Manuel Mayr

Infections, Immunity, and Atherosclerosis Associations of Antibodies to Chlamydia pneumoniae, Helicobacter pylori, and Cytomegalovirus With Immune Reactions to Heat-Shock Protein 60 and Carotid or Femoral Atherosclerosis

BACKGROUNDnAtherogenesis involves inflammatory processes in which infections are incriminated as possible contributors.nnnMETHODS AND RESULTSnWe evaluated cardiovascular risk factors as well as seropositivity to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus in a population-based study. A significant association between prevalence and severity of atherosclerosis in carotid and femoral arteries and IgA antibodies to C pneumoniae was demonstrated that was not substantially altered after adjustment for established risk factors. For anti-H pylori IgG antibodies, significant correlations to vascular disease were restricted to low social status and lesions in carotid arteries. In addition, the study design allowed us to monitor lesion progression over time. In this prospective analysis, C pneumoniae seropositivity emerged as a significant risk predictor. Antibody titers against cytomegalovirus were not a marker for prevalence or incidence of atherosclerosis in this population. Further infection parameters added to the predictive value of chlamydial serology in risk assessment: Mean odds ratios for the prevalence of carotid atherosclerosis were 4.2 and 6.3 for seropositive subjects with elevated C-reactive protein levels and clinical evidence for chronic respiratory infection, respectively. For subjects with all 3 infection parameters, the odds ratio of carotid atherosclerosis reached 10.3 (P<0.0001). Concomitantly, serum antibodies to mycobacterial heat-shock protein 65 (mHSP65) correlated with seropositivity to C pneumoniae and H pylori but not to cytomegalovirus.nnnCONCLUSIONSnThis prospective population-based study provides strong evidence for a potential atherogenic role of persistent bacterial infection, especially C pneumoniae, as indicated by serological and clinical data and demonstrates a correlation between immune reactions to mHSP65 and bacterial infections in atherogenesis.

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

BACKGROUNDnPrevious 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.nnnMETHODS AND RESULTSnA 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).nnnCONCLUSIONSnThese findings indicate a sustained existence of anti-hsp65 antibodies in subjects with severe atherosclerosis, which is predictive for mortality.

Cyclic Strain Stress-induced Mitogen-activated Protein Kinase (MAPK) Phosphatase 1 Expression in Vascular Smooth Muscle Cells Is Regulated by Ras/Rac-MAPK Pathways

Recently, we demonstrated that mechanical stress results in rapid phosphorylation or activation of platelet-derived growth factor receptors in vascular smooth muscle cells (VSMCs) followed by activation of mitogen-activated protein kinases (MAPKs) and AP-1 transcription factors (Hu, Y., Bock, G., Wick, G., and Xu, Q. (1998) FASEB J. 12, 1135–1142). Herein, we provide evidence that VSMC responses to mechanical stress also include induction of MAPK phosphatase-1 (MKP-1), which may serve as a negative regulator of MAPK signaling pathways. When rat VSMCs cultivated on a flexible membrane were subjected to cyclic strain stress (60 cycles/min, 5–30% elongation), induction of MKP-1 proteins and mRNA was observed in time- and strength-dependent manners. Concomitantly, mechanical forces evoked rapid and transient activation of all three members of MAPKs, i.e.extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal protein kinases (JNKs), or stress-activated protein kinases (SAPKs), and p38 MAPKs. Suramin, a growth factor receptor antagonist, completely abolished ERK activation, significantly blocked MKP-1 expression, but not JNK/SAPK and p38 MAPK activation, in response to mechanical stress. Interestingly, VSMC lines stably expressing dominant negative Ras (Ras N17) or Rac (Rac N17) exhibited a marked decrease in MKP-1 expression; the inhibition of ERK kinases (MEK1/2) by PD 98059 or of p38 MAPKs by SB 202190 resulted in a down-regulation of MKP-1 induction. Furthermore, overexpressing MKP-1 in VSMCs led to the dephosphorylation and inactivation of ERKs, JNKs/SAPKs, and p38 MAPKs and inhibition of DNA synthesis. Taken together, our findings demonstrate that mechanical stress induces MKP-1 expression regulated by two signal pathways, including growth factor receptor-Ras-ERK and Rac-JNK/SAPK or p38 MAPK, and that MKP-1 inhibits VSMC proliferation via MAPK inactivation. These results suggest that MKP-1 plays a crucial role in mechanical stress-stimulated signaling leading to VSMC growth and differentiation.

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.

Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases

The present study was designed to investigate whether apoptosis occurs in early‐stage vein grafts and to determine the mechanisms by which mechanical stress contributes to apoptosis in vascular smooth muscle cells (SMCs). Apoptosis in vessel walls of mouse vein grafts was confirmed by morphological changes and by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL). TUNEL+ cells in vein grafts 1, 4, and 8 wk postoperatively was 13%, 29%, and 21%, respectively, and apoptosis occurred mainly in veins grafted to arteries, remaining unchanged in vein‐to‐vein grafts. When mouse, rat, and human arterial SMCs were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐ and strength‐dependent manner. All three types of SMCs showed apoptotic death as confirmed by TUNEL, propidium iodide, and annexin V staining. To further study the signal pathways leading to apoptosis, activities of p38, a subfamily of mitogen‐activated protein kinases (MAPKs), were determined. Mechanical stress resulted in p38 MAPK activation, reaching high levels within 8 min. SB 202190, a specific inhibitor for p38 MAPKs, prevented SMC apoptosis in response to mechanical stress. SMC lines stably transfected with a dominant negative rac, an upstream signal transducer, or overexpressing MAPK phosphatase‐1, a negative regulator for MAPKs, completely inhibited mechanical stress stimulated p38 activation and abolished mechanical stress‐induced apoptosis. Thus, we provide solid evidence that one of the earliest events in venous bypass grafts is apoptosis, in which mechanical stress‐induced p38‐MAPK activation is responsible for transducing signals leading to apoptosis.—Mayr, M., Li, C., Zou, Y., Huemer, U., Hu, Y., Xu, Q. Biomechanical stress‐induced apoptosis in vein grafts involves p38 mitogen‐activated protein kinases. FASEB J. 15, 261–270 (2000)

Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells

Recently, we demonstrated that biomechanical stress induces apoptosis of vascular smooth muscle cells (SMCs) (Mayr et al., FASEB J. 2000; 15:261.270). In this article we investigated the molecular mechanisms of mechanical stress‐induced apoptosis. When SMCs were subjected to cyclic strain, tumor‐suppressor p53 was activated as evidenced by gel mobility shift assays and Western blot analyses. p53 activation was largely attenuated if SMCs were pretreated with SB202190, a specific p38MAPK inhibitor, or were stably transfected with dominant negative rac, an upstream signal transducer of p38MAPK pathways. Kinase assays provided direct evidence that p38MAPKs phosphorylated p53 within 30 min of cyclic strain. Additionally, mechanical stress resulted in oxidative DNA damage as detected by the presence of 8‐oxoguanine. Treatment with the antioxidant U‐74389G abrogated p53 activation. p53 activation was followed by expression and mitochondrial translocation of the proapoptotic protein Bax. Likewise, mechanical stress resulted in up‐regulation of anti‐apoptotic Bcl‐2 proteins, including Bcl‐2 and Bcl‐xL. However, a marked loss of mitochondrial membrane potential occurred in wild‐type, but not in p53−/−, SMCs. The latter lost their ability to express Bax and showed no apoptosis in response to cyclic strain. Taken together, our data provide the first evidence that SMC apoptosis induced by mechanical stress is p53‐dependent.

Increased Risk of Atherosclerosis Is Confined to CagA-Positive Helicobacter pylori Strains Prospective Results From the Bruneck Study

Background and Purpose— Accumulating evidence indicates that a variety of infections contribute to the pathogenesis of atherosclerosis, but there is controversy concerning the impact of Helicobacter pylori infections in atherosclerosis. Methods— We evaluated seropositivity to H pylori and to its cytotoxin-associated gene A (CagA) product in a large, prospective, population-based study (n=684). Intima-media thickness and atherosclerosis of carotid arteries were thoroughly assessed by high-resolution duplex scanning. Results— In our study population, H pylori infections defined by seropositivity have no relationship with levels of classic cardiovascular risk factors or markers of systemic inflammation, except for elevated levels of immune reactions to mycobacterial heat shock protein 65. The latter showed a trend toward highest levels in those harboring virulent H pylori strains (P =0.08). Common carotid artery intima-media thickness—both absolute values and changes between 1995 and 2000—were significantly enhanced in subjects seropositive to CagA but not in those infected with CagA-negative H pylori strains. There was a clear dose-response relation between anti-CagA antibodies and both intima-media thickness and atherosclerosis risk. Notably, the risk of atherosclerosis associated with CagA seropositivity was amplified by elevated C-reactive protein levels. Conclusions— Infections with virulent CagA-bearing H pylori strains may contribute to the pathogenesis of early atherosclerosis by aggravating immune-inflammatory reactions.

Smooth muscle cell apoptosis in arteriosclerosis

Arteriosclerosis, a paradigmatic age-related disease, encompasses (spontaneous) atherosclerosis, restenosis after percutaneous transluminal coronary angioplasty, autologous arterial or vein graft arteriosclerosis and transplant arteriosclerosis. In all types of arteriosclerosis, vascular smooth muscle cell (SMC) accumulation in the intima is a key event, but abundant evidence also indicates the importance of SMC apoptosis in the development of arteriosclerosis. Because SMC proliferation and apoptosis coincide in arteriosclerotic lesions, the balance between these two processes could be a determinant during vessel remodeling and disease development. Various stimuli, including oxidized lipoproteins, altered hemodynamic stress and free radicals, can induce SMC apoptosis in vitro. As risk factors for arteriosclerosis, these stimuli may also lead to vascular cell apoptosis in vivo. The presence of apoptotic cells in atherosclerotic and restenotic lesions could have potential clinical implications for atherogenesis and contributes to the instability of the lesion. Based on the progress in this field, this review focuses on the mechanism and impact of SMC apoptosis in the pathogenesis of arteriosclerosis and highlights the role of biomechanical stress in SMC apoptosis.

Rapid Development of Vein Graft Atheroma in ApoE-Deficient Mice

Several animal models manifesting lesions resembling neointimal hyperplasia of human vein grafts have been developed, but no spontaneous atheromatous lesions in their vein grafts have been observed. We developed and here characterize a new animal model of vein graft atheroma, a maturated atherosclerotic plaque, in apoE-deficient mice. The lesion displayed classical complex morphological features and heterogeneous cellular compositions and consisted of a fibrous cap, infiltrated mononuclear cells, foam cells, cholesterol crystal structure, necrotic core with calcification, and neovasculature. Cell component analysis revealed smooth muscle cells (SMCs) localized in the cap region, macrophages which made up a large portion of the lesions, and CD4+ T cells scattered under the cap. Importantly, apoptotic/necrotic cells determined by TUNEL assay in vein grafts into apoE−/− mice were significantly higher than wild-type mice, although a similar number of proliferating cell nuclear antigen-positive cells in both types of lesions was found. Interestingly, vascular SMCs cultivated from aortas of apoE-deficient mice showed a high rate of spontaneous apoptosis/necrosis and a higher rate of cell death stimulated by a nitric oxide donor, sodium nitroprusside, H 2 O 2 , and oxidized low density lipoprotein (LDL), although no difference in proliferation of both SMCs incubated with platelet-derived growth factor, angiotensin II, LDL, and oxidized LDL was seen. Thus, the pathogenic mechanisms of vein graft atheroma involve increased intimal cell death initiated by biomechanical stress and amplified by hypercholesterolemia, which leads to continuous recruitment of blood mononuclear cells to constitute atheromatous lesions. This mouse model resembling human vein graft disease has many advantages over other animal models.

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.