Cardiovascular Research 74 (2007) 19 – 28 www.elsevier.com/locate/cardiores

Review

Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease Alireza Shamaei-Tousi a,⁎, Julian P. Halcox b , Brian Henderson a a

Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, United Kingdom b Department of Cardiology, Institute of Child Health, University College London, United Kingdom Received 13 July 2006; received in revised form 9 October 2006; accepted 30 October 2006 Available online 1 November 2006 Time for primary review 22 days

Abstract

Keywords: Basic science research; Atherosclerosis

All biomedical scientists will be familiar with the relationship between the ‘shape’ of a protein and its biological function, and be aware that proteins undergo denaturation and lose biological activity. Biologically active proteins are said to be ‘correctly folded’ and it is only within the last twenty years that protein folding has been recognised to be a major controlling factor in cellular regulation and in human diseases, including cardiovascular disease.

⁎ Corresponding author. Present address: Medical Microbiology, Department of Cellular and Molecular Medicine, St Georges' University London. London, SW17 0RE, United Kingdom. Tel.: +44 20 7915 1190; fax: +44 20 7915 1127. E-mail address: [email protected] (A. Shamaei-Tousi).

1. A brief history of protein folding Four decades ago, the heat shock or cell stress response was discovered as a puffing pattern in the polytene chromosomes of Drosophila [1]. It was also recognised early on that stress such as exposure to heavy metals, metabolic poisons, ischemia/reperfusion and free radicals would have same the effect as elevated temperature, leading to production of heat shock proteins (Hsps), also known as cell stress proteins (CSP) [2]. To make the terminology more confusing, some Hsps have also been known as molecular chaperones. A molecular chaperone is defined as ‘‘one of a large and diverse group of proteins that share the property of assisting the non-covalent assembly/disassembly of other macromolecular structures, but which are not permanent components of these structures

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It is only some forty years since the discovery of the heat shock or cell stress response and just over twenty years since the heat shock/cell stress response was linked to protein misfolding. The plethora of intracellular proteins which promote correct protein folding in the cell, variously termed molecular chaperones, heat shock proteins, or cell stress proteins, have only been identified in the last fifteen years. During this period it has also been discovered that: (i) molecular chaperones are potent immunogens with immunomodulatory activity and (ii) they can be secreted by cells and exhibit intercellular signaling actions. These various functions of molecular chaperones are increasingly being linked to the pathology of the cardiovascular system. Molecular chaperones within cells can exhibit cardioprotection if their levels are artificially elevated, suggesting that these proteins may have therapeutic activity. In contrast, there is evidence that atherogenesis may be linked to immunity to one specific molecular chaperone, Hsp60. This may offer the possibility of treating atherosclerosis by vaccination. However, there is also growing evidence that secreted molecular chaperones have pro- or anti-inflammatory actions that are relevant to cardiovascular pathology. This review brings these various strands of research together to provide an overview of the role of molecular chaperones in cardiovascular disease. © 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

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when these are performing their normal biological functions” [3]. Today there are more than 25 families of molecular chaperones, with more than 100 proteins participating in folding events in the mammal. A number of the eukaryotic molecular chaperones are listed in Table 1. These include the proteins that will be discussed throughout this review. For clarity, the term cell stress protein (CSP) has been used throughout this article to describe molecular chaperones and stress proteins.

pressive protein found in the blood in the initial stages of pregnancy and later identified as chaperonin (Hsp) 10. Since the late 1980s it has been discovered that CSP can act as intercellular signalling molecules [11] which may exist on the surface of cells and may be secreted [12]. Proteins that have more than one function are now known as moonlighting proteins [13] and many of the stress proteins of cells have the ability to moonlight. 1.3. CSP as cardioprotectants

1.1. CSP — the immunological connection

1.2. CSPs go moonlighting In 1977, a year before the conception of CSPs, a molecular chaperone was discovered in the blood of pregnant women. This was early pregnancy factor (EPF) [10], an immunosup-

Some of the earliest studies of chaperones in the cardiovascular field suggested an intracellular role as cardioprotectants. This has been reviewed extensively by others [14–16] and this section will provide a literature update. Through numerous studies in a variety of model systems, it is now a well documented fact that increased expression of certain CSPs protect the ischemic myocardium. However, the exact molecular mechanism for such protection remains elusive. Two CSPs have been extensively studied. It is believed that Hsp70 inhibits caspase-dependent and caspaseindependent apoptotic stimuli [17]. It has also been shown that Hsp27 inhibits cytochrome c-dependent activation of procaspase 9 [18] and stabilises cytoskeletal structures [19]. The latter could be important, since major cytoskeletal lesions could occur during irreversible ischemic injury in the myocardium and therefore stabilisation and protection of these structures probably could prevent lethal cell injury. Cell culture studies showed enhanced synthesis of CSPs in cultured cardiac myocytes exposed to hemodynamic overload [20] and exposure of isolated hearts to ischemia or elevated perfusion temperature caused the induction of Hsp70 [21]. Experimental occlusion of the coronary artery to induce myocardial ischemia also resulted in elevation of Hsp70 in heart tissues [22]. These experiments led to the

Table 1 Some eukaryotic cell stress protein families Protein

Function

Reference

Nucleoplasmins/nucleophosmins α-crystallins Hsp27/28 Peptidyl–prolyl isomerases PDI⁎ and thioredoxin-related proteins Chaperonins (Cpn10/Cpn60/TCP-1) Hsp40 Hsp47 Hsp70 Hsp90 Hsp100 Calnexin and calreticulin NAC complex Prefoldin Tim complexes Clusterin

Nucleosome and ribosome assembly/disassembly Prevention of protein aggregation Prevention of protein aggregation Promotion of protein folding Prevention of misfolding in ER Folding of newly synthesized and denatured polypeptides Protein folding/transport, assembly of protein oligomers Folding of pro-collagen in ER Protein folding/transport, assembly of protein oligomers Controlling cell functions such as cell cycle, signal transduction, hormone action Dissolution of insoluble protein aggregates Folding of glycoproteins in ER Folding of newly synthesized proteins Folding of newly synthesized proteins Prevention of aggregation of imported mitochondrial proteins Extracellular chaperone

[112] [113] [113] [114] [73] [3] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124]

⁎PDI — protein disulphide isomerase.

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CSP are highly conserved proteins. Analysis of immune responses to bacteria in the 1970s identified what was termed a “common antigen” in many species [4]. Patients infected with Mycobacterium tuberculosis or M. leprae exhibited significant antibody responses to a 65 kDa antigen [5]. Subsequent work identified this antigen and the ‘common antigen’ as the molecular chaperone, Hsp60 [5]. Since these early studies it has been amply confirmed that the CSPs of infectious agents (Hsp10, Hsp60, Hsp70 and Hsp90) are powerful immunogens [6] and immunomodulators in experimental models of arthritis, diabetes and atherosclerosis [7,8]. Such findings have led to the first clinical trials of CSP, or derived peptides, in human diseases such as type I diabetes [9]. The reason for the strong immune responses to CSPs is not clear. One possible explanation lies in the finding (to be discussed) that many CSPs are potent activators of immune cells. Thus they may be acting both as adjuvants as well as immunogens.

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1.4. CSPs and ‘cardio-immunology’ Over the past decade Wick and co-workers have generated experimental data to support the hypothesis that atherogenesis is driven by cross-reactive immunity to bacterial Hsp60 proteins. In an initial study they immunized rabbits with human atherosclerotic plaques (reviewed in Ref. [8]). They found that such immunisation in normocholesterolemic rabbits caused atherosclerosis but that this was due to the Freund's complete adjuvant (FCA) and not the plaque proteins. They also showed the major component of the Table 2 Cell stress proteins conferring cardioprotection Molecular chaperone

Reference

αB crystallin HspB2 Hsp20 Hsp27 Hsp32 CHIP Hsp70

[44] [44] [45] [46] [47] [48] [40] and text

FCA, M. tuberculosis with its dominant immunogenic Hsp65 protein, was the atherogenic agent. These results established the foundation of the hypothesis that crossreactive immunity between bacterial Hsp60 proteins and host Hsp60 expressed on stressed vascular endothelial cells is the main atherogenic stimulus. This pathological mechanism was propounded before it was fully understood that CSPs could have intercellular signalling actions. The first evidence that human Hsp60 is found in the circulation was reported by Graham Pockley [29]. Wick's group then reported that the Hsp60 levels in participants in the Bruneck study (a prospective study of cardiovascular risk factors) showed a correlation with atherosclerosis as assessed by carotid artery intima-media (IMT) thickness [30]. A study of 293 Whitehall participants (a longitudinal study of atherosclerosis risk factors) has revealed a strong correlation between plasma Hsp60 levels and psychological distress in women [31]. A follow on, as yet unpublished, study of around 900 Whitehall participants has confirmed the results of the earlier study and revealed a correlation between plasma Hsp60 levels and measures of psychological distress in both males and females. In another study of 855 diabetes mellitus patients a significantly higher proportion of the patients with cardiovascular disease (CVD) had measurable levels of plasma Hsp60 compared with those with no evidence of CVD [32]. This association was more profound among those with a history of myocardial infarction. What is striking about these various studies of Hsp60 is the enormous range of Hsp60 concentrations in blood — from low nanograms to milligram amounts of Hsp60/ml. As will be discussed, it is now established that human Hsp60 at levels N 10 μg/ml is able to activate human monocytes and vascular endothelial cells (VECs) and modulate the activity of other cell populations [33]. Thus individuals with high Hsp60 levels would be expected to have activated myeloid, lymphoid and VEC populations. Are circulating levels of Hsp60 simply a marker for some other process that is driving the atherogenic programme or are they driving the process in their own right? Wick examined 141 17–18 year old males for immunological reactivity to Hsp60 and vessel wall thickness by high resolution ultrasound. This small study revealed that circulating T cell reactivity to human Hsp60 was an independent predictor of early atherosclerosis [34]. The authors have recently conducted a study of 256, 13–16 year olds, to ascertain circulating Hsp60 levels and blood vessel function as assessed by flow-mediated vasodilatation (FMD). This has revealed that subjects with Hsp60 in their circulation exhibited vascular dysfunction as assessed by FMD [35]. These studies are suggestive that Hsp60 in the circulation contributes to the aetiopathogenesis of atherosclerosis. 1.5. Secreted CSP as intercellular signals Reports that CSP are on the surface of or are secreted from a range of cells including myeloid, lymphoid, epithelial

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question — would artificial upregulation of CSP levels in the heart protect against pathophysiological stress such as occurs in myocardial infarction? This was answered by exposing rats to elevated temperature before removing the hearts and exposing them to ischemia. Compared to animals kept at physiological temperature, the hearts of rats exposed to elevated temperature had reduced tissue damage and exhibited quicker recovery from the ischemic episode [23]. Similar results were found in the rabbit [24]. These studies suggested that Hsp70 played a major role in cardioprotection. However, other CSPs would also be upregulated. To show that Hsp70 is cardioprotective, transgenic mice overexpressing Hsp70 were generated and these animals were, indeed, found to be protected against ischemic injury [25]. In the last decade a growing number of other CSPs have been reported to be cardioprotective, including members of the small heat shock protein family, heme oxygenase and CHIP (carboxyl terminus of Hsp70-interacting protein) (Table 2). As each protein provides some degree of protection it is likely that the general induction of the cell stress response would be beneficial for patients with cardiovascular disease. The question is how could this be done? Gene transfer of Hsp70 has been shown to reduce post-ischemic infarct size in the rabbit heart [26]. Another strategy being investigated is the use of agents that enhance the cellular expression of CSPs. Such agents include the antiulcer compound genanyl–geranyl–acetone which has been shown to inhibit post-ischemic heart damage [27] and a hydroxylamine derivative, bimoclomol whose cardioprotective actions are closely approximated with increased expression of Hsp70 [28]. Clinical trials to determine clinical efficacy will presumably follow [28].

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Table 3 Cell stress proteins found on the cell surface and/or secreted by cells and/or found in extracellular fluids Protein

Hsp10 Trx80 Thioredoxin Thioredoxin reductase Glutaredoxin TRANK⁎ Peroxiredoxin Cyclophilin A Cyclophilin B Hsp27 PDI Calnexin Calreticulin Hsp54 Hsp60 Hsp70 Hsp72 Grp74 BiP Hsp90 Gp96 Clusterin

Cell surface Secreted by location cultured cells

+ + + + + + + + + + + + + + + + + + +

Found in extracellular fluid

Reference

+ + + +

[10] [79] [73,76] [93]

+

[125] [73] [73] [99] [126] [127] [128] [129] [129] [42] [8,130] [131,132] [133] [134] [135] [136] [137] [138]

+ + +

+

+ +

+

+ + +

⁎TRANK — thioredoxin peroxidase-related activator of NF-κB.

evidence suggested that secreted CSPs caused activation of myeloid cells and VECs [43] and therefore these proteins acted as pro-inflammatory stimuli. The initial reports of bacterial Hsp60 revealed that myeloid cell activation [44,45] and VEC stimulation [45] was distinct from that induced by the major Gram-positive pro-inflammatory component, lipopolysaccharide (LPS). Thus the Hsp60 from the oral Gramnegative bacterium Actinobacillus actinomycetemcomitans was able to stimulate bone resorption from toll-like receptor (TLR) 4-negative mice [44] and the E. coli homologue was unaffected by neutralising antibodies to the LPS receptor, CD14 [45]. The M. tuberculosis Hsp60 protein induced cultured human VEC to synthesis vascular adhesion proteins by a mechanism that did not involve the early response cytokines IL-1 and TNFα [46]. Later studies of CSP concentrated on the Hsp60 and Hsp70 proteins from Homo sapiens and Hsp60 protein from Chlamydia pneumoniae. The pro-inflammatory actions of the human Hsp60 [47] and Hsp70 proteins [48] and the C. pneumoniae Hsp60 protein [49] were blocked by antibodies to CD14 or TLR2/TLR4 or only activated in cells transfected with the genes encoding these proteins. This has led to controversy in the literature as to whether the activity of the human Hsp60 and Hsp70 is an inherent property of these proteins or simply due to LPS contamination [50]. The major problem is that many workers have used commercially available recombinant proteins, which are heavily contaminated with LPS. A number of groups have removed the LPS from recombinant chaperones and have shown that the biological activity of these proteins are: (i) blocked by heating; (ii) completely inhibited after proteolysis; (iii) replicated by synthetic peptides free from LPS contamination and (iv) able to promote different cell signalling events from that induced by LPS [51]. More recently, it was shown that Hsp60 can downregulate adaptive immune responses. This TLR2 mediated signaling modulated CD4+ CD25+ regulatory T cell function [52]. As inflammation is clearly a major component of atherosclerosis the ability of secreted CSPs to modulate macrophage activation is of obvious importance. Recent reports reveal that human Hsp60 can modulate T cell functionality, suggesting that this particular protein has important functions in controlling inflammation [53,54]. 1.6. CSPs and receptors What receptors transduce the activity of secreted CSPs? The receptor(s) for Hsp60 proteins depends on the source of the Hsp60, with some bacterial proteins not binding to CD14/ TLR4, while the human and some bacterial Hsp60s do bind to this receptor complex [33]. Hsp70 binds to a variety of cell surface receptors including CD14, TLR4, CD40 and LOX-1 [51]. To some extent, this reflects the fact that individual groups are comparing oranges to lemons. That is, they are using Hsp70 proteins from different sources. As the degree of sequence similarity between Hsp70 proteins is only around 50% there is sufficient sequence divergence to allow the

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and mesenchymal cells have been appearing over the past 20 years [36]. In spite of the relatively large number of reports of CSP in bodily fluids, the hypothesis that these proteins are secreted has been criticised for the lack of any mechanism. Specifically, the CSPS generally do not have a signal sequence which is involved in one pathway of the secretion of proteins from eukaryotic cells. Critics ignore the fact that the same criticism can be levelled at a number of important signalling proteins such as the early response cytokine, interleukin (IL)-1 and a range of other proteins [37]. In recent years at least four so-called non-classical pathways of protein secretion have been elucidated in which signal sequences are not involved [37]. Indeed, it is only recently that the mechanism of release of IL-1 has been elucidated [37]. However, one mechanism by which CSPs are released from eukaryotic cells has been identified. This is via exosomes, which are 50–100 nm membrane vesicles that are secreted by cells of hematopoietic origin [38–41]. Mast cell exosomes contain Hsp60 [40]. Importantly, stress increases monocytes exosome/Hsp70 release [39]. There are 21 eukaryotic CSPs which are found on the cell surface, released by cultured cells or are found in extracellular fluids (Table 3). Indeed, a recent proteomic study of cancer cells has identified a very large number of CSPs on the cell surface [42]. The potential biological and medical importance of cell surface and secreted CSPs is their ability to modulate the activity of myeloid cells, T lymphocytes and VECs. The early

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different proteins to bind to different receptors or different parts of the same receptor. Cyclophilin A binds to cell surface heparans and signals via cell surface CD147 [55]. Curiously, CSPs are also able to function as receptors or as part of receptor complexes. As discussed, great care has to be taken to exclude LPS from CSP preparations. Ironically, it turns out that the receptor for LPS contains two CSPs — Hsp70 and Hsp90 whose presence is required for transduction of the signal from LPS [56]. These same two CSPs are part of the receptor for Dengue virus [57]. The receptor for the major outer membrane protein of E. coli is a homologue of the Hsp90 family member, gp96 [58]. Thus, in addition to acting in solution as cell-modulating agonists, certain CSPs can function, as part of a cell membrane receptor complex for specific agonists, which may be of bacterial origin. In addition to functioning as receptors for infective agents, the human Hsp60 protein has been reported to be a high affinity receptor for ApoA-II [59]. 1.7. Secreted CSPs and cardiovascular disease

1.7.1. Hsp60 and Hsp70 As mentioned earlier circulating concentrations of human Hsp60 can be as high as milligram amounts per ml of plasma [31,32,35], that would certainly activate circulating leukocytes and VEC [33,46]. This presumably accounts for the reports that circulating Hsp60 levels are associated with atherosclerosis [30,32,34,35]. The same argument can be made for Hsp70. The cardioprotective effect of the artificial upregulation of Hsp70 was described. Hsp70 gene transcription and levels of this protein have been reported to be upregulated at atherosclerotic sites in atherosclerosis-susceptible mice [65,66]. Two studies have measured Hsp70

levels in atherosclerosis. In 218 subjects with established hypertension whose carotid intima-media thickness was measured over a four year period the smallest changes in this measure were found in patients who had high serum Hsp70 [67]. A cross-sectional study of 421 individuals examined by coronary angiography reported that serum Hsp70 concentrations are significantly higher in those with no evidence of coronary artery disease [68]. Circulating levels of Hsp70 may reflect intracellular levels of this protein which, the evidence suggests, are protective. In contrast, Hsp70 levels were elevated in individuals with peripheral and renal vascular disease [69] and in patients with chronic heart failure [70]. 1.7.2. CSPs involved in regulation of thiol:dithiol redox balance Reactive oxygen species (ROS) and oxidative stress generally are involved in the initiation and progression of cardiac disease [71,72]. A number of CSPs control the thiol (SH-):dithiol (-S-S-) redox balance within the cell and in its local environment. These proteins include thioredoxin, thioredoxin reductase, thioredoxin peroxidase, thioredoxininteracting protein and glutaredoxin [73]. Thioredoxin [74] and glutaredoxin [75] within cells interact with several important transcription factors such as NF-κB and AP-1. Thioredoxin (Trx) is released by transformed human T lymphocytes and has both cytokine [76] and chemokine-like activity [77]. High circulating levels of Trx in HIV-infected patients with low CD4 cell counts is associated with high mortality rates, which is believed to be due to the failure in such individuals of leukocytes to traffic to sites of infection because of the large chemotactic activity in the blood [78]. High Trx levels in the circulation may be deleterious in terms of mounting anti-inflammatory responses but may be beneficial as it would inhibit macrophage recruitment into atheroma. A naturally occurring N-terminal truncated form of Trx, termed Trx80, which lacks thiol oxidoreductase activity, is naturally secreted from human monocytes. In the blood, Trx80 is recognised as a potent alternative activator of monocytes [79]. There are now a number of reports suggesting a role for Trx in cardiovascular disease. Blood levels of Trx are reported to be an indicator of oxidative stress [80,81]. Levels of Trx are elevated in atherosclerotic plaques and in endothelial cells during neointimal regeneration following ballon injury [82]. Overexpression of Trx in transgenic mice ameliorates focal ischemic brain damage or adriamycininduced cardiotoxicity [83] and administration of soluble Trx inhibits ischemia reperfusion injury in various experimental models [84–87]. Do soluble forms of the other cardioprotective CSPs block ischemic injury? Patients with coronary spastic angina have higher serum Trx levels than controls and this is associated with lower circulating vitamin E [88]. Interestingly, nitroglycerin administration causes an increase in plasma Trx in this group of patients [89]. Significantly elevated plasma Trx

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Proteomic analysis of human (VEC) has identified the prominence of CSPs in the protein profile of these cells [60]. Vascular endothelial growth factor (VEGF) plays an important role in the development and physiology of the vasculature. Using differential proteomics it has been found that exposure of quiescent human VEC to VEGF results in the upregulation of 85 proteins of which 17 (20%) are CSPs [61]. As CSPs represent only a small fraction of the human proteome, the finding that such a large proportion of the proteins elevated in VEGF-stimulated cells are CSPs suggests the importance of the cell stress protein machinery in the modulation of VEC function. Using proteomics have also revealed that Hsp27 was released from normal but not diseased arteries. This was reflected in the lower levels of Hsp27 in the blood of patients with atherosclerosis relative to healthy controls [62]. Hsp27 induces human monocytes to produce large amounts of the anti-inflammatory cytokine, IL-10 relative the pro-inflammatory TNFα [63] and inhibits TLR4 expression on monocytes and monocyte differentiation into dendritic cells [64]. This raises the question of whether Hsp27 is a naturally circulating CSP with antiinflammatory/anti-atherosclerotic properties?

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1.7.3. Peptidylprolyl isomerases Another family of enzymic CSPs are the peptidyl prolyl isomerases (PPIases) which catalyse the interconversion between cis and trans forms of the peptide bond preceeding proline residues in proteins [96]. Three families of PPIases exist: cyclophilins, FK506 binding proteins (FKBs) and parvulins. When injected into the footpads of LPSinsensitive C3H/HeJ mice, cyclophilin A stimulated a significant inflammatory response [97] and one of the actions of this protein on human circulating neutrophils and monocytes was as a chemoattractant [97,98]. Human cyclophilin A has been detected at concentrations as high as 0.7 μM in rheumatoid synovial fluid [99]. Berk and colleagues are exploring the hypothesis that reactive oxygen species are involved in the pathogenesis of atherosclerosis by, in part, promoting vascular smooth muscle cell (VSMC) growth. Exposure of cultured rat VSMC to the naphthoquinolinedione, LY83583, which is a generator of oxygen radicals, resulted in the secretion of a range of proteins which have been termed secreted oxidative stress-induced factors (SOXF). Three of these proteins have been identified as Hsp90-α, cyclophilin A and cyclophilin B. Hsp90-α and cyclophilin A are potent activators of VSMC ERK1/2 and proliferation [100,101]. Cyclophilin A is an

activator of VECs able to upregulate the adhesiveness of these cells and stimulate key signalling systems such as NF-κB and MAP kinases [102–104]. The mechanism by which cyclophilin A stimulates cells has been established. This protein binds to cell surface heparans but signals through the cell surface receptor CD147 [55]. 2. Conclusions and future considerations The circulatory system, with its enormous surface area, exposed to: (i) a complex and changeable admixture of molecules including toxins and radicals; (ii) shear stress and (iii) mechanical stresses, may be one of the most stressed cellular systems in the body. It is perhaps for this reason that attention has focused on the role of CSPs in cardiovascular pathology. The finding that upregulation of Hsp70 expression was protective of the ischemic heart is of obvious interest. However, it is clear that many CSPs have cardioprotective effects. Is there a hierarchy of cardioprotection with particular CSPs being much better than others, or do all CSPs have the same protective effect? This is a question which needs answering. It may be that induction of a general cell stress response is all that is required for cardioprotection. How is this to be achieved clinically? Would it be possible to prophylactically elevate CSP levels in patients at risk? The answer to this question is a tentative yes [28]. This, of course, may run foul of other aspects of the biological function of CSPs as these molecules are accomplished moonlighting proteins with many and varied functions. Many bacterial and parasitic homologues of human CSPs are potent immunogens which is unexpected given their sequence homology. Elevating levels of human CSPs may carry with it the price of the development of autoimmunity. Equally, if Georg Wick's hypothesis is correct [8], and atherosclerosis is an autoimmune disease with Hsp60 as the major cross-reactive immunogen, then treatment of atherosclerosis could take the form of selected vaccination. This may not be as far fetched as it seems, as Irun Cohen's group in Israel have reported a double blind placebo controlled phase II study of a Hsp60 peptide, p277. Individuals with newly diagnosed type I diabetes were immunised with either placebo or peptide p277 derived from human Hsp60. Insulin levels, assessed by assay of C-peptide, were measured at 10 months after immunisation. Levels had fallen in the placebo group but were maintained in the p277administered group and the requirement for insulin was lower in the latter group. In addition, those receiving p277 showed an enhanced Th2-helper phenotype suggesting the peptide had induced a shift from Th1 to Th2 responses to Hsp60 [105]. Therefore, immunisation against atherosclerosis is a possibility. It is not clear what controls secretion of many of the CSPs but there is evidence that, at least for mitochondrial proteins, increased translation results in increased secretion [106]. We are entering into a fascinating phase of the study of atherosclerosis and its relationship to cell stress. In a world in

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levels have also been reported in patients with acute myocardial infarction [90] and those with unstable angina [91]. Patients with peripheral vascular disease who were treated by angioplasty had significantly elevated plasma Trx levels within 4 h of the procedure, with levels returning to normal by 24 h [92]. Mammaliam thioredoxin reductase (TrxR) is a seleniumcontaining protein secreted by a number of cells types including monocytes through the classical Golgi pathway and secretion is markedly stimulated in monocytes exposed to pro-inflammatory stimuli such as LPS, IFNγ and IL-1α. The concentration of TrxR in the blood of healthy volunteers was measured and ranged from 6 ng/ml to 0.6 μg/ml [120]. Interestingly, no relationship was found between the release of TrxR and of Trx [93]. In human atherosclerotic plaques, TrxR is upregulated principally in foam cells. Using human monocyte-derived macrophages, oxidised LDLs, but not native LDLs dose-dependently increased the levels of intracellular TrxR mRNA. Promoter analysis revealed that this increase was due to the phospholipid component of the oxidised LDLs [94]. Analysis of thioredoxin-interacting protein (TXNIP) which binds and inhibits Trx, reveals that there is a complex signalling network in endothelial cells in which fluid shear stress controls TXNIP expression, in turn regulating Trx activity and thereby controlling the inflammatory behaviour of VECs [95]. In the aortae of TXNIP-deficient mice there was a significant downregulation in the ability of the major pro-inflammatory cytokine, TNFα, to induce VCAM-1 expression [95]. It would be of obvious interest to determine the response of other CSP coding genes to fluid shear stress.

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which Biology is transmogrifying into Systems Biology and the search for emergent functions, it is sensible to try to view the relationship between CSPs and atherogenesis in systems terms. Thus one should view the stressed cell as a particular system state in which pathology only occurs if this normally transient state fails to return to the pre-existing ‘normal’ level. Evidence is emerging that the cell stress response in the endoplasmic reticulum results in marked changes in cell behaviour due to the upregulation of specific signalling systems [107]. This is called the unfolded protein response (UPR) and similar responses occur in other cell compartments although they have not been so well studied. The UPR is under the control of an Hsp70 homologue termed BiP [107] which is now known to be a secreted protein with potent anti-inflammatory activity able to cause macrophage deactivation [108]. Evidence is now emerging that there is a feedback relationship between the endoplasmic reticulum UPR and altered cholesterol metabolism [109–111]. Thus the challenge for the next decade is to elucidate the systems biology of the cell stress response in its totality encompassing the regulation of CSP transcription, translation and intracellular destruction and the intracellular roles played by these proteins. Linked to this has to be the new paradigm that CSPs can enter onto the cell surface and act as either ligands or ligand receptors and can be released from cells to act as immunogens and as directly-acting intercellular signalling proteins. The possession of this knowledge will aid significantly in our understanding and ability to treat atherosclerosis and other cardiovascular diseases.

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