLDL and estrogen cause the secretion of HSP27 into the extracellular space, where it binds the scavenger receptor A to prevent acLDL uptake and inflammatory foam cell formation.
A logical question arose from our initial observations on serum HSP27 levels in mice and humans: what is the mechanism by which HSP27 exits the cell? Given that HSP27 is primarily described as an intracellular protein and does not contain signal sequences that would sort it to a traditional secretory vesicle we reviewed our initial observations and hypothesized that perhaps estrogens combined with components of the atherogenic diet played a role in HSP27 release. Although the promoter region of HSP27 does contain a functional estrogen response element and that HSP27 expression is regulated by estrogen,30,31 its secretion into the extracellular space is not known to be induced by ovarian hormones. Hence we approached the question of HSP27 release from the cell by examining the following 3 features. First, we looked at the structural means by which HSP27 might exit the cell, and found that, like HSP70 it was sorted in the cell into lysosome-like vesicles. Second, we demonstrated that estrogen, in both a dose- and time-dependent fashion, enhanced the secretion of HSP27 by macrophages. Finally, acLDL also promoted HSP27 release from the cell—and in an apparent synergistic manner with E2. Given that there was no change in membrane integrity or cell viability between treatments and controls (as measured by the LDH-release assay) we can conclude that these observed increases in HSP27 secretion into the media are not simply a result of cell necrosis or changes in membrane permeability, but rather by an active and potentially regulated secretion mechanism. Interestingly, cultured human smooth muscle cells did not secrete HSP27 in response to E2, whereas cultured human endothelial cells showed minimal release of HSP27 in response to E2.
Perhaps the most intriguing data from our studies is the discovery of the interaction between HSP27 and the SR-A. Not only is this interaction specific, as demonstrated using a competitor for SR-A as well as SR-A−/− macrophages, but HSP27 reduces the ability of SR-A on the surface of macrophages to engulf acLDL and acquire the foam cell phenotype. To our knowledge, this is the first evidence of a cell-surface receptor for HSP27. Previous reports demonstrate that some members of the heat shock protein family are capable of binding a variety of receptors, namely those involved in antigen recognition and immune signaling (reviewed by32). For example, HSP70 can bind toll-like receptors 2 and 4 (TLR-2 and -4) and thereby induce NFκB activation, IL-6 production, as well as the secretion of IL-1β and IL-12—both proinflammatory cytokines.22,33 Other reports show that SR-A is capable of binding Gp96, an endoplasmic reticulum–bound HSP on antigen presenting cells.34 However, we now show that HSP27 may have effects opposite to those observed for other extracellular HSPs. When added to macrophages in vitro, HSP27 reduced acLDL-induced secretion of IL-1β and increased the secretion of IL-10, implying that HSP27 primarily results in antiinflammatory cytokine induction. Indeed, apoE−/− HSP27o/e macrophages showed decreased cell adhesion and migration relative to peritoneal macrophages from apoE−/− mice. Taken together, these data suggest a unique atheroprotective mechanism by which extracellular HSP27 is capable of preventing the uptake of atherogenic lipids and reducing foam cell-induced inflammation.
Although the available data appear to indicate that HSP27 is a novel biomarker of atherosclerosis and is atheroprotective, mechanistic insights remain elusive. From our previous studies we noted that HSP27 is an ERβ associated protein and, at least in vitro, is a corepressor of estrogen mediated signaling.1,35 However, these studies focused purely on the intracellular role for HSP27 in estrogen transcriptional signaling. Currently, we are working toward understanding whether ERs are involved in the regulation of HSP27 release from cells. Experiments involving ovariectomy and specific estrogen receptor modulation in HSP27o/eapoE−/− mice will help to resolve this question. Furthermore, it remains unclear whether the levels of extracellular HSP27 are partly reflective of the intracellular levels of this protein, which may be released into the extracellular space. It is possible that an increase in intracellular HSP27 precedes an increase in extracellular HSP27, which can serve to prevent atherogenic lipid uptake and inflammation, resulting in protection from atherosclerosis development. Hence, we hope to develop novel therapies to modulate HSP27 levels either intra- or extracellularly or attenuate levels of circulating anti-HSP27 autoantibodies.7,14
Naturally, certain limitations apply to our study. First, although we observed an increase in circulating levels of HSP27 in female mice after a high-fat diet, we can only make conclusions about relative and not absolute levels of HSP27. As seen in patients with normal coronary arteries, there are relatively high levels of HSP27 in healthy males and females.11,13 It is possible that in healthy mice, their levels of HSP27 are below the sensitivity of our assay and therefore reported as undetectable. Regardless, we still note a relative increase in serum HSP27 levels in female mice on a high-cholesterol diet compared to males or mice on a normal diet. Second, although we observe the colocalization of HSP27 within lysosomal-like vesicles after treatment with acLDL and estrogen, the specific participation of the lysosome in this pathway is still unknown. The exact cellular mechanisms by which HSP27 exits the cell certainly warrant further study.
In summary, we propose a novel mechanism of HSP27 atheroprotection that involves estrogen-induced attenuation of inflammation and inhibition of cholesterol uptake via the interaction of HSP27 with SR-A. Whereas the atheroprotective effects of estrogens are well-recognized in animal models, our data suggest that estrogen-induced release of HSP27 may in fact be an important mechanism by which estrogens exert their beneficial effects on atherogenesis.36 Clearly the implications of this unique role for HSP27 are far-reaching and highlight the possibility that HSP27 may be a new target for therapeutic modulation. Although intracellular HSP27 protein levels are known to be induced by estrogen,31 there may be important subtleties with regards to how selective estrogen receptor modulation may trigger the release of atheroprotective HSP27; hence this is an area of ongoing research.
Acknowledgments
We acknowledge the Animal Care and Veterinary Services at the UOHI, as well as Drs Yves Marcel and Stewart Whitman for providing the SR-A−/− mice.
Sources of Funding
This work was supported by the Canadian Institute for Health Research (CIHR) operating grant #80204. E.O.B. holds a Research Chair from CIHR-Medtronic, and K.R. was supported by studentships from both the Heart and Stroke Foundation of Ontario and CIHR/Institute of Gender and Health/Ontario Women’s Health Council.
Disclosures
None.
Footnotes
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↵*These authors contributed equally to this study.
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Original received October 18, 2007; resubmission received January 22, 2008; revised resubmission received May 8, 2008; accepted June 11, 2008.
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