Skip to main content
NCBI home page
International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2016 Feb 8;97(1):56–65. doi: 10.1111/iep.12167

Increased expression of phosphorylated forms of heat‐shock protein‐27 and p38MAPK in macrophage‐rich regions of fibro‐fatty atherosclerotic lesions in the rabbit

Shahida Shafi 1,✉, Rosalind Codrington 2, Lewis Michael Gidden 1, Gordon Ashley Anthony Ferns 3
PMCID: PMC4840240  PMID: 26853073

Summary

We aimed to assess the expression and distribution of Hsp27, pHsp27 (Ser82), p38MAPK and p‐p38MAPK in fibro‐fatty atherosclerotic lesions and the myocardium of hypercholesterolaemic rabbits. Male New Zealand white rabbits were fed a high‐cholesterol diet for 18 weeks, maintaining serum cholesterol at approximately 20 mmol/l over this period. Aortic arch and myocardial tissues were analysed by Western blot, immunohistochemistry and double immunofluorescence. Plasma Hsp27 levels were measured by ELISA. There was a significant increase in the expression of monomeric and dimeric forms of Hsp27, together with pHsp27 (Ser82), p38MAPK and p‐p38MAPK in the fibro‐fatty atherosclerotic lesions (P < 0.01; P < 0.05; P < 0.001; and P < 0.001, respectively) and the myocardial tissues (P < 0.001) from the cholesterol‐fed rabbits compared with equivalent tissues from controls when the plasma concentration was low. Immunohistochemical analysis of the fibro‐fatty lesions showed marked increases in Hsp27 and pHsp27 (Ser82) immunoreactivity. Double immunostaining showed intense expression of pHsp27 and p‐p38MAPK in regions that were rich in macrophages, suggesting a close association with these inflammatory cells, whereas, in regions rich in smooth muscle cells, only p‐p38MAPK was found to be strongly expressed. An increased expression of pHsp27 (Ser82) was spatially associated with increased p‐p38MAPK within fibro‐fatty atherosclerotic lesions and was colocalized to regions rich in macrophages. The initial increase in plasma Hsp27 levels may reflect the increase in systemic inflammation and oxidative stress in the early phases of disease. The falling concentrations subsequently may be coincident with the development of the advanced atherosclerotic lesions.

Keywords: atherosclerosis, macrophages, phosphorylated Hsp27 (Ser82) and p38MAPK, plaques

Atherosclerosis is now recognized as a chronic inflammatory disease with strong evidence of an underlying autoimmune response. The heat‐shock proteins (Hsps) are constitutively expressed molecular chaperones that protect cells against injury, restoring denatured proteins to their normal configuration and function. However, it appears that some Hsps (60/65, 70/72 and 90) promote atherogenesis possibly through autoimmune mechanisms (Berberian et al. 1990; George et al. 1999; Xu 2002; Ghayour‐Mobarhan et al. 2007; Businaro et al. 2009). There are less data for the involvement of the small heat‐shock proteins, including Hsp27 and its phosphorylated forms in atherogenesis.

There have been a small number of studies on the expression of Hsp27 and its phosphorylated forms in human atherosclerotic tissue, but these have been inconsistent, possibly reflecting the heterogeneity of the atherosclerotic lesions examined, with respect to severity and site (Martin‐Ventura et al. 2006; Park et al. 2006; Garcia‐Arguinzonis et al. 2010). The expression and phosphorylation state of Hsp27 are altered in response to several stimuli including oxidative stress, some cytokines and growth factors, ischaemia, inflammation and infection (Wu et al. 2013). An upregulation in the expression of Hsp27, like several other Hsps, is mediated by heat‐shock transcription factor HSF1, which is activated by p38MAPK (Hung et al. 1998) and binds to the heat‐shock element (HSE) in the promoter region of the Hsp genes (Wu et al. 2013).

Hsp27 is phosphorylated on three serine residues (at positions 15, 78 and 82) via stress factor‐activated p38MAPK pathways that involve several kinases. These kinases phosphorylate p38MAPK (p38α), which in turn activates MAPKAPK kinases 2/3/5 (Moens et al. 2013). Other kinases including PKB, PKC, PKD and PKG also known to phosphorylate Hsp27 independently (Boivin et al. 2012). The two serine residues at positions 78 and 82 are known to be the predominant sites of phosphorylation in Hsp27, and Ser82 is conserved throughout the animal kingdom, whereas Ser78 is replaced by asparagine in number of animals (Landry et al. 1992). The phosphorylation of Hsp27 promotes the disassociation of large Hsp27 multimers (dimers–oligomers) into monomers and dimers (Lambert et al. 1999), which have several distinct functions (Martin et al. 1999). Dimers are the minimum size of phosphorylated Hsp27.

There have been no previous studies investigating the phosphorylation of both Hsp27 and p38MAPK in advanced atherosclerotic lesions. It is possible that crosstalk between Hsp27 and p38MAPK within cells of the artery wall exposed to stressors may lead to the activation and phosphorylation of p38MAPK and subsequent enhanced Hsp27 expression and phosphorylation (Ser82). This may be involved in modulating the functions of monocytes/macrophages, endothelial cells and smooth muscle cells during the development of atherosclerotic lesions.

Materials and methods

Animals, induction of atherosclerosis, blood sampling and tissue harvesting

All experiments were performed in accordance with the UK Home Office licence (Scientific Procedures) Act of 1986 and were approved by the Ethics Committee of the University of Surrey, Guildford, UK. New Zealand white male rabbits (2.8–3.6 kg; B & K Universal Ltd, Hull, UK) were acclimatized for 1 week and then divided into two groups of six. One group was maintained on normal chow and the other group on a 0.3–2% cholesterol‐enriched diet (Special Diet Service, Essex, UK) for 16 weeks. Blood total cholesterol concentration was determined at weekly intervals and maintained between 20 and 30 mmol/l (levels comparable to those found in patients with homozygous familial hypercholesterolaemia) by altering the diet.

Blood samples were collected into heparinized tubes at fortnightly intervals over the time course of the experiment for plasma Hsp27 measurements. After 16 weeks, the aortic arch and myocardial tissues were excised, embedded in paraffin as described previously (Shafi et al. 2010) or frozen immediately in liquid nitrogen for subsequent Western blot analysis.

Protein extraction and Western blot analysis

Aortic arch and myocardial tissues (~0.1 g) were crushed in a Mikro‐Dismembrator U (Braun, Melsungen, Germany) at 2000 rpm for 60 seconds and lysed in precooled RIPA buffer (1 ml) {(100 mM Tris–HCl (pH 8), 150 mM NaCl, 5 mM EDTA (pH 8), 1 mM NaF, 1 mM Na3VO4, 1% Na deoxycholate, 1% Nonidet P‐40, 0.1% SDS and 1% Triton‐X100)} containing protease inhibitor cocktail (Pierce; Thermo Scientific, Winsford, UK). Total protein was quantified using the Pierce bicinchoninic acid (BCA) assay. Western blots were performed with soluble protein (10 or 20 μg), and with Hela cell lysate as a positive control. Samples from both the controls and cholesterol‐fed rabbits were loaded onto the same gels so that a direct comparison could be made. The blots were probed with the following antibodies: anti‐Hsp27 (#AF1580; R & D systems, Oxfordshire, UK), anti‐pHsp27 {(Ser82); #9709)}, anti‐p38MAPK (#9212), anti‐p‐p38MAPK (#9215), anti‐Hsp60 (#12165), GAPDH (#MCA4739; AbD Serotech, Oxfordshire, UK) and β actin (#ab8224; Abcam, Cambridgeshire, UK); all were purchased from Cell Signalling Technology Inc., (Danvers, MA, USA), except where indicated. Peroxidase‐ or phosphatase‐conjugated IgG secondary antibodies were used, and the blots were developed with enhanced chemiluminescent substrates (ECL or CPD‐star; Invitrogen, Paisley, UK).

Following analysis, the PVD membranes were stripped and reprobed with a control antibody (GAPDH or β‐actin) to allow standardization for loading. Quantification of protein band density was performed using the Syngene Gene‐Genius Bioimaging system (Cambridge, UK) and standardized to the GAPDH bands using Gene Snap and Gene Tools image analysis software.

Immunohistochemistry and double immunofluorescence

The following primary antibodies were used: specific for rabbit Hsp27 (mouse monoclonal, 1:20), pHsp27 (Ser82, 1:60), p‐p38MAPK (1:200), macrophage‐specific mouse RAM 11 (1:100; Dako, Cambridgeshire, UK) and VSMC‐specific α‐actin HHF‐35 (1:200; Dako). Unless indicated, the antibodies were purchased from Cell Signalling Technology, Inc. For immunolocalization of Hsp27 or pHsp27 (Ser82) in atherosclerotic lesions, single immunostaining was performed on fixed serial sections as described previously using Vector Elite ABC kit and visualized with DAB (Shafi et al. 2010). Negative controls for immunostaining and immunofluorescence microscopy included incubation with class‐ and species‐matched antibodies or omitting the primary antibody.

For colocalization of phosphorylated forms of Hsp27 (Ser82) and p38MAPK with monocytes/macrophages and SMCs, double immunofluorescence staining was performed. Briefly, tissue sections were probed with macrophage‐ or SMC‐specific antibodies (detailed above), washed and incubated with the secondary antibody anti‐mouse IgG F(ab)2 conjugated to a Alexa 488 (green) as described above. Sections were then incubated with anti‐pHsp27 (Ser82) or anti‐p38‐pMAPK followed by appropriate secondary antibody anti‐rabbit IgG F(ab)2 Alexa Fluor 647‐conjugate (Red) or anti‐mouse IgG Cy3 F(ab)2. Nuclear staining was performed with DAPI (Cell Signalling Technology, Inc.). Fluorescent images were visualized and captured with a laser scanning confocal microscope (Nikon A1M DS‐SU).

Plasma Hsp27 enzyme‐linked immunosorbent assay

Plasma Hsp27 levels were measured using an optimized in‐house ELISA as described previously (Alshammari et al. 2010). The coating antibody used was mouse monoclonal anti‐Hsp27 antibody (clone IAP‐28; Abcam, Cambridge, UK) at a concentration of 1.5 μg/ml in phosphate‐buffered saline (PBS). Non‐specific binding sites were blocked with blocking buffer (3% BSA in PBS, 200 μl/well). Plasma samples (1:20 diluted in blocking buffer) from cholesterol‐fed and normal chow diet control rabbits were added into both the antigen‐coated (target‐) and uncoated, antigen‐free (negative control) wells. The captured Hsp27 antigen was detected using a rabbit anti‐human/anti‐mouse polyclonal antibody (1:8000 dilution in blocking buffer; SPA‐803; Cambridge Bioscience, Cambridge, UK). The secondary antibody used was goat anti‐rabbit HRP‐conjugated antibody (1:12,000; A0545; Sigma‐Aldrich, Poole, UK) followed by an enzymatic substrate. Every plate included positive control plasma with known high IgG Hsp27 absorbance. Each sample was assayed in duplicate and with corresponding uncoated wells to estimate the non‐specific binding. After correction for the non‐specific background absorbance (subtracting the absorbance of uncoated wells from the corresponding antigen‐coated wells), the Hsp27 levels were expressed as the specific binding in optical density units. The intra‐ and interassay coefficients of variations was 8.5 and 14%, respectively.

Statistical analysis

Differences in the mean tissue protein levels (relative band densities) were analysed using unpaired Student's t‐test. Comparisons of plasma Hsp27 levels between cholesterol‐fed and normal control rabbits were performed with grappad prism 6.0 software using a two‐way anova followed by multiple Bonferroni post hoc tests. The results were expressed as means ± SEMs, and a P value < 0.05 was considered statistically significant.

Results

Hsp27, p38MAPK and their phosphorylated (p) forms are increased in fibro‐fatty atherosclerotic lesions and the myocardium of hypercholesterolaemic rabbits

Figure 1 (a–c & e–g) shows increased expression (relative band densities) of Hsp27, p38MAPK and their phosphorylated forms in the atherosclerotic lesions and myocardial tissues compared with their relevant normal controls. Both Hsp27 and pHsp27 (Ser82) were found in two forms with molecular masses of 27 kDa and ~53 kDa corresponding to the monomeric and predominant dimeric forms, respectively. The levels of monomeric and dimeric forms of Hsp27 (Figure 1a) and pHsp27 (Ser82) (Figure 1b) were significantly higher in the atherosclerotic lesion compared with the control aorta (P < 0.0001 and P < 0.01 for Hsp27; P < 0.02 and P < 0.01 for pHsp27 (Ser82), respectively). The relative increase in the atherosclerotic lesions of the dimeric forms of Hsp27 and pHsp27 (Ser82) was greater than for their monomeric counterparts (2.24 and 11.30 fold increase, respectively, P < 0.003). The ratio of dimers of phospho‐Hsp27 (Ser82) to total Hsp27 was significantly higher in the atherosclerotic lesions compared with the normal aortic arch (Table 1, P < 0.05).

Figure 1.

Figure 1

Open in a new tab

Western blotting analysis showing significant increases in Hsp27 (a, e), pHsp27‐Ser82 (b, f), p38MAPK, p‐p38MAPK (c, g) and Hsp60 (d) in the fibro‐fatty atherosclerotic lesion and myocardium tissues from cholesterol‐fed rabbits compared with the relevant controls: ****P < 0.0001; ***P < 0.001; **P < 0.01 and *P < 0.05. Results are means ± SEMs for n = 4 rabbits/group.

Table 1.

Monomeric and dimeric expression of phosphorylated Hsp27 (Ser82) normali‐zed to total Hsp27 in tissues from controls and cholesterol‐fed rabbits by Western blot

Myocardium Aortic arch
Monomer Dimer Monomer Dimer
Controls 8.15 ± 2.1 0.95 ± 0.09 0.00 ± 0.00 1.51 ± 0.41
Cholesterol‐fed 1.14 ± 0.32a 0.63 ± 0.02a 0.70 ± 0.17 2.98 ± 0.45a
Open in a new tab

Data are means ± SEMs from n = 4 rabbits/group.

a

Significance of difference compared to controls P < 0.05.

Furthermore, the expression of p38MAPK and p‐p38MAPK was also both significantly higher in atherosclerotic lesion when compared to the normal aorta tissue, Figure 1c (P < 0.001). The activation ratio of MAPK (p‐p38/p38) in the atherosclerotic lesions was higher than for the control aorta (2:1).

Figure 1 also shows substantial increase in the dimeric forms of Hsp27 (e) and pHsp27 (Ser82) (f) and in p‐p38MAPK (g) in the myocardium of cholesterol‐fed rabbits when compared to the control tissue (P < 0.001; P < 0.01; P < 0.001, respectively). When pHsp27 (Ser82) was normalized to Hsp27, there were significant decreases in both the monomeric and dimeric ratios in the myocardium of cholesterol‐fed rabbit compared with the controls (P < 0.05; Table 1). However, the monomers were the predominant forms of the pHsp27 (Ser82) in the myocardium of the control rabbits as shown in Table 1. No significant changes were observed in p38MAPK between the myocardial tissues from the two groups of animals.

Hsp60 expression was increased in atherosclerotic lesions

The expression of Hsp60 (Figure 1d) was increased in the atherosclerotic lesions but not in the myocardium (Figure 1h) when compared to the corresponding tissues from normal controls: this difference was not statistically significant (P = 0.08).

Fibro‐fatty lesions

Fibro‐fatty atherosclerotic lesions were evident in aortic arch of the cholesterol‐fed rabbits at 16 weeks consisting of abundant macrophage‐ and SMC‐derived foam cells, cholesterol crystals and fibrotic cap (Figure 2c and f). The control aortic arch showed normal morphology with an intact endothelial monolayer lying directly on internal elastic lamina (a and b).

Figure 2.

Figure 2

Open in a new tab

Representative H & E‐stained cross sections of a normal aortic arch from controls and of fibro‐fatty atherosclerotic lesion of aortic arch from cholesterol‐fed rabbits over 16 weeks. The control aortic arch shows a normal morphology (a, b), and the fibro‐fatty atherosclerotic lesions (c) represents an advanced lesions with pathological changes consisting of accumulated macrophage‐ and SMC‐derived foam cells in the centre (d), cholesterol crystals (e) and a fibrotic cap consisting of SMCs (f). Arrows indicate endothelial cells, and arrowheads indicate cholesterol crystals. IEL, internal elastic lamina. Scale bars, 30 μm (a and c), 100 μm (b, d–f).

Hsp27 and pHsp27 (Ser82) localization

Figure 3 shows immunolocalization of Hsp27 (b and c) and pHsp27 (Ser82) (f and g) in these lesions and in normal aortic arch tissues (a and e, respectively). Hsp27 immunoreactivity (brown staining) was homogenously distributed throughout the lesion (3b and c) except in the superficial region that contains SMCs predominantly, where the staining was intense (3c). In contrast, the pHsp27 (Ser82) immunoreactivity was strongly expressed in the core region, rich in monocyte/macrophages; and to lesser extent in the superficial areas of the lesion (f and g). Hsp27 and pHsp27 (Ser82) staining was observed within the SMCs of medial layer and in endothelial cells of the aortic arch sections from normal rabbits (a and e).

Figure 3.

Figure 3

Open in a new tab

Immunolocalization of Hsp27 (a–c) and pHsp27‐Ser82 (e–g) in fibro‐fatty atherosclerotic lesions of the rabbit aortic arch and in normal aortic arch. (c) Higher magnification of (b), showing Hsp27 homogenously expressed throughout the intima. In contrast, pHsp27 (brown staining) distribution is highly expressed in the intima areas (f) corresponding to the regions rich in macrophages and to lesser extent in the superficial layer (g, high magnification). (a and e) Normal control aortic arch with positive staining (brown staining) for Hsp27 and pHsp27 within the SMCs of medial layers and in endothelial cells. No positive staining is observed in negative controls (mouse IgG1) for Hsp27/p‐p38MAPK (d and h). Arrows indicate IEL. Scale bars, 50 μm (b, d, f), 100 μm (a, c, e, g, h).

The immunostaining pattern in the myocardium tissues of cholesterol‐fed rabbits (Figure 4) shows increased expression of Hsp27 (Figure 4d) and pHsp27 (Ser82) (Figure 4e) compared with the controls and this corresponded well with the results of Western blot analysis. Phospho‐Hsp27 (Ser82) was also localized in the endothelial cells of the myocardium blood vessel (Figure 4b and c) and the nucleus (Figure 4f).

Figure 4.

Figure 4

Open in a new tab

Representative confocal laser scanning images of the expression of Hsp27 (a and d, green) and pHsp27‐Ser82 (b and e, red) and their colocalization (yellow areas, c and f) in myocardial tissues and coronary artery from control and cholesterol‐fed rabbits. Phospho‐Hsp27 (Ser82) was also localized in the endothelial cells of the myocardium blood vessel (b and c, red) and the nucleus (f). An image (f) shows colocalization of Hsp27 with pHsp27 (Ser82) in the cardiomyocytes (yellow areas. Arrows indicate the expression of pHsp27 in the nucleus (purple areas). Scale bars, 50 μm.

Phosphorylated Hsp27 and p38MAPK are highly expressed in lesional macrophages

Phosphorylated Hsp27 and p38MAPK were both abundantly expressed in regions rich in macrophages (Figure 5b and f) and were found to be colocalized with monocytes/macrophages (merged images Figure 5c, d and g), strongly suggesting that these phosphorylated forms are highly expressed in macrophages of the fibro‐fatty atherosclerotic lesions. Furthermore, p‐p38MAPK was particularly highly expressed in SMC‐rich areas; the superficial area of the intima, and in the medial layers (Image m). Surprisingly, pHsp27 (Ser82) was not detectable in SMCs (image j). The expression of p‐p38MAPK in SMCs was predominant in the cytoplasm with apparently weaker nuclear staining (insert image m).

Figure 5.

Figure 5

Open in a new tab

Representative confocal laser scanning images of the expression of pHsp27‐Ser82 and p‐p38MAPK (with anti‐pHsp27 and anti‐p‐p38MAPK; red, Alexa 647/Cy3, b, f, i and l), macrophages (Mφ, a and e), SMCs (green, Alexa 488, h, k) and nuclei (DAPI‐positive blue) showing co‐localization in the areas of atherosclerotic lesions with abundant macrophages (yellow areas; merged c; d, enlarged view of the merged image c). Further studies of confocal images (h–m) showed strong co‐localization of p‐p38MAPK with SMCs (yellow areas; merged m). Insert in image m showed p‐p38MAPK expression in SMCs was predominantly in cytoplasmic and some in the nucleus (purple‐pink). No, co‐localization of pHsp27‐Ser82 with SMCs (merged j). Scale bars; 50 μm (d), 100 μm (k–m) and 200 μm (a–j).

Circulating Hsp27 levels

Plasma levels of Hsp27 increased rapidly two weeks after the commencement of the cholesterol diet and remained at this level until week 6 (Figure 6); these levels of Hsp27 were significantly higher than for the control rabbits (P < 0.01 and P < 0.05). After six weeks of cholesterol diet, the circulating levels of Hsp27 decreased rapidly, reaching a plateau by a week 10 after which they were similar in both groups.

Figure 6.

Figure 6

Open in a new tab

Changes in plasma Hsp27 levels in cholesterol‐fed (n = 6) and control (normal diet; n = 5) rabbits over 16 weeks. Each time point represents mean ± SEM. Two‐way anova with Bonferroni post hoc test. *P < 0.05 control vs cholesterol‐fed.

Discussion

We have found that Hsp27, p38MAPK and their phosphorylated forms are highly expressed in fibro‐fatty atherosclerotic lesions, and the myocardium of cholesterol‐fed hypercholesterolaemic rabbits. The regions in which there was an increased expression of pHsp27 (Ser82), were also found to express increased levels of p‐p38MAPK and were co‐localized to regions that were rich in macrophage. Furthermore, p‐p38MAPK was expressed at high levels in SMCs comprising the superficial layer of the plaque and the medial layer of the arterial wall. The expression of pHsp27 (Ser82) was low in SMCs within and adjacent to the core of the plaque; the increased uptake of cholesteryl esters into these cells under hypercholesterolaemic conditions may be associated with dephosphorylation, oligomerization and subcellular relocalization of Hsp27. This has been proposed to be a protective mechanism but may also play a critical role in the progression of atherosclerotic lesions (Garcia‐Arguinzonis et al. 2010). We found that the plasma Hsp27 concentrations were significantly increased at early stages of atherogenesis and then decreased with the progression of atherosclerosis.

We confirmed previous reports of an increased expression of Hsp60 in fibro‐fatty atherosclerotic lesions; it has been established that Hsp60 is positively related to the severity of the lesions in man (Xiao et al. 2005). However, in contrast to our findings for Hsp27, the expression of Hsp60 did not differ significantly between the myocardia/atherosclerotic lesions from normo‐ and cholesterol‐fed rabbits.

We have used a well‐characterized experimental model of atherosclerosis, in which the lesions develop predictably in the arterial tree; initially in the aortic arch, then in the thoracic aorta and progressively extend to the abdominal aorta (Yu et al. 2012; Fan et al. 2015). At the time point investigated, the lesions in the aortic arch were more fibro‐fatty than elsewhere in the aorta, containing abundant foam cells that were derived from macrophages and smooth muscle cells; a macrophage‐rich core with cholesterol crystal clefts; and in some lesions abundant SMCs that formed a ‘cap‐’like superficial layer (Kolodgie et al. 1996).

In this study, we were interested in post‐translational modified Hsp27 at serine 82 residues. Limited number of studies have suggested Ser78 and 82 are the major sites of phosphorylation and their expression was high in human diseased arteries compared with Hsp27 phosphorylated at Ser15 (Landry et al. 1992; Robinson et al. 2010). Ser82 is highly conserved throughout the animal kingdom, and Ser78 is replaced by asparagine in number of animals (Kostenko & Moens 2009).

Falling plasma Hsp27 concentrations were coincident with the development of fibro‐fatty atherosclerotic lesions

Plasma Hsp27 levels rose quickly after starting the high‐cholesterol diet, and then fell to near normal concentrations after some weeks. At this time, the expression of Hsp27 within the fibro‐fatty atherosclerotic lesions was relativ‐ely high, and as we have previously shown, plasma Hsp27 antibody titres are also high. These findings are in contrast to those of Park et al. (2006) and others (Martin‐Ventura et al. 2006; Robinson et al. 2010), who have reported a reduced expression of Hsp27 in human atherosclerotic plaques. The hypercholesterolaemic rabbit is an acute and accelerated model of atherosclerosis that may better represent the early phases of atherogenesis, whereas the human lesions evolve over a much longer time period. Rayner et al. (2008) have shown that the expression of Hsp27 diminishes with the progression of the disease. Furthermore, it has been reported that low serum Hsp27 levels in human studies may indicate the presence of atherosclerotic lesions in coronary artery as well as other arteries (Seibert et al. 2013; Jin et al. 2014). The mechanisms responsible for the fall in plasma Hsp27 are unknown. However, previous studies have shown that the concentration of circulating Hsp27 antibodies are high in patients with coronary heart disease (Ghayour‐Mobarhan et al. 2007; Shams et al. 2008; Pourghadamyari et al. 2011). These anti‐Hsp27 antibodies may form immune complexes with Hsp27 antigen that are cleared from the circulation via the FC receptors of the cells of the reticuloendothelial system (Rahsepar et al. 2013) which provides a potential explanation for the reduction in circulating Hsp27 levels and eventually to low concentrations in the atherosclerotic lesions.

Significance of increased expressions of Hsp27, p38MAPK and their phosphorylated forms in atherosclerotic lesions and the myocardium

We found that the expression of pHsp27 (Ser82) was increased in atherosclerotic lesions and that this was spatially related to p‐p38MAPK expressed within these lesions; the phosphorylation of p38MAPK and Hsp27 may be important events in atherogenesis. Some previous studies have reported lower levels of Hsp27 and pHsp27 (Ser82) in plaque secretions and plaques (Martin‐Ventura et al. 2006; Park et al. 2006; Robinson et al. 2010). This discrepancy may be due to differences in the complexity and stage of the plaques being investigated, or the arteries involved (Galis et al. 1994; Lijnen 2001).

This study suggested that the Hsp27 has propensity to dimerize, and hence, the results showed increases in pHsp27 (Ser82) dimers in atherosclerotic lesions, where there are increased number of necrotic cells and the presence of plasma; these may provide reducing or oxidising environment for Hsp27 to dimerize. The dimeric form of pHsp27 probably enhances the chaperone activity, as this unit is the active component of Hsp27 (Jovcevski et al. 2015). The functional significance of pHsp27 (Ser82) dimerization is a part of the mechanism to recruit other mediators to the atherosclerotic lesions such as monocyte recruitment into the lesional area, thereby leading to the increase in number of macrophages that may enhance atherogenesis via oxidative stress. In addition, this phosphorylated form may also be important in regulating proliferation or apoptosis of cells within atherosclerotic lesions. Phosphorylated Hsp27 has been reported to inhibit Daxx in a Fas‐mediated apoptosis, thereby contributing to the cell survival (Charette et al. 2000).

Previous studies have shown that activated p38MAPK can induce the production of pro‐inflammatory cytokines (IL‐1, IL‐6 and TNF‐α) by activating SMCs, T cells and macrophages, thereby leading to the chronic inflammation and potentially cell death (Lee et al. 1994; Ohashi et al. 2000). The increased expression of p‐p38MAPK that we have observed in the fibro‐fatty atherosclerotic lesions and its colocalization with SMCs and macrophages therefore suggest a prominent role in the development of atherosclerosis. We believe that further in vivo and in vitro research is needed to explore the interactions between the phosphorylated forms of Hsp27 and p38MAPK with other intracellular proteins and their relationship with markers of plaque inflammation.

The functional importance of increase in phosphorylated Hsp27 may be to stabilize cytoskeleton and to protect the heart against ischaemic injury. In the myocardial cells, the phosphorylated Hsp27 was localized not only in the cytosol but also in the nucleus and this may be important in protection against actin fragmentation and degradation of microtubules (Dana et al. 2000). The increases in phosphorylated Hsp27 and p38MAPK in the myocardium induced by hypercholesterolaemia may be associated with increase in oxidative stress, inflammation, induction of apoptosis and immune responses and may account for the myocardium injury and necrosis (Osipov et al. 2009) by initiating the internal signalling cascades or providing resistance against ischaemia. Gaitanaki et al. (2003) showed inhibition of p38MAPK by an inhibitor (SB203580) abolished increases in phosphorylated Hsp27 and loss of cardioprotection, thereby providing further evidence that increases in the phosphorylation of p38MAPK and Hsp27 are important in cardioprotection. Weinbrenner et al. (1997) studies also showed that the activation of p38MAPK in rabbit was important for the cardioprotection. Here, we suggest for the first time that hypercholesterolaemia may cause a mild chronic stressful, stimulus to myocardium inducing upregulation of endogenous Hsp27 and pHsp27 (Ser82) by activating p38MAPK signalling pathways which may in turn in the early stages of atherosclerosis development enhance heart resistance to ischaemic injury. Furthermore, the significant decrease in the monomers and dimers in terms of the ratios of pHsp27 (Ser82) to Hsp27 in myocardium of cholesterol‐fed rabbits also suggests a protective role. Animal studies have previously shown increases in activated p38MAPK, Hsp27 and pHsp27 (Ser82) in response to acute ischaemia, reperfusion injury or cardioplegic arrest, suggesting a cardioprotective role (Martin et al. 1999; Clements et al. 2011; Wu et al. 2013). However, Gorog et al. (2009) have reported that the phosphorylation of Hsp27 is not necessarily associated with cardioprotection. We observed an increased expression of p‐p38MAPK. It is therefore possible that p38MAPK activation is associated with the phosphorylation of Hsp27 in conditions of oxidative stress and that these events may be important in cardioprotection.

Significance of increased expression of pHsp27 (Ser82) and p‐p38MAPK in macrophages

Our second major finding was the increased expression of p‐p38MAPK in macrophage‐rich regions of fibro‐fatty atherosclerotic lesions. This may be implicated in Hsp27 phosphorylation and may itself be involved in the oxidative defence mechanisms (Rao 2001; Seimon & Tabas 2009). However, in vitro studies have shown that the activation of p38MAPK is involved in foam cell formation in the early stages of atherosclerosis (Mei et al. 2012). The co‐expression of pHsp27 (Ser82) and p‐p38MAPK in the inflammatory cells may have profound effects on atherogenesis via several mechanisms that can enhance atherogenesis, including effects on cell proliferation, differentiation and apoptosis (Rao 2001; Seimon & Tabas 2009; Mei et al. 2012).

Activation and phosphorylation of p38MAPK in atherosclerotic lesions do not induce Hsp27 phosphorylation in smooth muscle cells

Phosphorylated p38MAPK was expressed at high levels in SMCs in specific regions of the atherosclerotic lesions. This included the superficial layers overlying these lesions and the tunica media. However, this was not accompanied by an increased phosphorylation of Hsp27; no pHsp27 (Ser82) was detected in the SMCs of the atherosclerotic lesions. It is possible that this is due to the process of LDL‐stimulated dephosphorylation of Hsp27 (Garcia‐Arguinzonis et al. 2010). The reduced pHsp27 (Ser82) may be associated with a loss of caspase‐3 activation leading to increased SMC apoptosis and an enhanced risk of plaque rupture (Walsh et al. 2000). A small number of studies of human atherosclerotic plaques have also demonstrated diminished expression of pHsp27 (Ser82) in SMCs (Martin‐Ventura et al. 2004; Park et al. 2006; Garcia‐Arguinzonis et al. 2010). Apoptosis has been previously reported in VSMCs in association with increased phosphorylation of p38MAPK induced by mechanical stress (Cornelissen et al. 2004). An increased or sustained expression of p‐p38MAPK may result in a chronic inflammatory state.

Further studies are required to determine the potential mechanisms for the increased expression of pHsp27 (Ser82) and p‐p38MAPK and their role in lesion development. This may include in vitro studies on macrophages and VSMCs using selective inhibitors of p38MAPK (SB203580 or SB202190) to block the phosphorylation of p38MAPK and Hsp27 or knocking‐down Hsp27 by specific siRNA, thereby preventing the phosphorylation of Hsp27 in the inflammatory cells of the atherosclerotic lesions. Moreover, assessment of the expression of proinflammatory cytokines or other inflammatory markers in atherosclerotic plaques in conjunction with Hsp27 and p38MAPK expression may reveal their role in the development of atherosclerotic lesions.

Authors' contributions

SS designed and performed experiments, collected data, analysed and interpreted and generated the figures. SS and LG performed Western blots; RC advised on Western blot and result analysis; GF and SS drafted the manuscript, and GF was involved in the final discussion on the outcome of the study. All authors gave final approval for the submission and publication of this manuscript.

Disclosures

None.

Acknowledgments

This work was supported by British Heart Foundation project grant (PG/09/081). We would like to thank Professor Susanna Hourani for proof reading the manuscript and valuable suggestions.

References

  1. Alshammari E., Shafi S., Nurmi‐Lawton J., Burut D.F., Lanham‐New S. & Ferns G. (2010) Markers of inflammation, endothelial activation and autoimmunity in adolescent female gymnasts. J. Sports Sci. Med. 9, 538–546. [PMC free article] [PubMed] [Google Scholar]
  2. Berberian P.A., Myers W., Tytell M., Challa V. & Bond M.G. (1990) Immunohistochemical localization of heat shock protein‐70 in normal‐appearing and atherosclerotic specimens of human arteries. Am. J. Pathol. 136, 71–80. [PMC free article] [PubMed] [Google Scholar]
  3. Boivin B., Khairallah M., Cartier R. & Allen B.G. (2012) Characterization of hsp27 kinases activated by elevated aortic pressure in heart. Mol. Cell. Biochem. 371, 31–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Businaro R., Profumo E., Tagliani A. et al (2009) Heat‐shock protein 90: a novel autoantigen in human carotid atherosclerosis. Atherosclerosis 207, 74–83. [DOI] [PubMed] [Google Scholar]
  5. Charette S.J., Lavoie J.N., Lambert H. & Landry J. (2000) Inhibition of Daxx‐mediated apoptosis by heat shock protein 27. Mol. Cell. Biol. 20, 7602–7612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clements R.T., Feng J., Cordeiro B., Bianchi C. & Sellke F.W. (2011) p38 MAPK‐dependent small HSP27 and alphaB‐crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest. Am. J. Physiol. Heart Circ. Physiol. 300, H1669–H1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cornelissen J., Armstrong J. & Holt C.M. (2004) Mechanical stretch induces phosphorylation of p38‐MAPK and apoptosis in human saphenous vein. Arterioscler. Thromb. Vasc. Biol. 24, 451–456. [DOI] [PubMed] [Google Scholar]
  8. Dana A., Skarli M., Papakrivopoulou J. & Yellon D.M. (2000) Adenosine A(1) receptor induced delayed preconditioning in rabbits: induction of p38 mitogen‐activated protein kinase activation and Hsp27 phosphorylation via a tyrosine kinase‐ and protein kinase C‐dependent mechanism. Circ. Res. 86, 989–997. [DOI] [PubMed] [Google Scholar]
  9. Fan J., Kitajima S., Watanabe T. et al (2015) Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine. Pharmacol. Ther. 146C, 104–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gaitanaki C., Konstantina S., Chrysa S. & Beis I. (2003) Oxidative stress stimulates multiple MAPK signalling pathways and phosphorylation of the small HSP27 in the perfused amphibian heart. J. Exp. Biol. 206, 2759–2769. [DOI] [PubMed] [Google Scholar]
  11. Galis Z.S., Sukhova G.K., Lark M.W. & Libby P. (1994) Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J. Clin. Invest. 94, 2493–2503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garcia‐Arguinzonis M., Padro T., Lugano R., Llorente‐Cortes V. & Badimon L. (2010) Low‐density lipoproteins induce heat shock protein 27 dephosphorylation, oligomerization, and subcellular relocalization in human vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 30, 1212–1219. [DOI] [PubMed] [Google Scholar]
  13. George J., Shoenfeld Y., Afek A. et al (1999) Enhanced fatty streak formation in C57BL/6J mice by immunization with heat shock protein‐65. Arterioscler. Thromb. Vasc. Biol. 19, 505–510. [DOI] [PubMed] [Google Scholar]
  14. Ghayour‐Mobarhan M., Lamb D.J., Tavallaie S. & Ferns G.A. (2007) Relationship between plasma cholesterol, von Willebrand factor concentrations, extent of atherosclerosis and antibody titres to heat shock proteins‐60, ‐65 and ‐70 in cholesterol‐fed rabbits. Int. J. Exp. Pathol. 88, 249–255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gorog D.A., Jabr R.I., Tanno M. et al (2009) MAPKAPK‐2 modulates p38‐MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38‐MAPK activation during myocardial ischemia. Cell Stress Chaperones. 14, 477–489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hung J.J., Cheng T.J., Lai Y.K. & Chang M.D. (1998) Differential activation of p38 mitogen‐activated protein kinase and extracellular signal‐regulated protein kinases confers cadmium‐induced HSP70 expression in 9L rat brain tumor cells. J. Biol. Chem. 273, 31924–31931. [DOI] [PubMed] [Google Scholar]
  17. Jin C., Phillips V.L., Williams M.J., van Rij A.M. & Jones G.T. (2014) Plasma heat shock protein 27 is associated with coronary artery disease, abdominal aortic aneurysm and peripheral artery disease. Springerplus 3, 635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jovcevski B., Kelly M.A., Rote A.P. et al (2015) Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity. Chem. Biol. 22, 186–195. [DOI] [PubMed] [Google Scholar]
  19. Kolodgie F.D., Katocs A.S. Jr, Largis E.E. et al 1996. Hypercholesterolemia in the rabbit induced by feeding graded amounts of low‐level cholesterol. Methodological considerations regarding individual variability in response to dietary cholesterol and development of lesion type. Arterioscler. Thromb. Vasc. Biol. 16, 1454–1464. [DOI] [PubMed] [Google Scholar]
  20. Kostenko S. & Moens U. (2009) Heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology. Cell. Mol. Life Sci. 66, 3289–3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lambert H., Charette S.J., Bernier A.F., Guimond A. & Landry J. (1999) HSP27 multimerization mediated by phosphorylation‐sensitive intermolecular interactions at the amino terminus. J. Biol. Chem. 274, 9378–9385. [DOI] [PubMed] [Google Scholar]
  22. Landry J., Lambert H., Zhou M. et al (1992) Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen‐activated kinases that recognize the same amino acid motif as S6 kinase II. J. Biol. Chem. 267, 794–803. [PubMed] [Google Scholar]
  23. Lee J.C., Laydon J.T., McDonnell P.C. et al (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739–746. [DOI] [PubMed] [Google Scholar]
  24. Lijnen H.R. (2001) Plasmin and matrix metalloproteinases in vascular remodeling. Thromb. Haemost. 86, 324–333. [PubMed] [Google Scholar]
  25. Martin J.L., Hickey E., Weber L.A., Dillmann W.H. & Mestril R. (1999) Influence of phosphorylation and oligomerization on the protective role of the small heat shock protein 27 in rat adult cardiomyocytes. Gene Expr. 7, 349–355. [PMC free article] [PubMed] [Google Scholar]
  26. Martin‐Ventura J.L., Duran M.C., Blanco‐Colio L.M. et al (2004) Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis. Circulation 110, 2216–2219. [DOI] [PubMed] [Google Scholar]
  27. Martin‐Ventura J.L., Nicolas V., Houard X. et al (2006) Biological significance of decreased HSP27 in human atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 26, 1337–1343. [DOI] [PubMed] [Google Scholar]
  28. Mei S., Gu H., Ward A. et al (2012) p38 mitogen‐activated protein kinase (MAPK) promotes cholesterol ester accumulation in macrophages through inhibition of macroautophagy. J. Biol. Chem. 287, 11761–11768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moens U., Kostenko S. & Sveinbjornsson B. (2013) The role of Mitogen‐Activated Protein Kinase‐Activated Protein Kinases (MAPKAPKs) in inflammation. Genes (Basel) 4, 101–133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ohashi N., Matsumori A., Furukawa Y. et al (2000) Role of p38 mitogen‐activated protein kinase in neointimal hyperplasia after vascular injury. Arterioscler. Thromb. Vasc. Biol. 20, 2521–2526. [DOI] [PubMed] [Google Scholar]
  31. Osipov R.M., Bianchi C., Feng J. et al (2009) Effect of hypercholesterolemia on myocardial necrosis and apoptosis in the setting of ischemia‐reperfusion. Circulation 120, S22–S30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Park H.K., Park E.C., Bae S.W. et al (2006) Expression of heat shock protein 27 in human atherosclerotic plaques and increased plasma level of heat shock protein 27 in patients with acute coronary syndrome. Circulation 114, 886–893. [DOI] [PubMed] [Google Scholar]
  33. Pourghadamyari H., Moohebati M., Parizadeh S.M. et al (2011) Serum antibody titers against heat shock protein 27 are associated with the severity of coronary artery disease. Cell Stress Chaperones. 16, 309–316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rahsepar A.A., Mirzaee A., Moodi F. et al (2013) Changes in anti‐heat shock protein 27 antibody and C‐reactive protein levels following cardiac surgery and their association with cardiac function in patients with cardiovascular disease. Cell Stress Chaperones. 18, 65–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rao K.M. (2001) MAP kinase activation in macrophages. J. Leukoc. Biol. 69, 3–10. [PubMed] [Google Scholar]
  36. Rayner K., Chen Y.X., McNulty M., Simard T., Zhao X., Wells D., de Belleroche J., O'Brien E.R. (2008) Extracellular release of atheroprotective heat shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to scavenger receptor. A. Circ. Res. 18, 133–141. [DOI] [PubMed] [Google Scholar]
  37. Robinson A.A., Dunn M.J., McCormack A., dos Remedios C. & Rose M.L. (2010) Protective effect of phosphorylated Hsp27 in coronary arteries through actin stabilization. J. Mol. Cell. Cardiol. 49, 370–379. [DOI] [PubMed] [Google Scholar]
  38. Seibert T.A., Hibbert B., Chen Y.X. et al (2013) Serum heat shock protein 27 levels represent a potential therapeutic target for atherosclerosis: observations from a human cohort and treatment of female mice. J. Am. Coll. Cardiol. 62, 1446–1454. [DOI] [PubMed] [Google Scholar]
  39. Seimon T. & Tabas I. (2009) Mechanisms and consequences of macrophage apoptosis in atherosclerosis. J. Lipid Res. 50(Suppl), S382–S387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shafi S., Lamb D., Modjtahedi H. & Ferns G. (2010) Periadventitial delivery of anti‐EGF receptor antibody inhibits neointimal macrophage accumulation after angioplasty in a hypercholesterolaemic rabbit. Int. J. Exp. Pathol. 91, 224–234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shams S., Shafi S., Bodman‐Smith K., Williams P., Mehta S. & Ferns G.A. (2008) Anti‐heat shock protein‐27 (Hsp‐27) antibody levels in patients with chest pain: association with established cardiovascular risk factors. Clin. Chim. Acta 395, 42–46. [DOI] [PubMed] [Google Scholar]
  42. Walsh K., Smith R.C. & Kim H.S. (2000) Vascular cell apoptosis in remodeling, restenosis, and plaque rupture. Circ. Res. 87, 184–188. [DOI] [PubMed] [Google Scholar]
  43. Weinbrenner C., Liu G.S., Cohen M.V. & Downey J.M. (1997) Phosphorylation of tyrosine 182 of p38 mitogen‐activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J. Mol. Cell. Cardiol. 29, 2383–2391. [DOI] [PubMed] [Google Scholar]
  44. Wu J., Jiang S., Ding Z. & Liu L. (2013) Role of heat shock protein 27 in cardiovascular disease. J. Biochem. Pharmacol. Res. 1, 43–50. [Google Scholar]
  45. Xiao Q., Mandal K., Schett G. et al (2005) Association of serum‐soluble heat shock protein 60 with carotid atherosclerosis: clinical significance determined in a follow‐up study. Stroke 36, 2571–2576. [DOI] [PubMed] [Google Scholar]
  46. Xu Q. (2002) Role of heat shock proteins in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 22, 1547–1559. [DOI] [PubMed] [Google Scholar]
  47. Yu Q., Li Y., Waqar A.B. et al. (2012) Temporal and quantitative analysis of atherosclerotic lesions in diet‐induced hypercholesterolemic rabbits. J. Biomed. Biotechnol. 2012, 506159. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from International Journal of Experimental Pathology are provided here courtesy of Wiley

RESOURCES