Granulocytes in coronary thrombus evolution after myocardial infarction--time-dependent changes in expression of matrix metalloproteinases.

BACKGROUND: Remodeling of extracellular matrix is a key process during wound healing, which is strictly regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors [tissue inhibitors of metalloproteinases (TIMPs)]. In this study, we evaluated intrathrombotic MMPs and TIMPs and their cellular origin during thrombus evolution after disruption of coronary atherosclerotic plaque. MATERIALS AND METHODS: Thrombectomy materials (N=120) obtained from patients with acute myocardial infarction were histologically classified in three groups based on thrombus age: fresh (<1day), lytic (1-5days), or organized (>5days) thrombi; materials showing a heterogeneous composition were classified according to oldest part. Presence and cellular origin of MMPs (MMP-1, MMP-2, MMP-8, MMP-9, and MMP-14) and TIMPs (TIMP-1, TIMP-2, and TIMP-3) was evaluated with immunostains (double) and with polymerase chain reaction. RESULTS AND CONCLUSION: MMPs and TIMPs were present in all the thrombectomy samples. A distinct temporal change in extent and cellular origin of MMPs and TIMPs during thrombus evolution was observed. In the early (fresh and lytic) stages of thrombus, high numbers of neutrophilic granulocytes occupy the thrombus mass and produce large amounts of MMPs and TIMPs. However, with progression of thrombus evolution (organizing stage) and diminishment of neutrophil granulocytes, there is disappearance of MMP-8 and MMP-9, steep decline of MMP-1 and TIMP-2, and progressive decrease of TIMP-3. In contrast, intrathrombotic MMP-2 and MMP-14 are present at a constant high level during the entire process of thrombus evolution. These temporal changes indicate a complex time-dependent function of MMPs, which are largely granulocyte derived, in the healing process of thrombus after plaque disruption.

Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction.

Neutrophils are important cellular sources of interleukin (IL) 17A and -F. Moreover, upon activation neutrophils are able to excrete chromatin embedded with components from their cytoplasmic granules to form 'neutrophil extracellular traps' (NETs). Recent studies suggested that NETs contribute to thrombosis by promoting fibrin deposition and platelet aggregation. IL17A may also promote thrombosis by enhancing platelet aggregation. In the present study we investigated the presence of neutrophils, NETs and IL17A and -F in coronary thrombosuction specimens obtained from patients after acute myocardial infarction. Neutrophils and NETs were identified using histochemical (HE, Feulgen procedure) and immunohistochemical stainings (Histone H1, myeloperoxidase, neutrophil elastase) in 15 fresh, 15 lytic and 15 organised thrombi. The presence and distribution of IL17A and -F was studied using (immuno)histochemical double staining and spectral image analysis, rtPCR and Western blot. High numbers of neutrophils are present (10-30% of the thrombus mass) in fresh and lytic, but not in organized thrombus. NETs were frequently observed in fresh (4/15) and lytic (12/15), but never in organised thrombus specimens. Double staining combining the Feulgen reaction with Histone-H1, MPO or neutrophil elastase confirmed colocalisation with DNA. Cytoplasmatic IL17A/F staining was found in the majority of the neutrophils, extracellularly and in NETs. Western blotting confirmed the presence of IL17A and IL17F in thrombus specimens. In conclusion, a large burden of neutrophils, neutrophil extracellular traps and IL17A and -F are important constituents of fresh and lytic thrombus after acute myocardial infarction. The specific colocalisation of these indicates a role during thrombus stabilisation and growth.

Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruch's membrane.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by mineralization of connective tissue, which leads to pathology in eye, skin and blood vessels. The disease is caused by mutations in ABCC6. To learn more about PXE eye pathology, we analyzed Bruch's membrane (BM) of the eye of an Abcc6 knockout mouse. With age, BM differences between Abcc6-/- and wild type mice became apparent. At two years of age, von Kossa staining indicated clear calcification of BM in Abcc6-/- mice, and not in healthy controls. Electron microscopy revealed BM changes as early as at 10 months of age: Fibrous structures with abnormal high electron-density were present in the central layers of BM of Abcc6-/- mice. EDX (Energy Dispersive X-ray) analysis demonstrated that these structures contained elevated levels of Ca, P and O. Since some of these electron-dense structures showed a banding pattern with periodicity of about 50 nm, they most likely represent calcified collagen fibers. Immunoelectron microscopy showed that the calcified structures were positive for collagen III. Remarkably, the elastic layer of BM appeared to have a normal ultrastructure, even in 2.5 year old Abcc6-/- mice. Our results suggest that Abcc6 deficiency in the mouse causes calcification of BM. While PXE is considered to affect primarily the elastic fibers, we found predominantly mineralization of collagen fibers.

Proliferation and maturation of microvessels in arteriovenous malformations--expression patterns of angiogenic and cell cycle-dependent factors.

BACKGROUND: Areas of microvascular proliferation have been observed in a subpopulation of symptomatic congenital vascular malformations later in life. We investigated whether this angiogenic response is followed by a stage of maturation. METHODS: Resections of vascular malformations (n = 15), infantile hemangiomas (IHs) (n = 8) and pyogenic granulomas (PGs) (n = 5) were studied. Histopathologically, all lesions were screened for presence of foci of immature and/or mature microvessels. These areas were further studied immunohistochemically for differential expression of several angiogenic factors, cell cycle-dependent proteins, p53 and active caspase3. Immunostains were scored semiquantitatively. RESULTS: Immature microvessel areas were present in five vascular malformations (all of the arteriovenous type), five IHs and five PGs; these lesions also contained transitions between immature and mature microvessels. Conglomerates of mature microvessels were found in 19 cases (6 vascular malformations, 5 PGs and 8 IHs). Expression of vascular endothelial growth factor-A, angiopoietin-1, Ki-67, p16 and p21/27 ratios were overall significantly lower in mature areas than in immature areas including those in vascular malformations. P53 and caspase3 expression was scarce in all lesions. CONCLUSIONS: Microvascular areas in vascular malformations appear to follow the same pattern of vascular proliferation and maturation as seen in other microvascular lesions of skin and soft tissue.

Smooth muscle homeostasis in human atherosclerotic plaques through interleukin 15 signalling.

Interleukin (IL)-15 is a cytokine that has a broad tissue distribution and is important in maintaining homeostasis of cells and stability of tissues. When II-15 is also expressed by vascular smooth muscle cells (SMC), which are the dominant type of cells in most atherosclerotic plaques, it could be important in maintaining plaque tissue integrity and hence resistance of plaques towards development of clinically relevant complications such as plaque rupture and thrombosis. In this study, IL-15 and IL-15Rα in vitro expression by coronary artery SMC was investigated using RT -PCR and FACS analysis. Immunohistochemistry was used to study in situ expression of IL-15 and IL-15R by SMC of human carotid artery atherosclerotic plaques. Multiplex ligand-dependent probe amplification (MLPA) was used to investigate the mRNA expression of 40 pro- and anti inflammatory genes after stimulating coronary SMC with IL-15. We found that atherosclerotic SMC express both IL-15 and its receptor IL-15R, and TNF-γ and TNF-α enhance IL-15R expression in cultured SMC. MLPA studies on SMC revealed enhanced expression of PDGF beta mRNA after IL15 stimulation. In conclusion, our data suggest that IL-15 may contribute to atherosclerotic plaque integrity by stimulation of smooth muscle cells, probably in a PDGF dependent fashion.

Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy.

BACKGROUND: Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions. METHODOLOGY/PRINCIPAL FINDINGS: The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm(2) respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3(+) regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3(+)/mm(2) p<0.05), but relative numbers, expressed as a percentage of the total number of CD3(+) T cells ((FOXP3(+)/CD3(+)) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB(+) cells were rare in NA, but significantly increased numbers of GrB(+) cells were found in AV lesions (85±24 and 0.2±0.1 GrB(+)/mm(2), p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3(+) cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB(+) cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05). CONCLUSIONS: Similar to NA, AV is characterized by a low frequency of intimal FOXP3(+) regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.

IGF-1 has plaque-stabilizing effects in atherosclerosis by altering vascular smooth muscle cell phenotype.

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype.

The prolactin receptor is expressed in macrophages within human carotid atherosclerotic plaques: a role for prolactin in atherogenesis?

Atherosclerotic vascular disease is the consequence of a chronic inflammatory process, and prolactin has been shown to be a component of the inflammatory response. Additionally, recent studies indicate that prolactin contributes to an atherogenic phenotype. We hypothesized that this may be the result of a direct effect of prolactin on atherogenesis through activation of the prolactin receptor. Human carotid atherosclerotic plaques were obtained from patients by endarteriectomies. The mRNA of prolactin receptor, but not of prolactin, was detected in these atherosclerotic plaques by quantitative real-time PCR. In situ hybridization confirmed the expression of the prolactin receptor in mononuclear cells. Analysis at the protein level using immunohistochemistry and immunoelectron microscopy revealed that the prolactin receptor was abundantly present in macrophages near the lipid core and shoulder regions of the plaques. Our findings demonstrate that the prolactin receptor is present in macrophages of the atherosclerotic plaque at sites of most prominent inflammation. We therefore propose that prolactin receptor signaling contributes to the local inflammatory response within the atherosclerotic plaque and thus to atherogenesis.

Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis.

BACKGROUND: Although recent studies indicate a crucial role for IL-17A and IL-22 producing T cells in the pathogenesis of psoriasis, limited information is available on their frequency and heterogeneity and their distribution in skin in situ. METHODOLOGY/PRINCIPAL FINDINGS: By spectral imaging analysis of double-stained skin sections we demonstrated that IL-17 was mainly expressed by mast cells and neutrophils and IL-22 by macrophages and dendritic cells. Only an occasional IL-17(pos), but no IL-22(pos) T cell could be detected in psoriatic skin, whereas neither of these cytokines was expressed by T cells in normal skin. However, examination of in vitro-activated T cells by flow cytometry revealed that substantial percentages of skin-derived CD4 and CD8 T cells were able to produce IL-17A alone or together with IL-22 (i.e. Th17 and Tc17, respectively) or to produce IL-22 in absence of IL-17A and IFN-γ (i.e. Th22 and Tc22, respectively). Remarkably, a significant proportional rise in Tc17 and Tc22 cells, but not in Th17 and Th22 cells, was found in T cells isolated from psoriatic versus normal skin. Interestingly, we found IL-22 single-producers in many skin-derived IL-17A(pos) CD4 and CD8 T cell clones, suggesting that in vivo IL-22 single-producers may arise from IL-17A(pos) T cells as well. CONCLUSIONS/SIGNIFICANCE: The increased presence of Tc17 and Tc22 cells in lesional psoriatic skin suggests that these types of CD8 T cells play a significant role in the pathogenesis of psoriasis. As part of the skin-derived IL-17A(pos) CD4 and CD8 T clones developed into IL-22 single-producers, this demonstrates plasticity in their cytokine production profile and suggests a developmental relationship between Th17 and Th22 cells and between Tc17 and Tc22 cells.

Anti-human vascular endothelial growth factor (VEGF) antibody selection for immunohistochemical staining of proliferating blood vessels.

Nine commercially available vascular endothelial growth factor (VEGF) antibodies were investigated for their ability to immunostain vascular malformations (VMs) with or without immature capillary proliferation. First, all antibodies were optimized for their performance in IHC, with placenta and colon adenocarcinoma as positive control tissues. Five antibodies were regarded as unfit for VEGF immunostaining based on poor immunostaining criteria. Subsequently, Western blot analysis using VEGF rabbit polyclonal antibody (Thermo RB-9031) revealed a clear 45-kDa band in tissue extracts from VMs with immature capillary proliferation and a high Ki67-labeling index, whereas tissue extracts from mature VMs without microvascular proliferation and no Ki67-labeling index demonstrated only a very weak 45-kDa band. In contrast, two VEGF antibodies, including the popular Santa Cruz A-20, revealed bands at 45 kDa of similar intensity in tissue extracts from both types of VMs. Staining characteristics of the 45-kDa band were reflected in the results obtained in IHC.

Differential expression of interleukin-17 family cytokines in intact and complicated human atherosclerotic plaques.

In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL-17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL-17A, -E and -F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL-17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL-17A, -E and -F were expressed in the majority of plaques under investigation. IL-17A/F was expressed by mast cells in all stages of plaque development. IL-17A/F(+) neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL-17A/F(+) Tcells ('TH17') were never observed. IL-17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL-17E and its functional receptor (IL-17RB). The constitutive expression of IL-17E by resident plaque cells, and the additional presence of IL-17E(+) B cells and IL-17A/F(+) neutrophils in advanced and complicated plaques indicates a complex contribution of IL-17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease.

Adequate synapse formation between leukemic B cells and effector T cells following stimulation with artificial TCR ligands.

Artificial T cell receptor (TCR) ligands can be used to direct virus-specific cytotoxic T lymphocytes (CTL) towards tumor cells. Because of their size, these constructs may differ from cognate peptides in their ability to induce T cell activation. We here analysed signalling outcomes upon synapse formation between human cytomegalovirus (CMV)-specific CTL and chronic lymphocytic leukemia (CLL) cells through targeted complexes (TC) containing anti-CD20 single-chain variable fragment and biotinylated major histocompatibility complex class I molecules presenting peptides from CMVpp65. TC coated CLL cells were effectively lysed by CMVpp65-specific CTL but induced less interferon gamma (IFN-gamma) production than peptide loaded targets. Confocal microscopy revealed that TC induced a redistribution of TCR/CD3 but not CD2 towards the immunological synapse. Furthermore, morphological examination of immunological synapses showed smaller and 'patchy' interactions between TC coated B cells and CTL as compared with peptide coated targets. Finally, pre-incubation of CTL with CD2 antibodies led to an Fc-dependent redistribution of CD2 into TC-induced synapses and restored IFN-gamma production by CMV-specific CTL. Thus, redistribution of CD2 towards the immunological synapse appears to be essential for full T cell activation. However, CD2 triggering is not required for efficient lysis of tumor cells, demonstrating that CTL require only minimal stimulation to release their cytotoxic content.

Selective expansion of influenza A virus-specific T cells in symptomatic human carotid artery atherosclerotic plaques.

BACKGROUND AND PURPOSE: Evidence is accumulating that infection with influenza A virus contributes to atherothrombotic disease. Vaccination against influenza decreases the risk of atherosclerotic syndromes, indicating that inflammatory mechanisms may be involved. We tested the hypothesis that influenza A virus-specific T cells contribute to atherosclerotic plaque inflammation, which mediates the onset of plaque rupture. METHODS: T-cell cultures were generated from atherosclerotic segments and peripheral blood of 30 patients with symptomatic carotid artery disease. The response of plaque and peripheral blood T cells to influenza A virus was analyzed and expressed as a stimulation index (SI). Selective outgrowth of intraplaque influenza A-specific T cells was calculated by the ratio of plaque T cell SI and peripheral blood T cell SI for each patient. Accordingly, the patients were categorized as high- (SI ratio >or=5), intermediate- (5

Low numbers of FOXP3 positive regulatory T cells are present in all developmental stages of human atherosclerotic lesions.

BACKGROUND: T cell mediated inflammation contributes to atherogenesis and the onset of acute cardiovascular disease. Effector T cell functions are under a tight control of a specialized T cell subset, regulatory T cells (Treg). At present, nothing is known about the in situ presence of Treg in human atherosclerotic tissue. In the present study we investigated the frequency of naturally occurring Treg cells in all developmental stages of human atherosclerotic lesions including complicated thrombosed plaques. METHODOLOGY: Normal arteries, early lesions (American Heart Association classification types I, II, and III), fibrosclerotic plaques (types Vb and Vc) and 'high risk' plaques (types IV, Va and VI) were obtained at surgery and autopsy. Serial sections were immunostained for markers specific for regulatory T cells (FOXP3 and GITR) and the frequency of these cells was expressed as a percentage of the total numbers of CD3+ T cells. Results were compared with Treg counts in biopsies of normal and inflammatory skin lesions (psoriasis, spongiotic dermatitis and lichen planus). PRINCIPLE FINDINGS: In normal vessel fragments T cells were virtually absent. Treg were present in the intima during all stages of plaque development (0.5-5%). Also in the adventitia of atherosclerotic vessels Treg were encountered, in similar low amounts. High risk lesions contained significantly increased numbers of Treg compared to early lesions (mean: 3.9 and 1.2%, respectively). The frequency of FOXP3+ cells in high risk lesions was also higher compared to stable lesions (1.7%), but this difference was not significant. The mean numbers of intimal FOXP3 positive cells in atherosclerotic lesions (2.4%) was much lower than those in normal (24.3%) or inflammatory skin lesions (28%). CONCLUSION: Low frequencies of Treg in all developmental stages of human plaque formation could explain the smoldering chronic inflammatory process that takes place throughout the longstanding course of atherosclerosis.

Immunohistochemical analysis of regulatory T cell markers FOXP3 and GITR on CD4+CD25+ T cells in normal skin and inflammatory dermatoses.

Regulatory T cells (Treg) are a subset of T lymphocytes that play a central role in immunologic tolerance and in the termination of immune responses. The identification of these cells in normal and inflammatory conditions may contribute to a better understanding of underlying pathology. We investigated the expression of FOXP3 and GITR in normal skin and in a panel of different inflammatory dermatoses. Immunohistochemical double stainings in skin tissue sections revealed that FOXP3 and GITR were almost exclusively present on T cells that express both CD4 and CD25. Further, immunohistochemical double staining, as well as fluorescence-activated cell sorter analysis, on peripheral blood T cells showed that most FOXP3(+) cells expressed GITR and vice versa, whereas a minority were single-positive for these markers. The mean frequency of FOXP3(+) T cells in spongiotic dermatitis, psoriasis, and lichen planus was in the same range (25-29%), but the frequency of these cells in leishmaniasis appeared to be lower (approximately 15%), although this was not statistically significant. The mean frequency of GITR(+) T cells was fairly similar in all conditions studied (14-20%). Normal human skin also contained FOXP3(+) and GITR(+) cells in the same frequency range as in diseased skin, but the absolute numbers were, of course, much lower. In conclusion, frequencies of FOXP3(+) and GITR(+) T cells were similar in all inflammatory skin diseases studied and normal skin, despite the well-known differences among the inflammatory conditions under investigation.

Ultrastructural pathology of aortic dissections in patients with Marfan syndrome: Comparison with dissections in patients without Marfan syndrome.

Despite the discovery in 1990 that mutations in the fibrillin-1 gene cause the Marfan syndrome, the pathogenesis of the life-threatening dissections associated with this disease is far from elucidated. Both the massive number of known fibrillin-1 mutations that result in a heterogeneous patient population and the strongly heterogeneous histology of patients' aortae presumably contribute to this lack of knowledge. We performed a detailed ultrastructural immunoelectron microscopic and histochemical analysis of the dissected media of ascending aortae of 10 patients with Marfan syndrome and compared them with those of 6 patients without Marfan syndrome and 77 individuals without known aortic disease. Relatively similar abnormalities were found in both patient groups, although they were more numerous and more diffusely spread in the patients with Marfan syndrome than in the patients without Marfan syndrome. The most conspicuous ultrastructural defects were the formation of abrupt transverse tears in thick and compact elastic lamellae and the local breaking up of smooth muscle cell-elastic lamella connections (that largely consist of microfibrils and elastic extensions, protruding from the elastic lamellae). This breaking up was characterized by a strongly reduced number of microfibrils and a severe shortening of the elastic extensions. Finally, the elastic extensions detached from the lamellae to ultimately degenerate and disappear. These changes were found mainly in the oldest group of patients with Marfan syndrome, indicating that they represented a loss of previously normally developed structures. We also compared our findings with those from a recently developed murine Marfan model (Pereira L, Lee SY, Gayraud B, Andrilopoulos K, Shapiro SD, Bunton T, Biery NJ, Dietz HC, Sakai LY, Ramirez F. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci. U. S. A. 1999: 96: 3819-3823). Next to similarities, several striking differences existed, demonstrating that this model is not fully representative of the human Marfan syndrome.

Epstein Barr virus specific T-cells generated from unstable human atherosclerotic lesions: Implications for plaque inflammation.

OBJECTIVE: T-cell activation is an essential feature of atherosclerotic plaque inflammation, which eventually may lead to plaque rupture. In this study, we investigated if EBV, a common herpes virus, is capable of stimulating atherosclerotic plaque derived T-cells and thus could contribute to atherosclerotic plaque inflammation. METHODS: Plaque derived T-cell cultures were established from symptomatic carotid atherosclerotic plaques of 19 patients. B-cells from the same patients were transformed with EBV to form lymphoblastoid cell lines (B-LCL) that served as antigen presenting cells. The proliferation of T-cells in the presence of autologous B-LCL was analyzed using 3H-thymidine incorporation. The presence of EBV in atherosclerotic material was analyzed by PCR. RESULTS: Of the 19 cell obtained T-cell cultures, 11 responded to EBV (58%, mean stimulation index: 10.1+/-3.1). PCR analysis showed that EBV DNA was present in 15 of the tissue samples (79%). All the specimens that contained EBV responding T-cells also contained EBV. EBV specific T-cells secreted granzymes, as indication of functional cytotoxic potential. CONCLUSIONS: EBV-specific cytotoxic T-cells and EBV DNA can be frequently observed in human atherosclerotic plaques. This suggests that a T-cell response against EBV could contribute to plaque inflammation, and thus to the onset of acute clinical symptoms.

Apoptosis via the B cell antigen receptor requires Bax translocation and involves mitochondrial depolarization, cytochrome C release, and caspase-9 activation.

Various routes to apoptosis can be active during B cell development. In a model system of mature B cells, differences in caspase-3 processing have suggested that antigen receptor (BCR)-mediated apoptosis may involve a zVAD-insensitive initiator protease(s). In search of the events leading to caspase-3 activation, we now establish that both CD95- and BCR-mediated apoptosis depend on Bax activation and cytochrome C (cytC) release. Nevertheless, the timing and caspase-dependence of mitochondrial membrane depolarization differed considerably after CD95- or BCR-triggering. To delineate events subsequent to cytC release, we compared apoptosis induced via BCR triggering and via direct mitochondrial depolarization by CCCP. In both cases, partial processing of caspase-3 was observed in the presence of zVAD. By expression in 293 cells we addressed the potential of candidate initiator caspases to function in the presence of zVAD, and found that caspase-9 efficiently processed caspase-3, while caspase-2 or -8 were inactive. Finally, retroviral expression of dominant-negative caspase-9 inhibited both CD95- and BCR-mediated apoptosis. In conclusion, we obtained no evidence for involvement of a BCR-specific protease. Instead, our data show for the first time that the BCR-signal causes Bax translocation, followed by mitochondrial depolarization, and cytC release. Subsequent caspase-9 activation can solely account for events further downstream.

Association between complex coronary artery stenosis and unstable angina and the extent of plaque inflammation.

PURPOSE: Patients with unstable coronary syndromes often have complex morphology of coronary stenoses at angiography. We evaluated the association between qualitative assessment of coronary stenoses and plaque inflammation determined by immunohistochemistry. METHODS: A total of 79 patients with unstable (n = 46) or stable angina (n = 33) underwent directional coronary atherectomy for culprit lesions. Qualitative analysis of coronary angiograms was performed using a modified Ambrose classification. Coronary lesions were categorized as either simple (concentric and eccentric type I, n = 29) or complex (eccentric type II and multiple irregularities, n = 50). Cryostat sections of retrieved atherosclerotic specimens were stained immunohistochemically with monoclonal antibodies, alpha-actin (smooth muscle cells), CD68 (macrophages), and CD3 (T lymphocytes). The extent of atherosclerotic inflammation within each coronary lesion was determined by the percentage of immunopositive macrophages per total tissue area (including smooth muscle cells) and the number of T lymphocytes per mm(2). RESULTS: The mean (+/- SD) percentage of macrophages in atherectomy specimens from patients with unstable angina was greater than in specimens from patients with stable angina (21% +/- 14% vs. 13% +/- 10%, P = 0.01); similar results were seen when complex coronary lesions were compared with simple lesions (23% +/- 13% vs. 9% +/- 8%, P <0.001). In multivariate linear regression models, the combination of unstable angina and lesion complexity was strongly associated with the percentage of plaque macrophages. CONCLUSION: The extent of atherosclerotic plaque inflammation is associated with angiographic grading of coronary lesion complexity and unstable angina.

Increased expression of T cell activation markers (CD25, CD26, CD40L and CD69) in atherectomy specimens of patients with unstable angina and acute myocardial infarction.

Atherosclerotic plaques contain a chronic immune mediated inflammation in which T cells play an important role. A previous study revealed that the numbers of interleukin-2 receptor-positive T cells is increased in culprit lesions of patients with acute coronary syndromes; a finding of considerable interest since it indicates a recent change in the intraplaque T cell mediated immune response. Confirmation of this observation is important, because it could provide insight into the onset of the acute event. We have, therefore, expanded our earlier work by using a panel of different T cell activation markers (CD25, CD26, CD40L, CD69). The study is based on 58 culprit lesions from patients who underwent coronary atherectomy. There were four groups of patients: chronic stable angina (n=13), stabilized unstable angina (n=16), refractory unstable angina (n=15), and acute myocardial infarction (AMI; n=14). Activated T cells were expressed as a percentage of the total of CD3-positive cells. CD25, CD26, CD40L, and CD69/CD3 percentages increased with the severity of the coronary syndrome. In patients with AMI all percentages were significantly higher than in patients with chronic stable angina. CD25, CD26, CD40L, and CD69/CD3 percentages in patients with an unstable condition (refractory unstable angina and AMI) were significantly higher than those in patients with a stable condition (chronic stable or stabilized unstable angina) The finding that the percentage of T cells with recent onset activation is significantly increased in the culprit lesions of patients with acute coronary syndromes suggests strongly that a recent change in pathogenic stimulation has occurred leading to local T cell activation.