Bernhard Metzler

Association of Serum Antibodies to Heat-Shock Protein 65 With Carotid Atherosclerosis Clinical Significance Determined in a Follow-Up Study

BACKGROUND Previous work has proved that increased titers of antibodies against heat-shock protein (hsp) 65 are associated with atherosclerotic lesions independently of other established risk factors. The present follow-up study was designed to further scrutinize the association of hsp antibodies and atherosclerosis and evaluate the possible predictive value of these antibodies for the development and/or progression of lesions in the same population. METHODS AND RESULTS A total of 750 subjects 45 to 74 years old were recruited, and the rate of participation was 93.6%; 58 subjects died between 1990 and 1995. All participants were subjected to determination of serum antibodies against hsp65 and sonography to assess carotid atherosclerotic lesions and evaluate other risk factors, ie, age, sex, body mass index, blood cholesterol, apolipoprotein B, apolipoprotein A, triglycerides, lipoprotein(a), fibrinogen, leukocyte number, antithrombin III, ESR, ferritin, hypertension, smoking, and diabetes mellitus. Our data show that hsp65 antibody titers in the population emerged as highly consistent over a 5-year observation period (r=0.78, P<0.0001). Titers were significantly elevated in subjects with progressive carotid atherosclerosis and correlated with intima/media thickness. Multiple linear regression analysis documented these associations to be independent of age, sex, and other risk factors. Subanalyses revealed a preferential association of hsp65 antibody titers with advanced lesions (odds ratio, 1.42; 95% CI, 1.02 to 1.98; P=0.039). Other risk factors neither confounded nor modified this association. Finally, hsp65 antibody titers significantly predicted the 5-year mortality (hazard ratio, 1.52; 95% CI, 1.14 to 2.03; P<0.001). CONCLUSIONS These findings indicate a sustained existence of anti-hsp65 antibodies in subjects with severe atherosclerosis, which is predictive for mortality.

Mouse Model of Venous Bypass Graft Arteriosclerosis

Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18+) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (alpha-actin+) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases.

Atherosclerosis, autoimmunity, and vascular-associated lymphoid tissue.

Atherosclerosis is a multifactorial disease induced by the effects of various risk factors on appropriate genetic backgrounds. It is characterized by vascular areas containing mononuclear and proliferating smooth muscle cells,as well as extracellularmatrix (ECM)2 components resulting in hardening and thickening (arteriosclerosis) of the arterial wall. In a strict sense, atherosclerotic lesions are localized in the inlima; they also contain foam cells and deposits of cholesterolcrystalsmanifested as fatty streaks and, finally, as atheroscleroticplaques. Fatty streaks are whitish, cushion-like lesions of the arterial intima harboring abundant lipid-laden macrophages, so-called foam cells, which are considered precursors of the fully developed plaques that may finally exulcerate and even calcif’. The main theories of atherogenesis are the “response to injury” (1) and the “response to altered lipoprotein” (2) hypotheses. The response to injury hypothesis postulates an alteration of the intima by various risk factors (mechanical injury, chemically altered low density lipoproteins [LDL], viruses, toxins) that initiates a primary endothelial dysfunction and subsequent changes in permeability, expression of adhesion molecules, and release of chemotactic and growth factors. Consequently, platelets and monocytes become activated and attach to these endothelial cells. Bloodderived monocytes transmigrate into the subendothelial space and transform into macrophages; smooth muscle cells (SMC) are attracted from the media to the same site. Both monocytes/macrophages and SMC possess the so-called scavenger receptor, which binds chemically altered (oxidized LDL {oxLDL]), but not native, LDL in a nonsaturable fashion. By uptake of oxLDL, macrophages and SMC develop into foam cells, and the deposition of collagenous and noncollagenous ECM components, especially in the peripheral “shoulder” region and the superficial “cap” area, complete the pathohistological appearance of fatty streaks and atherosclerotic plaques, respectively. The response toaltered lipoprotein hypothesis is based on the concept that lipoproteins can be chemically modified and are then able to induce foam cell formation by monocytes/macrophages and SMC. In recent years it has become evident that modification of lipoproteins does not occur primarily in the circulation or during transgression through the endothelium, but they rather accumulate in native form in the subendothelial space, where the lipoproteins are retained and oxidized. Accumulation of oxLDL, therefore, is not only the result of increased influx from the serum into the arterial intima but, conversely, is also due to a diminished efflux with subsequent foam cell formation. This “response to LDL retention” hypothesis (3) is thus a special variant of the response to altered LDL concept. Thus far, these latter theories have not explained why atherosclerotic lesions develop at certain arterial predilection sites or why the disease does not affect the venous vascular bed.

Epitope Specificity of Anti–Heat Shock Protein 65/60 Serum Antibodies in Atherosclerosis

Levels of specific antibodies (Ab) against mycobacterial and human heat shock protein (hsp) 65/60 are increased in the sera of patients with atherosclerotic lesions and have been demonstrated to be capable of mediating endothelial cytotoxicity. To clarify the antigen epitopes recognized by these serum Abs, Ab binding to hsp65 deletion mutants (Dms), as well as to overlapping 15-mer and 8-mer hsp65 peptides, was assessed. Western blotting of hsp65 Dms indicated the presence of at least one epitope between amino acid (aa) residues 171 and 276, recognized by both high-titer sera and affinity-purified anti-hsp65/60 Ab. Fluorescence immunoassays using 53 15-mer peptides and Pin ELISA using 526 7-mer peptides demonstrated three distinct, conserved sequences with high affinity to high-titer sera and purified anti-hsp65/60 Ab. Two N-terminal sequences, aa 97-109 and aa 179-187, and one C-terminal sequence, aa 504-512, were identified. These three epitopes recognized by anti-hsp65/60 Ab may serve as autoantigens in certain circumstances in vivo. This phenomenon could contribute to the initiation of atherosclerosis by an autoimmune reaction.

Myocardial injury leads to a release of heat shock protein (hsp) 60 and a suppression of the anti-hsp65 immune response

OBJECTIVE While atherosclerosis is associated with high titers of autoantibodies to bacterial hsp65 crossreacting with human hsp60 (anti-hsp60 autoantibodies), myocardial infarction entails decreased humoral immune response to hsp65. We previously hypothesized that myocardial ischemia and subsequent infarction not only induce myocardial hsp60 expression, but also trigger release of myocardial hsp60 into the circulation, influencing the systemic hsp immune response via immune complex formation. METHODS In the present study, organ culture of rat hearts under circulatory arrest provided a model of myocardiocyte injury due to ischemia. RESULTS Reperfusion of ischemic hearts confirmed the occurrence of myocardial injury by a rise of heart enzymes. Myocardial hsp60 expression was induced up to threefold in response to ischemia, and most of hsp60 expression was localized to the muscle fibers. Analysis of coronary eluate revealed release of hsp60 from myocardium. In addition, hsp60-containing, but not hsp60-free, coronary eluate was recognized by anti-hsp65 serum antibodies and induced proliferation of hsp65-specific T cells. When hsp60-containing coronary eluate was reinjected into an hsp65-primed rat, both humoral and cellular hsp65-immune responses were strongly downregulated. CONCLUSION Our findings demonstrate the release of highly immunogenic and crossreactive hsp60 into the circulation in response to myocardial ischemia and myocardiocyte injury.

Hyperexpression and Activation of Extracellular Signal–Regulated Kinases (ERK1/2) in Atherosclerotic Lesions of Cholesterol-Fed Rabbits

A hallmark of hyperlipidemia-induced atherosclerosis is altered gene expression that initiates cell proliferation and (de)differentiation in the intima of the arterial wall. The molecular signaling that mediates this process in vivo has yet to be identified. Extracellular signal-regulated kinases (ERKs) are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate the activity, abundance, and localization of ERK1/2 in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of ERK1/2, mainly localized in the cap and basal regions of atheromas. A population of ERK-enriched cells was identified as alpha-actin-positive smooth muscle cells (SMCs). ERK1 and 2 were heavily phosphorylated on tyrosyl residues and coexpressed with proliferating cell nuclear antigen in atherosclerotic lesions. ERK1/2 protein levels in protein extracts from atherosclerotic lesions were 2- to 3-fold higher than the vessels of chow-fed rabbits, and their activities were elevated 3- to 5-fold over those of the normal vessel. SMCs derived from atherosclerotic lesions had increased migratory/proliferative ability and higher ERK activity in response to LDL stimulation compared with cells from the normal vessel. Inhibition of ERK activation by PD98059, a specific inhibitor of mitogen-activated protein kinase kinases (MEK1/2), abrogated LDL-induced SMC proliferation in vitro. Taken together, our findings support the proposition that persistent activation and hyperexpression of ERK1/2 may be a critical element to initiate and perpetuate cell proliferation during the development of atherosclerosis.

Macrophage-lysis mediated by autoantibodies to heat shock protein 65/60.

Macrophages in atherosclerotic lesions have been shown to express high amounts of heat shock protein 60 (hsp60), a highly conserved protein. Patients with atherosclerosis have high titers of anti-hsp65/60 antibodies (Ab) recognizing macrophages in the lesions. To elucidate the role of anti-hsp65/60 Ab in macrophage cytotoxicity, human high titer serum and purified anti-hsp65/60 Ab were tested on in vitro heat-stressed cells of a human macrophage cell line (U937) and macrophages derived from peripheral blood. Application of heat stress at 42 degrees C for 30 min resulted in marked upregulation of hsp60 mRNA, followed by increased protein expression as determined by Northern blot and FACS-analysis, respectively. Compared to unstressed cells, high titer serum and anti-hsp65/60 Ab preferentially bound to the surface of stressed U937 macrophages, but not control antibodies. Furthermore, high titer serum and anti-hsp65/60 Ab exerted significant (P < 0.01) complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) on stressed 51Cr-labelled U937 and peripheral blood derived macrophages. Thus, macrophages expressing hsp60 can be lysed by autoantibodies against hsp65/60, which may contribute to cell death in atherosclerotic plaques in vivo.

Salivary Anti-hsp65 Antibodies as a Diagnostic Marker for Gingivitis and a Possible Link to Atherosclerosis

Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.

LDL Stimulates Mitogen-Activated Protein Kinase Phosphatase-1 Expression, Independent of LDL Receptors, in Vascular Smooth Muscle Cells

Low density lipoprotein (LDL) is a well-established risk factor for atherosclerosis, stimulating vascular smooth muscle cell (SMC) differentiation and proliferation, but the signal transduction pathways between LDL stimulation and cell proliferation are poorly understood. Because mitogen-activated protein kinases (MAPKs) play a crucial role in mediating cell growth, we studied the effect of LDL on the induction of MAPK phosphatase-1 (MKP-1) in human SMCs and found that LDL stimulated induction of MKP-1 mRNA and proteins in a time- and dose-dependent manner. Heparin, inhibiting LDL-receptor binding, did not influence LDL-stimulated MKP-1 mRNA expression, and human LDL also induced MKP-1 expression in rat SMCs and fibroblasts derived from LDL receptor-deficient mice, indicating an LDL receptor-independent process. Pretreatment of SMCs with pertussis toxin markedly inhibited LDL-induced MKP-1 expression. Depletion of protein kinase C (PKC) by phorbol 12-myristate 13 acetate or inhibition of PKC by calphostin C blocked MKP-1 induction, but the phospholipase C inhibitor U73122 had no effect. Pretreatment of SMCs with genistein or herbimycin A abrogated LDL-stimulated MKP-1 induction. The MAPK kinase inhibitor PD98059 abolished LDL-stimulated activation of extracellular signal-regulated protein kinases (ERKs) but not MKP-1 induction. Furthermore, constitutive expression of MKP-1 in vivo reduced LDL-induced expression of Elk-1-dependent reporter genes, and SMC lines overexpressing recombinant MKP-1 exhibited decreased ERK activities and retarded proliferation in response to LDL. Our findings demonstrate that LDL induces MKP-1 expression in SMCs via activation of PKC and tyrosine kinases, independent of LDL receptors and ERK-MAPKs, and that MKP-1 plays an important role in the regulation of LDL-initiated signal transductions leading to SMC proliferation.

The Role of Mast Cells in Atherosclerosis

Mast cells are characterized by numerous granules released extracellularly in response to stimuli, e.g. IgE and complement. These cells are believed to be crucial in the development of certain inflammatory or immune-mediated diseases, such as allergy and dermatitis, and it has recently been demonstrated that a large number of mast cells are present in atherosclerotic lesions. Atherosclerosis bears several similarities to chronic inflammation, characterized by T cell and monocyte infiltration, immunoglobulin-complement deposition, and lipid accumulation. The presence of mast cells in atherosclerotic lesions could be significant because they can release large amounts of chemotactic agents, inflammation activators, and granule remnants, and they may be responsible for mononuclear cell recruitment and smooth muscle cell proliferation. Furthermore, granule remnants nonspecifically bind to low-density lipoproteins, which can be phagocytosed by macrophages to form foam cells, a major cellular component of the early stage of atherosclerotic lesions. Thus, further elucidation of the role of mast cells in quantitative studies could enhance our understanding of the mechanism of atherogenesis, and may lead to new therapeutic strategies for atherosclerosis.