Emily A. Van Vré

Apoptotic Cell Death and Efferocytosis in Atherosclerosis

Apoptotic cell death is an important feature of atherosclerotic plaques, and it seems to exert both beneficial and detrimental effects depending on the cell type and plaque stage. Because late apoptotic cells can launch proatherogenic inflammatory responses, adequate engulfment of apoptotic cells (efferocytosis) by macrophages is important to withstand atherosclerosis progression. Several efferocytosis systems, composed of different phagocytic receptors, apoptotic ligands, and bridging molecules, can be distinguished. Because phagocytes in atherosclerotic plaques are very much solicited, a fully operative efferocytosis system seems to be an absolute requisite. Indeed, recent studies demonstrate that deletion of just 1 of the efferocytosis pathways aggravates atherosclerosis. This review discusses the role of apoptosis in atherosclerosis and general mechanisms of efferocytosis, to end with indirect and direct indications of the significance of effective efferocytosis in atherosclerosis.

Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease

Background Dendritic cells are potent antigen-presenting and immune modulating cells that have been implicated in the development of atherosclerosis. In human blood, two distinct lineages are distinguished: plasmacytoid dendritic cells and myeloid dendritic cells. Although dendritic cells have been described in atherosclerotic plaques, no information exists concerning circulating blood dendritic cells in atherosclerosis. This study aims to evaluate the number of circulating dendritic cells in patients with coronary artery disease. The relation with the extent of coronary artery disease, the clinical syndrome and with a marker of inflammation will be documented. Methods Patients with angiographically proven coronary artery disease (n=18) and age and sex-matched controls (n=18) were included. Myeloid dendritic cells and plasmacytoid dendritic cells were detected with the specific blood dendritic cell antigens, blood dendritic cell antigen-1 and blood dendritic cell antigen-2, respectively. Results Absolute and relative numbers of circulating plasmacytoid dendritic cells were significantly lower in patients with coronary artery disease (5722±601/ml and 0.08±0.01%) than in controls (12640±1289/ml and 0.21±0.02%). Plasmacytoid dendritic cells were more decreased in patients with troponin-positive unstable coronary syndromes than in patients with low troponin values, and tended to be lower in more extensive coronary artery disease. Absolute myeloid dendritic cells numbers tended to be reduced in patients, whereas relative numbers were significantly decreased: 11 857±1895/ml versus 15 226±928/ml and 0.17±0.03% versus 0.26±0.01% in controls. Conclusions The present study shows a significant decrease of circulating blood dendritic cell antigen-2 positive plasmacytoid dendritic cells in patients with coronary artery disease. The decrease tended to be more pronounced in unstable coronary syndromes and extensive coronary artery disease, suggesting a possible role of dendritic cells in plaque progression and rupture.

Dendritic Cells in Human Atherosclerosis: From Circulation to Atherosclerotic Plaques

Background. Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory infiltrates predominantly consisting of monocytes/macrophages and activated T cells. More recent is the implication of dendritic cells (DCs) in the disease. Since DCs were demonstrated in human arteries in 1995, numerous studies in humans suggest a role for these professional antigen-presenting cells in atherosclerosis. Aim. This paper focuses on the observations made in blood and arteries of patients with atherosclerosis. In principal, flow cytometric analyses show that circulating myeloid (m) and plasmacytoid (p) DCs are diminished in coronary artery disease, while immunohistochemical studies describe increased intimal DC counts with evolving plaque stages. Moreover, mDCs and pDCs appear to behave differently in atherosclerosis. Yet, the origin of plaque DCs and their relationship with blood DCs are unknown. Therefore, several explanations for the observed changes are postulated. In addition, the technical challenges and discrepancies in the research field are discussed. Future. Future studies in humans, in combination with experimental animal studies will unravel mechanisms leading to altered blood and plaque DCs in atherosclerosis. As DCs are crucial for inducing but also dampening immune responses, understanding their life cycle, trafficking and function in atherosclerosis will determine potential use of DCs in antiatherogenic therapies.

Immunohistochemical characterisation of dendritic cells in human atherosclerotic lesions: possible pitfalls

Background: Previously we demonstrated decreased blood myeloid (m) and plasmacytoid (p) dendritic cell (DC) counts in atherosclerotic patients. Therefore, we examined whether DCs, in particular DC precursors, accumulate in human plaques. Methods: Blood DC antigen (BDCA)-1, CD11c (mDCs), BDCA-2, CD123 (pDCs), langerin, fascin, S-100 (immature/mature DCs), and CD1a and CD83 (mature DCs) were investigated by immunohistochemistry of carotid arteries obtained by endarterectomy (EAS, frozen n = 11, fixed n = 11) or autopsy (fixed, n = 87). Results: Fascin and S-100 required formaldehyde fixation, other markers needed cryo-preservation. BDCA-1, BDCA-2, langerin, and S-100 appeared specific for intimal DCs, unlike CD123 and fascin (staining endothelial cells), CD11c and CD1a (staining monocytes, foam cells) or CD83 (staining lymphocytes). BDCA-1+ and BDCA-2+ cells were detected in EAS, preferentially near microvessels. S-100+ cells increased successively from intimal thickening, via pathological intimal thickening, fibrous cap atheroma and finally complicated plaques. Fascin+ cells followed the same pattern, but were more abundant. However, in lesions containing microvessels (complicated plaques, plaque shoulders and most EAS) this was partly explained by fascin positive endothelial cells. Even complicated plaques contained relatively few mature CD83+ DCs. Conclusions: Accumulation of BDCA-1 and BDCA-2 around neovessels showed that mDCs and pDCs are recruited to advanced plaques, which is in line with the previously described decline of circulating blood DCs in patients with coronary artery disease. Unexpectedly, several DC markers yielded false positive signals. Hence, some accounts on numbers, trafficking and activation of DCs in atherosclerotic plaques may require re-evaluation.

Human C-Reactive Protein Activates Monocyte-Derived Dendritic Cells and Induces Dendritic Cell-Mediated T-Cell Activation

Objective—Recent studies proposed a pathogenic role for C-reactive protein (CRP), an independent predictor of cardiovascular disease (CVD), in atherosclerosis. Therefore, we tested whether CRP may modulate dendritic cell (DC) function, because these professional antigen-presenting cells have been implicated in atherogenesis. Methods and Results—Human monocyte-derived immature DCs were cultured with human CRP (0 to 60 &mgr;g/mL) for 24 hours. Thereafter, activation markers were measured by flow-cytometry and DCs were cocultured with CFSE-labeled lymphocytes to measure T-cell proliferation and interferon (IFN)-γ secretion after 8 days. Exposure to 60 &mgr;g/mL CRP (n=5) induced an activated cell morphology and significant (CD40 increase MFI 5.23±0.28, P<0.01 paired t test; CD80 6.18±0.51, P<0.01) to modest (CD83 1.38±0.17, P<0.05, CCR7 1.60±0.29, P=0.05) upregulation of DC activation markers. The expression of CD86 and HLA-DR was high, but not affected. T-lymphocytes incubated with CRP-pulsed DCs displayed increased IFN-γ secretion and proliferation (P<0.001). DC activation was concentration-dependent and detected from 2 &mgr;g/mL CRP; the maximum effect was equivalent to that seen with 0.1 &mgr;g/mL lipopolysaccharide (LPS). Polymyxin B abolished the LPS response, without influencing CRP effects. Finally, immunohistochemistry could demonstrate DC/CRP colocalization in human atherosclerotic lesions. Conclusions—These findings suggest that CRP in plaques or found circulating in CVD patients can influence DC function during atherogenesis.

Changes in blood dendritic cell counts in relation to type of coronary artery disease and brachial endothelial cell function

BackgroundRecently we reported a decline of circulating myeloid (m) and plasmacytoid (p) dendritic cells (DCs) in patients with coronary artery disease (CAD). This study also determined the total blood DC numbers and focused on effects of extent (one vs. three-vessel disease) and type (stable vs. unstable) of CAD, and on endothelial cell function. MethodsPatients undergoing diagnostic coronarography were enrolled in four groups: control patients (atypical chest pain, <50% narrowing, n=15), stable one-vessel (n=15), stable three-vessel (n=15), and unstable one-vessel CAD (n=16). Total blood DCs were identified as lineage (lin)− and HLADR+, and DC subtypes with blood DC antigen (BDCA)-1+ for mDCs and BDCA-2+ for pDCs. Flow-mediated dilatation (FMD) was measured in the brachial artery. ResultsNumbers of total blood DCs, mDCs and pDCs declined in CAD patients compared with control patients, but without differences between the CAD groups. Interleukin-6 and high sensitivity C-reactive protein displayed inverse associations with mDCs. A FMD below the median of the study population, use of &bgr;-blockers or of lipid-lowering drugs was associated with increased mDCs, whereas pDCs were similar. Interestingly, the effects of drugs and FMD were additive with that of CAD. ConclusionThis study indicates that lower blood DCs do not result from medication intake or endothelial dysfunction, and are an overall systemic effect of atherosclerosis rather than CAD type (stable or unstable) or number of stenotic coronary arteries. In view of discrete associations with cytokines, FMD, &bgr;-blockers and statins, mDCs and pDCs seem to behave differently and may influence inflammation during atherosclerosis in different ways.

Expression of dendritic cell markers CD11c/BDCA-1 and CD123/BDCA-2 in coronary artery disease upon activation in whole blood.

OBJECTIVES Previous in vivo studies on dendritic cell (DC) enumeration in coronary artery disease (CAD) were not always consistent. Therefore, we investigated by flow cytometry whether this was due to CAD-related differences in expression of subset markers for myeloid (m)DCs (blood DC antigen (BDCA)-1, CD11c) and plasmacytoid (p)DCs (BDCA-2, CD123), before and after in vitro stimulation with Toll-like receptor ligands. RESULTS Our data showed that circulating DCs decline in CAD, irrespective of the DC subset marker that was used for enumeration. Upon in vitro activation, BDCA-2 was downregulated, whereas CD11c and CD123 were upregulated. This implies that the expression ratios CD11c/BDCA-1 and CD123/BDCA-2 can assess DC activation. Comparing these ratios between controls and CAD patients showed no differences in blood DC activation in both groups. CONCLUSIONS This study suggests that when different DC numbers are found between two study populations, the DC activation status from both groups always needs to be verified, since a decrease in BDCA-2(+) pDCs or an increase in CD11c(+) mDCs or CD123(+) pDCs can be due to the altered expression of these markers during activation. Given that CD11c, BDCA-1, CD123 and BDCA-2 are more abundantly expressed on blood DCs than typical activation markers like CD83, CD86 or CCR-7, the use of the ratios is an easy and reliable way to determine DC activation in whole blood assays.

Potential Use of Dendritic Cells for Anti-Atherosclerotic Therapy

The chronic inflammatory nature of atherosclerosis is nowadays widely accepted. Dendritic cells (DCs) are likely to play a crucial role in directing innate and adaptive immunity against altered (self-)antigens, such as oxidized low density lipoproteins (oxLDL). DCs are found in early lesions and their numbers become even higher when the lesion progresses. DCs are most abundant in areas of neovascularization where they are often found near T cells. All stages from precursors to fully mature DCs are present in human plaques. Treatment of atherosclerosis is currently based on reducing risk factors, e.g. by use of statins and beta-blockers. Some of these pharmacological agents also show anti-inflammatory properties and consequently can affect DC function. Yet, many patients remain at risk for acute coronary events, and new therapies to treat atherosclerosis are needed. One therapeutic strategy is based on isolation of patients DCs that are then pulsed with appropriate antigen(s) ex vivo, e.g. (immunogenic components of) oxLDL or total extract of atherosclerotic plaque tissue, and returned to the blood stream. Other approaches to ensure immune protection include generation of tolerogenic DCs, or using DCs to deplete detrimental Th1 or Th17 cells. However, the future lies in direct targeting of DCs by manipulating functions of different DC subsets. Therefore, it would be useful to isolate plaque-resident DCs to be able to identify unique antigen(s) on their surface. The challenge is to selectively identify regulatory molecules and novel therapies to inhibit DC migration and function during atherogenesis, without affecting normal DC function under physiological conditions.