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J Am Coll Cardiol, 2001; 38:1564-1569
© 2001 by the American College of Cardiology Foundation
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EXPERIMENTAL STUDY

Accelerated intimal thickening in carotid arteries of balloon-injured rats after immunization against heat shock protein 70

Jacob George, MD*, Shai Greenberg, MSc*, Iris Barshack, MD{dagger}, Madhavir Singh, PhD{ddagger}, Sara Pri-Chen, PhD*, Shlomo Laniado, MD* and Gad Keren, MD*,*

* Department of Cardiology and the Cardiovascular Research Laboratory, Tel Aviv Medical Center, Tel Aviv, Israel
{dagger} Institute of Pathology, Sheba Medical Center, Tel Hashomer, Israel
{ddagger} GBF-Braunschweig, Braunschweig, Germany

Manuscript received December 19, 2000; revised manuscript received June 14, 2001, accepted July 12, 2001.

* Reprint requests and correspondence: Dr. Gad Keren, Department of Cardiology and the Cardiovascular Research Laboratory, Ichilov Hospital, Elias Sourasky, Tel Aviv Medical Center, 6 Weizman Street, Tel Aviv, Israel
kereng{at}tasmc.health.gov.il


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
OBJECTIVES

The goal of this study was to test the hypothesis that induction of an immune response to heat shock protein (Hsp) 70 would increase intimal thickening in a rat carotid-injury model.

BACKGROUND

Restenosis resulting from intimal thickening poses a major limitation to the long-term success of coronary angioplasty. Several studies have proposed that infectious agents increase restenosis. Heat shock proteins are highly conserved structures, produced by all cells in response to nonspecific forms of stress. Infectious agents are known to contain Hsp70, which is markedly immunogenic and can elicit a strong immune response.

METHODS

To investigate whether Hsp70 immunity can affect neointimal thickening, we immunized rats with either Hsp70 (n = 11), bovine serum albumin ([BSA] n = 9) or with a control adjuvant (n = 10). Three weeks later, rats were boosted using the same regimen to achieve a sustained immune response to Hsp70 after which carotid injury was applied to all animals.

RESULTS

Arterial injury was associated with upregulation of Hsp70, 3, 7 and 14 days after induction of the injury as evidenced by Western blotting and immunohistochemistry. Intimal area and intimal/medial ratio was significantly increased in Hsp70-immunized rats in comparison with BSA or control-injected rats.

CONCLUSIONS

Our results imply that upregulation of Hsp70 in balloon-injured arteries can serve as a target for anti-Hsp70 immune response, thereby facilitating enhanced intimal thickening. These observations may provide a possible mechanism that explains the accelerated intimal thickening that has been associated with the occurrence of infectious pathogens.

Abbreviations and Acronyms
  BSA = bovine serum albumin
  ELISA = enzyme-linked immunosorbent assay
  Hsp = heat shock protein
  IFA = incomplete Freund’s adjuvant
  IgG = immunoglobulin G
  PBS = phosphate-buffered saline
  PI = protease inhibitor
  SMC = smooth muscle cell
  TBS = Tris-buffered saline

The long-term effectiveness of balloon angioplasty is still largely hampered by the occurrence of late lumen loss after intimal thickening (1,2). Although several experimental strategies have provided some success in reducing intimal thickening in animals (3,4), clinical trials in humans performed thus far failed to accomplish significant improvement. The reason for the limited effectiveness of appropriate prevention lies in the yet unresolved processes that mediate formation of the neoimtima and remodelling by the cellular participants (i.e., monocyte/macrophages, vascular smooth muscle cells [SMC], T lymphocytes) and their products.

In recent years, considerable data have accumulated to support a role for infectious agents in the progression of atherosclerosis and restenosis (5–7). The data derive from several lines of study: 1) evidence for the presence of the infectious agents within lesioned arteries (5); 2) the presence of antibodies to the pathogen in the sera of the patients (5,8); 3) experimental induction of lesions in infected animals (5,9); and 4) in vitro assays demonstrating the ability of the organism to influence the properties of the cellular components of the arterial wall (5,10,11).

As both atherosclerosis and restenosis basically display a form of response of the arterial wall to injury (12), an inflammatory phenotype set by an infectious agent is likely to influence the milieu within which atherosclerosis and restenosis occur. However, why certain infections appear to have a more detrimental effect on the response to injury than others is still controversial and requires further study.

Heat shock proteins (Hsp) are produced by all cells in response to several forms of stress (thermal, mechanical, irradiation, etc.) (13,14). Heat shock protein 70 members have been known to provide an essential function in preventing aggregation and assisting refolding of misfolded proteins. However, they also have a principal role under normal conditions including assisted folding of newly translated proteins, guiding translocating proteins across organellar membranes, disassembling oligomeric structures and facilitating proteolytic degradation of unstable proteins (13,14). It has recently been noticed that members of the Hsp family contained within infectious organisms are antigenic and immunodominant, thus eliciting strong cellular and humoral immune responses (15,16). This is specifically true for the Hsp70, which appears to trigger a particularly brisk immune response (17).

Several studies have pointed to an important role for Hsp 60 kd and 70 kd in the initiation of experimental autoimmune diseases (18–21). In a set of studies performed in the previous decade, it has been shown that Hsp60/65 could serve as an antigenic target of an immune-mediated response that serves to enhance atherosclerosis in experimental animals and in humans (22–25). The hypothesis claims that immunity against infectious agents cross-reacts with self-expressed Hsp60, thereby promoting lesion formation. Studies have also revealed expression of Hsp70 in atherosclerotic plaque (26,27), and similar mechanisms may be operative for Hsp70.

As Hsp70 is the predominant Hsp that is upregulated in response to stress on one hand (13,14) and that it is highly immunogenic (17) on the other hand, we tested the hypothesis that induction of an immune response to Hsp70 in an injured rat carotid artery would influence neointima formation. Such an effect could account for an important mechanism mediating infection-related acceleration of restenosis.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals.   Eight-week-old male Wistar rats (aged two to three months) were purchased from Tel Aviv University and raised at the local animal house. All animals were fed a normal diet and allowed free access to water and food.

Experimental design.   Rats were immunized with either 30 µg Hsp70 mycobacterial Hsp70 (GBF, Braunschweig, Germany; n = 11), bovine serum albumin ([BSA] n = 9) emulsified in incomplete Freund’s adjuvant (IFA) or with IFA alone (n = 10; "control" group). Rats were boosted after three weeks with the same antigen, and balloon injury of the left carotid artery was performed two weeks later.

Rat carotid injury model.   Animals were anesthetized by intraperitoneal injection of Ketamin (80 mg/kg) and Xylazine (5 mg/kg). Endothelial denudation and vascular injury were performed in the left common carotid artery, as described (28).

Histologic assessment of intimal lesions.   Serial cross sections (5 µm thick) were used throughout the entire length of the carotid artery for histologic analysis (average of five per animal). All samples were routinely stained with hematoxylin & eosin or Masson-Trichrome stains.

Quantification of intimal lesions in sections of carotid arteries.   Five equally spaced cross sections were used in all rats to quantify intimal lesions. Using image analysis software (Prof. I. Hammel, Department of Pathology, Tel Aviv University), total cross-sectional medial area was measured between the external and internal elastic laminae. Total cross-sectional intimal area was measured between the endothelial cell monolayer and the internal elastic lamina.

Detection of anti-Hsp70 antibodies.   Recombinant mycobacterial Hsp70 (1 µg/ml) in phosphate-buffered saline ([PBS] pH 7.2) was coated onto flat bottom 96-well enzyme-linked immunosorbent assay (ELISA) plates (Nunc, Maxisorp, Denmark) by overnight incubation at 4°C as previously described, and sera (dilution of 1:100) were probed using ELISA as previously described (25).

Western blot for detection of Hsp70 in rat carotid arteries.   Fresh balloon injured and normal carotid arteries were homogenized by homogenizer (polytron) in PBS containing 2 mmol of a protease inhibitor (i.e., phenylmethylsulfonylfluoride; Sigma, Rehovot, Israel). Supernatants of the respective injured and of nonmanipulated arteries were applied on 10% acrylamide SDS-gel under reduced conditions, transferred to nitrocellulose paper and probed with an anti-Hsp70 antibody. As a control marker, we used recombinant Hsp70.

Detection of Hsp70 by immunhistochemistry.   Immunohistochemical staining for Hsp70 was performed on 5 µm-thick frozen sections of the rat carotid arteries employing polyclonal mouse anti-Hsp70 antibody.

Elution and characterization of bound immunoglobulin G from rat carotid arteries.   In a separate experiment, rats (n = 6) were immunized with Hsp70, and arterial injury was performed as described in the preceding text. Tissue samples were obtained and homogenized by a Polytron homogenizer in Tris-buffered saline (TBS) supplemented with protease inhibitors ([PI] 0.02 µM, pH, 7.4). The homogenate was washed three times in TBS + PI by centrifugation. A minimal volume (0.3 to 0.5 ml) of 0.1 M Glycine-HCl buffer was (pH: 2.8 to 3.5) added to the pellet by vortexing. Subsequently, the tube was centrifuged, and the supernatant was collected and neutralized with 1 M of Tris (pH 7.4). The supernatant was later dialyzed against TBS overnight at 4°C. A bicinchoninic acid kit (Pierce, Rockford, Illinois) was used for protein determination.

Cell culture and measurement of SMC proliferation.   Smooth muscle cells were obtained from the carotid arteries of Wistar rats by use of the collagenase and elastase digestion method. Proliferation in the presence of immunoglobulin G (IgG) anti-Hsp70 or control rat antibodies (10 µg/ml and 100 µg/ml) was assessed by [3H]-thymidine incorporation into DNA was measured as described previously (29).

Statistical analysis.   Differences between groups were compared using a one-way analysis of variance test followed by Fisher protected least significant difference. P <0.05 was accepted as statistically significant. Values represent mean ± SD.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Kinetics of Hsp70 antibody production.   Our previous experience obtained from preliminary studies indicated, similar to others (17), that Hsp70 was highly immunogenic and elicited a pronounced immune response as manifested by a strong IgG anti-Hsp70 antibody response within 9 to 10 days of the initial immunization (Fig. 1). Hyperimmune sera from mycoabacterial Hsp70-immunized rats displayed cross-reactivity with human Hsp70 by ELISA (67% of the binding to mycobacterial Hsp70, data not shown).



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  		Figure 1 Kinetics of anti-heat shock protein (Hsp) 70 antibody production. Rats immunized with Hsp70, bovine serum albumin (BSA) or control were bled before (t = 0), 10 days after primary immunization (t = 1), at the time of injury (t = 2) or at sacrifice (t = 3). Anti-Hsp70 antibody levels were determined by enzyme-linked immunosorbent assay. Values represent mean ± SEM of OD from all animals in each group. Upward-pointing solid triangle = BSA; downward-pointing solid triangle = control; solid circle = Hsp70.

 
Detection of expressed Hsp70 in rat arteries by Western blot.   By employing a polyclonal mouse anti-Hsp70 antibody, we observed that constitutive Hsp70 expression was evident in relatively low levels in normal arteries, as has been shown by others (29). Three days after injury, Hsp70 was more abundantly expressed in the injured arteries as compared with the noninjured arteries (Fig. 2). Interestingly, injured arteries obtained seven days and 14 days after balloon injury still maintained higher expression levels of Hsp70 in comparison with the nonlesioned arteries from the same animals.



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  		Figure 2 Time dependence of heat shock protein (Hsp) 70 expression in injured rat carotid arteries. Injured or noninjured carotid arteries were harvested from animals at different time points before and after balloon injury. Heat shock protein 70 expression was determined by Western blot combined with enhanced chemiluminescence, employing polyclonal mouse anti-Hsp70 (A). Representative samples taken 3 and 14 days after arterial injury are shown. Densitometric analysis of reactivity with Hsp70 of injured arteries (average of three vessel per bar) from panel A (B).

 
Detection of Hsp70 by immunohistochemistry.   By using a high affinity antibody against Hsp70, we observed that noninjured arteries displayed mild Hsp70 expression that was principally distributed within the SMC in the medial regions (Fig. 3). However, not all normal carotid arteries appeared to express this protein. In the balloon-injured arteries, Hsp70 was abundantly present at the subendothelial regions and within SMC in the media.



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  		Figure 3 Immunohistochemical detection of heat shock protein (Hsp) 70 in rat carotid arteries. Frozen sections of injured (A) and noninjured (B) carotid arteries were frozen and embedded in OCT. Immunohistochemical detection of expressed Hsp70 was carried out employing mouse anti-Hsp70 polyclonal antibody as a primary antibody. The arrow indicates neointima-media border.

 
The extent of intimal thickening in rat arteries.   The immunization regimen did not influence animal weight, which was similar in all groups (280 ± 25 g for the Hsp70 group, 286 ± 24 g for the BSA group and 277 ± 28 g for the control group). Intimal area in the rats immunized against Hsp70 was considerably larger (mean ± SD: 0.18 ± 0.03 mm2) in comparison with the BSA (0.081 ± 0.025 mm2) control-immunized rats (0.075 ± 0.032 mm2; p < 0.001) (Figs. 4A and 5). Medial area did not differ significantly between the Hsp70-immunized, BSA and control-immunized rats (Fig. 4B). The intimal/medial ratio was also considerably higher in the animals immunized with Hsp70 (2.2 ± 0.33) as compared with their BSA (1.0 ± 0.12; p < 0.001) control-immunized littermates (1.05 ± 0.14; p < 0.001).



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  		Figure 4 Intimal thickening in rats immunized with heat shock protein (Hsp) 70. Rats were immunized and boosted with Hsp70, bovine serum albumin (BSA) or phosphate-buffered saline (PBS) (control). Two weeks after boost, balloon injury was applied to carotid arteries of both groups. Morphometric evaluation of intimal (A) and medial (B) area were performed using PhotoShop software. The ratio of intima to medial area was also calculated (C). Values represent mean ± SD of all animals in each group. *p = 0.001 as compared with control.

 


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  		Figure 5 Representative injured carotid arteries immunized with heat shock protein (Hsp) 70 or control. Representative hematoxylin & eosin staining of carotid arteries from Hsp70-immunized rats (A) as compared with control-injected rats (B) two weeks after balloon injury. Original magnification x25.

 
IgG deposition in rat carotid arteries.   Elution of IgG from rat arteries revealed that anti-Hsp70 IgG was present in the arteries from both injured and noninjured rats. This finding is not surprising, as constitutive Hsp70 is known to exist and has also been recently described by other authors (30). However, binding of eluted IgG anti-Hsp70 antibodies from injured carotid arteries (mean optical density ± SEM: 0.98 ± 0.066) was approximately twice as high as those eluted from normal arteries of Hsp70-immunized rats (0.445 ± 0.05; p < 0.0001) (Fig. 6). Immunoglobulin G antibodies recovered from Hsp70-immunized arteries did not appear to react with other irrelevant proteins such as BSA (OD: 0.016 for the injured rats, Hsp70 immunized rats and an OD of 0.01 for the noninjured immunized rats). No Hsp70 reactivity was evident in the small IgG amounts recovered from IFA-injected rats after carotid injury (data not shown).



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  		Figure 6 Heat shock protein (Hsp) 70 reactivity of immunoglobulin G fractions eluted from injured and noninjured carotid arteries of Hsp70 immunized rats. Immunoglobulin G eluted from carotid arteries of injured (striped bar) and noninjured (black bar) Hsp70-immunized animals were characterized by assessing their binding to solid phase coated Hsp70 or an irrelevant antigen (bovine serum albumin). Values represent mean ± SEM of three tissue samples in each group.

 
SMC proliferation assays.   No differences were evident with carotid artery SMC from all three groups with regard to their basal proliferation evident by thymidine uptake. Incubation of SMC with IgG Hsp70 (100 µg/ml) resulted in an increase of 58 ± 12% in proliferation in comparison with incubation with control IgG. No effect of SMC thymidine incorporation was evident by incubation with 10 µg/ml of anti-Hsp70 antibodies.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The following study tested the hypothesis that the elicitation of an immune response to Hsp70 would influence neointimal formation in the rat carotid artery. We have found that rats initially immunized with recombinant Hsp70 developed a brisk and sustained immune response towards the antigen. The strong reaction was evident by the production of IgG anti-Hsp70 antibodies starting from day 8 after the primary immunization. When compared with controls, injured carotid arteries from Hsp70-immunized rats exhibited a marked increase of neointimal area and a more pronounced increase in the intimal/medial ratio.

Infections, Hsp and restenosis.   Evidence has recently been put forward to incriminate infectious agents in the initiation and progression of atherosclerosis (5–7). As such, seroepidemiologic studies have pointed toward Herpes viruses and Chlamydia pneumoniae as candidate pathogens. Parallel animal and in vitro studies strengthened the notion that infectious agents can alter the properties of the arterial wall favoring increased lesion formation (5). In this context, it is worthwhile to investigate the role of Hsp in influencing the response to injury in the arterial wall. In a series of studies, it has been shown that experimental induction of arteriosclerosis could follow immunization with Hsp65 in rabbits (22–24). Lesions from rabbits have been shown to express mammalian Hsp60 and to contain rich infiltrates of T lymphocytes (23). These data were confirmed in mice fed a high fat diet and immunized against Hsp65 (25).

In humans, an association was noticed between anti-Hsp65 antibodies and sonographically confirmed atherosclerotic plaques (24). These results were substantiated by Birnie et al. (30) showing that anti-Hsp 65 titers correlated with the severity and extent of coronary atherosclerosis. Very recently, Zhu et al. (31) demonstrated that a significant association existed between levels of antihuman Hsp60 antibody levels and the presence and severity of coronary artery disease. These studies reinforce the association between humoral immunity to infectious agent-related Hsp65 (cross-reactive with human Hsp60) and carotid atherosclerosis.

Hsp expression in restenotic carotid arteries.   The rationale for favoring an immunization with Hsp70 over Hsp60 is that it is the preferential Hsp induced during mechanical stress (32). Indeed, we have found that Hsp70 was significantly expressed in the injured tissue as compared with the nonmanipulated arteries as evidenced by the Western blot studies and by immunohistochemistry. Interestingly, Hsp70 was expressed in the injured arteries 3, 7 and 14 days after balloon inflation in comparison with the normal vessels. We assume that the mechanical injury precipitated by the balloon injury elicited an ongoing low-grade inflammatory response with consequent local production of cytokine/chemokines and other mediators that may serve to constantly stress the SMCs and the leukocytes, thereby maintaining Hsp70 expression.

Immunity to Hsp70 and the association with intimal thickening.   We have also observed in this study that injured arteries of rats immunized with Hsp70 contained significantly larger Hsp70-reactive IgG in comparison with nonmanipulated arteries (from similarly immunized rats). Thus, a conceivable explanation for the larger deposition of Hsp70 IgG could be that injured tissues produced and expressed higher levels of endogenous Hsp70 that attracted circulating anti-Hsp70 antibodies. These antibodies may have functional effects on the endothelial cells as has been shown for anti-Hsp65 antibodies (33).

An additional possible mechanism for the acceleration of neointimal formation in the cellular immune response.   As Hsp70 is a powerful immunogen, it is possible that anti-Hsp70 T-cells were generated that localized to the areas of preferential Hsp70 expression (i.e., the arterial injury domains) where ligation of their receptor could have triggered a local production of cytokines that could promote smooth muscle migration and leukocyte chemoattraction.

Conclusions and possible implications of the study.   In conclusion, we have observed that immunization of rats with a recombinant Hsp70 led to a pronounced increase in neointimal formation in a balloon-injury model. By analogy, we assume that pathogens (harboring Hsp70) can elicit, upon infecting the host, an anti-Hsp70 immune response. When arterial injury is induced, self-Hsp70 is upregulated that eventually serves to direct trafficking of either anti-Hsp70 antibodies or T-cells to the lesioned area. The interaction of self-overexpressed Hsp70 with the cross-reactive anti-Hsp70 IgG or lymphocytes may act to facilitate events that result in SMC migration and enhanced intimal thickening. Additionally, IgG anti-Hsp70 antibodies may also influence SMC proliferation as shown in the cell culture assay, and this property may also explain a mechanism by which these antibodies could increase neointimal hyperplasia.

Our findings suggest that anti-Hsp70 antibody levels may prove as a marker for increased risk of restenosis in patients undergoing percutaneous transluminal coronary angioplasty. Studies to address this assumption are ongoing. Furthermore, generation of an anti-Hsp70 immune response may serve to explain a mechanism by which infectious pathogens alter the response to injury in the arterial wall.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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