Qingbo Xu

Heat-Shock Protein 60-Reactive CD4+CD28null T Cells in Patients With Acute Coronary Syndromes

Background—CD4+CD28null T cells are present in increased numbers in the peripheral blood of patients with acute coronary syndrome (ACS) compared with patients with chronic stable angina (CSA). The triggers of activation and expansion of these cells to date remain unclear. Methods and Results—Twenty-one patients with ACS and 12 CSA patients with angiographically confirmed coronary artery disease and 9 healthy volunteers were investigated. Peripheral blood leukocytes were stimulated with human cytomegalovirus (HCMV), Chlamydia pneumoniae, human heat-shock protein 60 (hHSP60), or oxidized LDL (ox-LDL). CD4+CD28null cells were separated by flow cytometry and assessed for antigen recognition using upregulation of interferon-&ggr; and perforin mRNA transcription as criteria for activation. CD4+CD28null cells from 12 of 21 patients with ACS reacted with hHSP60. No response was detected to HCMV, C pneumoniae, or ox-LDL. Incubation of the cells with anti-MHC class II and anti-CD4 antibodies but not anti-class I antibodies blocked antigen presentation, confirming recognition of the hHSP60 to be via the MHC class II pathway. Patients with CSA had low numbers of CD4+CD28null cells. These cells were nonreactive to any of the antigens used. Circulating CD4+CD28null cells were present in 5 of the 9 healthy controls. None reacted with hHSP60. Conclusions—We have shown that hHSP60 is an antigen recognized by CD4+CD28null T cells of ACS patients. Endothelial cells express hHSP60 either constitutively or under stress conditions. Circulating hHSP60-specific CD4+CD28null cells may, along other inflammatory mechanisms, contribute to vascular damage in these patients.

Autoimmunity to heat shock proteins in atherosclerosis

Current evidence lends increasing support to immunoinflammatory mechanisms as one of the prime pathogenic processes involved in the development and progression of atherosclerosis. It has been observed that most human beings have cellular and humoral reactions against microbial heat shock protein (HSP). Autoantibody levels against HSPs are significantly increased in patients with atherosclerosis and T lymphocytes specifically responding to HSPs have been demonstrated within atherosclerotic plaques. Most of the known risk factors for atherosclerosis, viz. oxidized low-density lipoprotein, hypertension, infections and oxidative stress, evoke increased expression of HSPs in endothelial cells, smooth muscle cells and macrophages, the main cellular constituents of atherosclerotic plaques. Evolutionary conservation has resulted in a high degree of sequence homology between microbial and human HSPs and hence the immune reactions against microbial HSPs carry a risk of being misdirected towards human HSPs expressed in the stressed cells of the blood vessels. HSPs and anti-HSP antibodies have been shown to elicit production of pro-inflammatory cytokines by macrophages and adhesion molecules by endothelial cells in various in vitro and animal model studies. These autoimmune reactions to HSPs expressed in the vascular tissue can contribute to both initiation and perpetuation of atherosclerosis.

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.

Progenitor cells in vascular disease.

Stem cell research has the potential to provide solutions to many chronic diseases via the field of regeneration therapy. In vascular biology, endothelial progenitor cells (EPCs) have been identified as contributing to angiogenesis and hence have therapeutic potential to revascularise ischaemic tissues. EPCs have also been shown to endothelialise vascular grafts and therefore may contribute to endothelial maintenance. EPC number has been shown to be reduced in patients with cardiovascular disease, leading to speculation that atherosclerosis may be caused by a consumptive loss of endothelial repair capacity. Animal experiments have shown that EPCs reendothelialise injured vessels and that this reduces neointimal formation, confirming that EPCs have an atheroprotective effect. Smooth muscle cell accumulation in the neointimal space is characteristic of many forms of atherosclerosis, however the source of these cells is now thought to be from smooth muscle progenitor cells (SMPCs) rather than the adjacent media. There is evidence for the presence of SMPCs in the adventitia of animals and that SMPCs circulate in human blood. There is also data to support SMPCs contributing to neointimal formation but their origin remains unknown. This article will review the roles of EPCs and SMPCs in the development of vascular disease by examining experimental data from in vitro studies, animal models of atherosclerosis and clinical studies.

Association of Anti-Heat Shock Protein 65 Antibodies With Development of Postoperative Atrial Fibrillation

Background—Atrial fibrillation (AF) is a frequently encountered arrhythmia after cardiac surgery, but its underlying mechanisms are still unclear. We hypothesize that autoimmune and inflammatory responses against heat shock protein 65 (HSP65) may be involved and hence examined the relationship between HSP65 autoantibodies and occurrence of postoperative AF. Methods and Results—A prospective study of 329 patients undergoing elective primary CABG was undertaken. Cardiovascular risk factors, ECG characteristics, medications, and intraoperative and postoperative features were documented. Anti-HSP65 antibodies and C-reactive protein levels were measured in all preoperative blood samples with ELISA. Postoperative AF was defined as the characteristic arrhythmia, lasting for at least 15 minutes and confirmed on 12-lead ECG and occurring within the first postoperative week. This occurred in 62 patients (19%). In univariate analysis, HSP65 antibodies were significantly higher in patients with postoperative AF (P=0.02). History of previous myocardial infarction, duration of bypass, number of distal anastomosis, and duration of ventilation were also associated with AF (P<0.05), but C-reactive protein levels were not (P=0.13). Multivariate analysis confirmed the positive association of HSP65 antibodies with postoperative AF (OR, 1.41; P=0.04) independent of age, sex, other cardiovascular risk factors, severity of coronary artery disease, duration of ventilation, duration of bypass, and left ventricular function. Conclusions—We report a novel association between anti-HSP65 antibodies and occurrence of postoperative AF, indicating a possible role for antibody-mediated immune response in its pathogenesis.

Autoimmune Mechanisms of Atherosclerosis

Accumulating evidence supports an autoimmune mechanism as one of the prime pathogenic processes involved in the development of atherosclerosis. So far, three proteins, including heat shock proteins (HSPs), oxidized low-density lipoprotein (oxLDL), and beta2 glycoprotein1 (beta2GP1) have been recognized as autoantigens. It has been demonstrated that risk factors for atherosclerosis, such as hypercholesterolemia, hypertension, infections, and oxidative stress, evoke increased expression of HSPs in cells of atherosclerotic lesions. Autoantibody levels against HSPs are significantly increased in patients with atherosclerosis and T lymphocytes specifically responding to these autoantigens have been demonstrated within atherosclerotic plaques. Subcutaneous immunization of animals with HSP65 induced atheroma formation in the arterial wall. Furthermore, circulating immunoglobulin (Ig) G and IgM oxidized low-density lipoprotein (oxLDL) antibodies are present in the plasma of animals and humans and form immune complexes with oxLDL in atherosclerotic lesions. These antibodies closely correlate with the progression and regression of atherosclerosis in murine models. Interestingly, recent reports demonstrated that pneumococcal vaccination to LDL receptor-deficient mice results in elevation of anti-oxLDL IgM Ab EO6, which is inversely correlated with the development of atherosclerosis. Finally, it has been observed that autoantigen beta2GP1 localizes in the atheroma and that autoantibodies to beta2GP1 are correlated with the incidence of atherosclerosis in patients. Hence, these autoimmune reactions to HSPs, oxLDL and beta2GP1 can contribute to the initiation and progression of atherosclerosis.

Association of serum soluble heat shock protein 60 with toll-like receptor 4 polymorphism and severity of coronary artery disease

Early atherosclerotic lesions are characterised by a relative abundance of inflammatory cells such as activated T lymphocytes, indicating involvement of immunoinflammatory process in the pathogenesis of atherosclerosis.1 One of the antigens stimulating recruitment of these T cells is believed to be heat shock protein (HSP). HSP60 (human) has been shown to enhance production of proinflammatory cytokines such as tumour necrosis factor α, interleukin 12, and interleukin 15 and to mediate monocyte adhesion to endothelial cells. In a large population based study, Xu et al 2 have shown a correlation between soluble HSP60 (sHSP60) and the severity of carotid atherosclerosis. The association of sHSP60 in patients with coronary artery disease (CAD), however, remains to be clarified.nnHuman HSP60 has been shown to require functional toll-like receptor 4 (TLR4), a receptor involved in innate immunity, for stimulating production of tumour necrosis factor α and macrophages. Recently, two polymorphisms in the TLR4 gene, Thr399Ile and Asp299Gly, have been associated with lower concentrations of proinflammatory cytokines and a reduced extent or progression of carotid atherosclerosis.3nnWe hypothesised that serum sHSP60 would be associated with CAD and interact with TLR4 variants, determining the severity of prevalent CAD. The present study was designed to assess the association of sHSP60 with TLR4 polymorphisms with the severity of CAD.nnA prospective study of 329 consecutive patients admitted for elective coronary artery bypass graft surgery under the care of one surgical unit between August 2002 and November 2003 was carried out. The median age of the cohort was 65 years (range 40–80 years) with a male to female ratio of 4:1. Exclusion criteria were a recent history of myocardial infarction; unstable angina; infection; associated valvar heart disease; a history of inflammatory disorders, such as systemic lupus erythematosus; and use of immunosuppresive drugs such as steroids. The presence …

Pitfalls of Proteomics

Lindholt et al1 used purified serum antibodies to C pneumoniae outer membrane protein (OMP) to probe protein extracts of abdominal aortic aneurysms separated by 1D and 2D electrophoresis. Although no specific signal for OMP was detected, strong staining was obtained for a 50-kDa protein, which subsequently was identified as immunoglobulin heavy chain through the use of mass spectrometry. The authors conclude that OMP antibodies cross-react with human immunoglobulins and that C pneumoniae might trigger an autoimmune reaction.nnWe agree that autoimmune reactions are a possible link between infections and atherosclerosis.2,3 However, additional controls are essential for supporting their conclusion, ie, a negative control probing the blots with horseradish peroxidase–conjugated secondary antibody only. It is well established that immunoglobulin deposits accumulate during progression of atherosclerosis.4 We frequently have observed strong positive staining for immunoglobulin chains in protein extracts of human vessels when the blot …

YIA1 Interaction between HDAC3 and XBP1 is critical in maintainence of endothelial integrity

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation through modulation of chromatin structure. The class I HDAC, HDAC3, is involved in maintaining endothelial cell integrity.1 Sustained activation of the x-box binding protein 1 (XBP1), an endoplasmic reticulum stress response transcription factor, results in the development of atherosclerosis in apoE−/− mice.2 HDAC3 and XBP1 are similarly expressed in the bifurcation regions of the aorta. In this study, we investigated whether cross-talk existed between HDAC3 and XBP1, and its role in the maintenance of endothelial cell integrity. Our study demonstrated that disturbed flow upregulated HDAC3 and unspliced XBP1 (XBP1u) protein production through the KDR/PI3K/Akt pathway. Knockdown of XBP1 by shRNA lentiviral transfection ablated disturbed flow-induced HDAC3 upregulation. Similarly to HDAC3, overexpression of XBP1u by adenoviral gene transfer increased Akt phosphorylation at Serine473 and haem oxygenase 1 gene transcription, which showed a protective role in hydrogen peroxide-induced apoptosis of endothelial cells. Co-immunoprecipitation assays demonstrated that HDAC3 physically associates with XBP1u and this could be enhanced by disturbed flow and VEGF treatment. The use of truncated HDAC3 constructs demonstrated that XBP1 binds to the central section of HDAC3. Further experiments indicated that overexpression of XBP1u increased the binding of HDAC3 to IRE1α, Akt and PI3K, especially after VEGF treatment. In contrast, sustained activation of spliced XBP1 decreased HDAC3 protein production through transcriptional suppression, leading to endothelial apoptosis. These results suggest that XBP1u protects endothelial cells from oxidative stress by interacting with HDAC3. Targeting this interaction may provide novel therapeutic strategies for vascular disease via maintaining endothelial integrity.

2HT02-2 Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells

BACKGROUNDnEndothelial regeneration and angiogenesis in the intima of the arterial wall are key events in the pathogenesis of transplantation arteriosclerosis. The traditional hypothesis that damaged endothelial cells are replaced by remaining cells of the donor vessel has been challenged by recent observations, but the cell origins of large arteries and microvessels are still not well established.nnnMETHODS AND RESULTSnAortic segments were allografted between Balb/c and TIE2-LacZ (C57BL/6) mice expressing beta-galactosidase (gal) in endothelial cells. beta-gal+ cells in TIE2-LacZ vessels grafted to Balb/c mice completely disappeared, whereas the positive cells found in Balb/c aorta allografted into TIE2-LacZ mice 4 weeks after surgery indicated a host origin. En face analysis of allograft vessels displayed a unique distribution of beta-gal+ cells on the surface at 3 days, 1 week, and 4 weeks. Interestingly, 35+/-19% beta-gal+ cells were found in arterial segments allografted into chimeric mice with TIE2-LacZ bone marrows. Furthermore, endothelial cells of microvessels within allografts had a beta-gal+ staining in the media at 1 week and in the neointimal lesions and adventitia at 4 weeks. Allograft studies in chimeric mice demonstrated that beta-gal+ cells of microvessels in transplant arteriosclerosis were derived from bone marrow progenitors.nnnCONCLUSIONSnWe provide strong evidence that endothelial cells of neointimal lesions in allografts are derived from circulating progenitor cells and that bone marrow-derived progenitors are responsible for angiogenesis of the allograft, that is, the formation of microvessels in transplant arteriosclerosis.