Heat Shock Protein 47 Is Expressed in Fibrous Regions of Human Atheroma and Is Regulated by Growth Factors and Oxidized Low-Density Lipoprotein
Background—Heat shock protein 47 (Hsp47) is a stress protein that may act as a chaperone for procollagen. Its involvement in atherosclerosis is unknown.
Methods and Results—Hsp47 expression in human coronary arteries was assessed by immunostaining. Strong focal expression was evident in atherosclerotic, but not normal, arteries and was prevalent in the collagenous regions. Double immunostaining revealed that all cells expressing type I procollagen also expressed Hsp47. Moreover, parallel regulation of proα1(I)collagen and Hsp47 mRNA expression occurred with cultured human smooth muscle cells stimulated with transforming growth factor-β1 or fibroblast growth factor-2. However, a proportion of Hsp47-expressing cells in plaque did not express type I procollagen, and this pattern could be reproduced in culture. Heat shock and oxidized LDL stimulated the expression of Hsp47 mRNA by smooth muscle cells, without a concomitant rise in proα1(I)collagen expression.
Conclusions—These findings identify Hsp47 as a novel constituent of human coronary atheroma. Its localization to the fibrous cap, regulation by growth factors in parallel with type I procollagen, and selective upregulation by stress raise the possibility that Hsp47 is a determinant of plaque stability.
The deposition of collagen fibers within human atheroma is a dominant process that can have important clinical consequences. Particularly critical is the amount and organization of collagen fibers within the rim of tissue, or cap, that overlies the necrotic core of lipid-rich lesions. Caps with a thin or weak structure are susceptible to tearing, which can lead to arterial thrombosis, myocardial infarct, and death.1
The major collagen species in human atherosclerotic plaque is type I collagen. The synthesis of type I collagen involves the productive association, within the endoplasmic reticulum, of 2 proα1(I)collagen chains and one proα2(I)collagen chain. The sorting and processing of procollagen chains into a native triple helix is not spontaneous; instead, it likely depends on the participation of molecular chaperones.2 Heat shock protein 47 (Hsp47) is a heat shock–inducible glycoprotein that binds nascent type I procollagen chains as they translocate into the endoplasmic reticulum.3 The duration of binding is longer if stable triple helix formation is inhibited,4 and the inhibition of Hsp47 expression has been associated with the decreased production of type I collagen.3 These findings suggest that Hsp47 may be a chaperone for type I procollagen.
Factors that control the efficiency and fidelity of procollagen folding could be critical to the clinical course of atherosclerosis. Therefore, we determined whether Hsp47 was expressed in human atheromas, and we ascertained its spatial and regulatory interactions with type I procollagen.
Gene expression was assessed using a mouse monoclonal antibody to rat Hsp47,5 a mouse monoclonal antibody to sheep proα1(I)collagen (SP1.D8, Developmental Studies Hybridoma Bank, University of Iowa), and partial cDNA clones for rat Hsp47 (pIP1) and human proα1(I)collagen (Hf677, ATCC). Human recombinant transforming growth factor (TGF)-β1 and fibroblast growth factor (FGF)-2 were purchased from R&D Systems and Gibco/BRL, respectively. Copper-oxidized LDL was kindly provided by Dr M. Huff of the Robarts Research Institute.
A total of 25 segments of the right coronary artery from 25 patients were obtained from postmortem tissue or native hearts harvested from cardiac transplant recipients at the London Health Sciences Center, London, Canada. The arteries were from patients aged 18 to 76 years. Specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of 4 μm were stained with hematoxylin and eosin and Movat’s pentachrome stain for morphological evaluation.
Deparaffinized tissue sections were subjected to microwave-based antigen retrieval; they were then incubated with primary antibodies overnight and then incubated with biotinylated horse anti-mouse IgG. Bound antibody was detected using the ABC Elite Kit (Vector Laboratories Inc) and visualized with 3,3′-diaminobenzidine (Sigma). Sections were counterstained with Harris’ hematoxylin. Human skin served as the control tissue. For both Hsp47 and proα1(I)-collagen, there was cytoplasmic staining of fibroblast-like cells in the dermis and no signal from epithelial cells. Expression in coronary artery sections was quantified by counting all positive cells in contiguous fields (×400). The entire section was evaluated.
For double immunolabeling, sections were immunostained for type I procollagen using SP1.D8 and developed using diaminobenzidine. They were then quenched with biotin solution, immunostained for Hsp47, and visualized using Vector SG peroxidase substrate, which yields a blue/gray color. Double-immunolabeled sections were not counterstained.
Cell Culture and Northern Blot Analysis
Primary cultures of human vascular smooth muscle cells (SMCs) were established from segments of the internal thoracic artery retreived at the time of coronary artery bypass surgery.6 SMCs were incubated in M199 containing 1% fetal bovine serum for 48 hours and then stimulated with TGF-β1, FGF-2, or oxidized LDL. SMCs were also subjected to heat shock (42°C for 4 hours), which was followed by the restoration of physiological temperature (37°C) for up to 6 hours. Total RNA was isolated, and Northern blot analysis was performed as previously described.6
Eight of the 25 arterial segments were normal or showed diffuse intimal thickening, and they were classified as nonatherosclerotic. Seventeen samples contained atherosclerotic plaque, which was subclassified as fibrous (n=12) or lipid-rich (n=5).
Nonatherosclerotic artery samples showed no or minimal Hsp47 expression (Figure 1⇓, A and B). In contrast, Hsp47 was readily detectable in 11 of the 17 atherosclerotic artery samples (Figure 1⇓, C and D). The strongest and most prevalent staining was in SMC-like cells within the dense fibrous plaques and within the fibrous cap of lipid-rich lesions (Figure 1E⇓). Cells within the necrotic lipid core did not express Hsp47.
Type I procollagen coexisted with Hsp47 in the same artery, and the 2 proteins localized to the same regions of the artery wall (Figure 1E⇑). Interestingly, however, Hsp47-positive cells were more prevalent than procollagen-expressing cells (Figure 1⇑, E through G). To further characterize this, selected sections were double-immunostained. Hsp47 and type I procollagen typically colocalized in a given cell, as evidenced by a mixture of brown and blue/gray color in the cytoplasm (arrows in Figure 1H⇑). Moreover, all cells that expressed type I procollagen also expressed Hsp47. However, a proportion of cells showed immunoreactivity only to Hsp47, as indicated by the blue/gray color alone (arrowhead in Figure 1H⇑).
To determine a basis for the generally close relationship between Hsp47 and type I procollagen expression, we studied cultured human SMCs. SMCs incubated with TGF-β displayed a dose-dependent increase in Hsp47 mRNA abundance and a parallel increase in proα1(I)collagen mRNA levels (Figure 2A⇓). Stimulation with FGF-2 yielded a dose-dependent decline in Hsp47 mRNA abundance and a parallel decline in proα1(I)collagen mRNA (Figure 2B⇓).
We also sought out conditions that might divergently regulate the expression of these 2 genes, given that some intimal cells selectively expressed Hsp47. As shown in Figure 2C⇑, 4 hours of heat stress (42°C) stimulated a 5.4-fold increase in Hsp47 mRNA expression but no significant change in proα1(I)collagen mRNA level. In addition, the incubation of SMCs with copper-oxidized LDL (150 μg/mL) yielded a 2-fold increase in Hsp47 mRNA abundance after 12 hours. In contrast, proα1(I)collagen mRNA expression declined.
We showed that the expression of the stress protein Hsp47 was increased in human atherosclerotic coronary arteries compared with normal coronary arteries and arteries with diffuse intimal thickening. Hsp47 was especially prominent in the fibrous/collagenous regions of atheromata, including the cap that overlies a lipid-rich core. This pattern is unique among the heat shock proteins that have been associated with atherosclerosis to date. Hsp70, for example, is concentrated around areas of necrosis and lipid accumulation.7 The relationship between Hsp47 and collagen was further strengthened by the regional colocalization of Hsp47 and type I procollagen and by double immunostaining, which established that all type I procollagen–producing cells expressed Hsp47.
The close association between Hsp47 and type I procollagen in atherosclerotic plaque and previous in vitro data suggesting a role for Hsp47 in collagen production3 imply that a mechanism must exist to ensure that Hsp47 is present within the cell when collagen is being produced. It must be recognized, however, that type I collagen and Hsp47 are distinctly different proteins encoded by dissimilar genes. The current study suggests that coordinate regulation in vascular disease may be based on parallel responsiveness to growth factors. TGF-β1 increased the expression of both Hsp47 and proα1(I)collagen mRNA in human SMCs, whereas FGF-2 decreased the expression of both genes.
A surprising finding was that, notwithstanding the evidence for coordinate regulation of Hsp47 and type I procollagen, evidence also existed for divergent regulation. Hsp47 was more prevalent in atheroma than type I procollagen, and double immunostaining established the existence of cells in which Hsp47, but not type I procollagen, was detectable. It is possible that a procollagen type other than type I was expressed by the Hsp47 single-positive cells; however, Hsp47 seems to be a very selective chaperone. It does not, for example, bind type III collagen.8 It is also possible that the anti-Hsp47 antibody had a higher affinity to Hsp47 than SP1.D8 did to type I procollagen or that the immunoreactivity of type I procollagen to SP1.D8 was masked by the interaction of procollagen with Hsp47. However, divergent regulation of Hsp47 and type I procollagen was supported by culture data. Heat shock stimulated Hsp47 mRNA expression by human SMCs, yet proα1(I)collagen mRNA abundance did not increase. Similarly, and perhaps of greater relevance to atherosclerosis, oxidized LDL selectively increased the expression of Hsp47 mRNA.
We speculate that the presence of Hsp47 in SMCs not producing immunodetectable type I procollagen may reflect a potential role of Hsp47 in trafficking abnormal procollagen. Up to 20% of newly synthesized procollagen is destined for intracellular degradation, and this fraction may increase under cellular stress.9 If Hsp47 is required to chaperone non-native procollagen to a degradation site, then stress conditions that yield aberrant procollagen might selectively stimulate Hsp47. In this regard, it is noteworthy that Hsp47 was expressed in parallel with procollagen in response to physiological stimuli (growth factors) but that it selectively increased in response to pathophysiological stressors (heat or oxidized LDL).
In summary, the current findings identify Hsp47 as a novel constituent of coronary atheroma and link this unique protein to the fibrous cap, to growth factor-mediated collagen production, and to atherogenic stress. The extent to which Hsp47 impacts the course of atherosclerosis, including plaque stabilization, seems to be a worthwhile avenue for study.
Supported by the Medical Research Council of Canada, a Career Investigator Award from the Heart and Stroke Foundation of Ontario (to J.G.P), and a Research Traineeship from the Heart and Stroke Foundation of Canada (to E.R). The authors thank Dr B.D. Sanwal for the anti-Hsp47 antibody and Hsp47 cDNA clone and for helpful comments.
- Received December 2, 1999.
- Revision received January 8, 2000.
- Accepted January 24, 2000.
- Copyright © 2000 by American Heart Association
Buchner J. Supervising the fold: functional principles of molecular chaperones. FASEB J. 1996;10:10–19.
Sauk JJ, Smith T, Norris K, Ferreira L. Hsp 47 and the translation-translocation machinery cooperate in the production of α1(I) chains of type I procollagen. J Biol Chem. 1994;269:3941–3946.
Nakai A, Satoh M, Hirayoshi K, Nagata K. Involvement of the stress protein hsp 47 in procollagen processing in the endoplasmic reticulum. J Cell Biol. 1992;117:903–914.
Pickering JG, Ford CM, Tang B, Chow LH. Coordinated effects of fibroblast growth factor-2 on expression of fibrillar collagens, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinases by human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997;17:475–482.
Johnson AD, Berberian PA, Tytell M, Bond MG. Differential distribution of 70-kD heat shock protein in atherosclerosis: its potential role in arterial SMC survival. Arterioscler Thromb Vasc Biol. 1995;15:27–36.
Jain N, Brickenden A, Lorimer I, Ball EH, Sanwal BD. Interaction of procollagen I and other collagens with collagen. Biochem J. 1994;304:61–68.