Reginald S. A. Lord

Mapping of vascular dendritic cells in atherosclerotic arteries suggests their involvement in local immune-inflammatory reactions

OBJECTIVE We previously demonstrated that vascular dendritic cells (VDCs) are present in the intima of large arteries and that their numbers are increased in atherosclerotic lesions. This study was undertaken to determine whether VDCs are involved in immune-mediated reactions in atherogenesis. METHODS Specimens of carotid artery and aorta were obtained at operation. VDCs were identified with anti-CD1a or with S-100. Co-localisation of VDCs with different intimal cells, including T-cells and macrophages, was studied using a double immunostaining procedure. In areas where the co-localising cells were detected, the peculiarities of expression of HLA-DR, ICAM-1, VCAM-1 were examined. RESULTS In all the atherosclerotic plaques, VDCs were seen in contact with T-cells, but these co-localising cells were irregularly distributed and were mainly found in zones of neovascularisation containing inflammatory infiltrates. In other areas, T-cell/VDC co-localisation was rarely detected but VDCs were often found in contact with macrophages. VDCs were detected also in the media beneath atherosclerotic lesions and in the adventitia, where they were mostly around vasa vasorum, especially in areas exhibiting signs of acute inflammation. In these areas VDCs expressed ICAM-1, VCAM-1 and were in contact with T-cells. In both plaques and in the adventitia, the areas with co-localising VDCs and T-cells corresponded to the areas with HLA-DR expression. CONCLUSIONS The results suggest that VDCs are involved in T-cell activation in atherogenesis. There are two regions within the arterial wall where VDC/T-cell co-localisation mostly occurs, namely, in zones of neovascularisation containing inflammatory infiltrates located within atherosclerotic lesions, and in areas with inflammatory infiltrates around vasa vasorum in the adventitia. Possibly, some intimal VDCs migrate through the media and adventitia to adjacent lymph nodes where they present atherosclerosis associated antigens. We also speculate that VDC/macrophage contacts are essential in processing immune information in atherogenesis.

Comparison of saphenous vein patch, polytetrafluoroethylene patch, and direct arteriotomy closure after carotid endarterectomy. Part I. Perioperative results

To evaluate the perioperative outcomes and the immediate increases in size after patch closure, 140 carotid endarterectomies were randomized into one of three groups: direct no-patch closure, saphenous vein patch closure, and polytetrafluoroethylene patch closure. Seven patients (4.4%) experienced signs of cerebral ischemia in the immediate postoperative period. In three cases this was transient and reversible. In the other four reexploration was undertaken and carotid thrombosis was corrected by thrombectomy. The condition of one of these patients deteriorated to a permanent stroke, whereas the other patients made a complete recovery. Neurologic complications were more frequent in the no-patch group, but the differences between the groups were not significant. The incidence of perioperative internal carotid stenosis, aneurysmal dilatation, and other morphologic abnormalities was assessed in 131 intravenous digital subtraction angiograms taken before the patient was discharged from the hospital. Eight (17.0%) of the endarterectomies in the no-patch group were narrowed by 30% to 50% diameter stenosis, whereas none of the patched arteries had more than 30% stenosis. In contrast, dilatation of the common or internal carotid artery to more than twice the measured diameter was absent in non-patched arteries but was present in seven (17.0%) saphenous patch closures and four (9.23%) polytetrafluoroethylene patch closures. We conclude that patch closure after carotid endarterectomy is less likely to cause stenosis in the perioperative period. Poly-tetrafluoroethylene patches resist dilatation better than do saphenous vein patches and are less likely to become aneurysmal.

S100A8 and S100A9 in Human Arterial Wall IMPLICATIONS FOR ATHEROGENESIS

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.

Vascular-associated lymphoid tissue (VALT) involvement in aortic aneurysm

Vascular-associated lymphoid tissue (VALT) consisting of accumulations of immunocompetent and antigen presenting cells has recently been recognised in the arterial wall. In this study, we investigated the involvement of VALT in immune responses in abdominal aortic aneurysms (AAAs). Tissue samples were collected during operations from 31 patients with atherosclerotic infrarenal abdominal aortic aneurysms ranging in diameters from 5 to 8 cm. The specimens were immediately frozen and examined using single and double immunohistochemical staining. T-cell subpopulations, B-cells, dendritic cells and macrophages were identified using cell type specific antibodies. Cell contacts were examined by electron microscopy. Most inflammatory infiltrates were found in the adventitia. T-cells were the predominant cell type in a majority of inflammatory infiltrates but in seventeen cases, typical lymphoid follicles with B-cells forming germinative centres were also observed. In eight cases, the lymphoid follicles aggregated in lymph node-like structures. Dendritic cells were present within all inflammatory infiltrates and contacted lymphocytes. The present observations show that in aortic aneurysm, VALT is involved in immune responses and its activation mostly occurs in the adventitia. The formation of lymphoid follicles and lymph node-like structures in the adventitia suggests that VALT might locally serve the entire complex of both cellular and humoral immune responses in the aneurysmal wall.

Selective Inhibition Of Nitric Oxide Production During Cardiac Allograft Rejection Causes A Small Increase In Graft Survival

Nitric oxide production is increased in allograft rejection and may have both beneficial and deleterious effects on graft function and survival. In animal models, conventional immunosuppressive agents have been shown to decrease nitric oxide production. The aim of our study was to determine what effect augmentation and selective inhibition of nitric oxide production may have on graft survival by using the model of heterotopic cardiac transplantation in the rat. L-Arginine, the naturally occurring substrate for nitric oxide production, was administered subcutaneously at 200 mg/kg/day. L-NG-monomethyl-L-arginine (L-NMMA) is a selective inhibitor of nitric oxide synthase and was administered at 500 mg/kg/day to allograft recipients from the day of operation. Endogenous nitric oxide production was quantified by analysis of urinary nitrate excretion, and time to rejection was determined by graft palpation. L-Arginine did not significantly alter urinary nitrate excretion by iso- or allografts, suggesting that nitric oxide production is not a substrate-limited process in this model. Graft survival in this group was unchanged. L-NMMA produced a small increase in graft survival from 5.1 +/- 0.1 to 6.3 +/- 0.3 days compared with control allografts (P = 0.001) and abolished the rise in urinary nitrate excretion seen with control allografts. Lower doses of L-NMMA produced dose-related decrements in urinary nitrate excretion, but did not alter graft survival. We found that allograft rejection can proceed to graft loss despite complete inhibition of the increase in nitric oxide production that occurs during untreated rejection. The small increase in graft survival suggests that nitric oxide plays a minor role as a cytotoxic effector molecule in this model of acute rejection.

Co-accumulation of Dendritic Cells and Natural Killer T Cells within Rupture-prone Regions in Human Atherosclerotic Plaques

We previously reported that CD1d, a molecule responsible for the presentation of lipid antigens, is expressed in atherosclerotic lesions and that its expression is restricted to dendritic cells. Recent studies demonstrating that CD1d-restricted natural killer T (NKT) cells are involved in atherogenesis prompted the present study investigating whether NKT cells are present in human atherosclerotic lesions and, if so, whether there is an association between NKT cells and dendritic cells. We found that NKT cells do accumulate in rupture-prone shoulders of atherosclerotic plaques and observed direct contacts of dendritic cells with NKT cells in rupture-prone regions of plaque.

Novel mutations in the SDHD gene in pedigrees with familial carotid body paraganglioma and sensorineural hearing loss

Paraganglioma (PGL) is a rare disorder characterized by tumors of the head and neck region. Between 10% and 50% of cases of PGL are familial, and the disease is autosomal dominant and subject to age‐dependent penetrance and imprinting. The paraganglioma gene (PGL1) has been mapped to 11q22.3–q23, and recently germline mutations in the SDHD gene have been identified. The SDHD region contains another gene, DPP2/TIMM8B, the homolog of which causes dystonia and deafness seen in Mohr‐Tranebjaerg syndrome. Using four PGL pedigrees, two of which exhibit coinheritance of PGL and sensorineural hearing loss or tinnitus, analysis of 14 microsatellite markers provided support for linkage to the PGL1 locus. Sequence analysis identified novel mutations in exon 1 and exon 3 of the SDHD gene, including a novel two base pair deletion in exon 3 creating a premature stop codon at position 67; a novel three base pair deletion in exon 3 resulting in the loss of Tyr‐93; a missense mutation in exon 3 resulting in the substitution of Leu‐81 for Pro‐81; and a novel G‐to‐C substitution in exon 1 resulting in the substitution of Met‐1 for Ile‐1. No base changes were detected in the DPP2/TIMM8B gene. There was no apparent loss of heterozygosity at the site of the SDHD mutations. However, RT‐PCR analysis of tumor samples showed monoallelic expression of the mutant (paternal) allele as expected for imprinting. This has not previously been shown for this disorder. The inheritance and expression of the SDHD gene is consistent with the PGL1 gene being subject to genomic imprinting.

Calcified deposit formation in intimal thickenings of the human aorta

The formation of calcified deposits in intimal thickenings of human aorta was studied by electron microscopy. Microzones of calcification were detected in about 20% of fatty streaks and were located predominantly in the deep musculoelastic layer of the intima. Calcified deposits formed only on previously existing structures including extracellular vesicles and unesterified cholesterol. Calcified deposits in the musculoelastic layer of the intima localised inside altered elastin fibres, but initiating the calcification of of elastin required the prior accumulation of cholesterol esters inside elastin fibres. Co-localization of calcified deposits and elastin fibres was followed by destruction of elastin. The present study suggests that at an early stage of development is atherosclerotic lesions, calcified deposits are formed by a physicochemical process which is not strongly controlled by the intimal cells. The recognition of calcified deposits in intimal thickenings support the hypothesis that a subset of fatty streaks might progress to fibrous plaques in human atherosclerosis.

Dendritic cells in the arterial wall express C1q: potential significance in atherogenesis.

OBJECTIVE Dendritic cells (DCs) accumulate in atherosclerotic lesions but their characteristics and their role in atherogenesis are poorly understood. C1q, an element of the first component of complement, is expressed by interdigitating dendritic cells and follicular dendritic cells in the spleen. It has been suggested that C1q is involved in capturing immune complexes in the lymphoid tissue. Immune complexes are also detected in atherosclerotic lesions. The present study investigated whether C1q is expressed by DCs in the arterial wall. Because DCs accumulating within atherosclerotic lesions might originate from monocytes that infiltrate the intima from very early stages of atherosclerosis, C1q expression was also examined in monocyte-derived DCs in vitro. METHODS Specimens of the aorta, carotid, mammary, popliteal and tibial arteries were obtained during operation. Expression of C1q in the arterial wall was studied by immunohistochemistry. The nature of cells expressing C1q was studied in sections double stained with antibodies to C1q and cell type specific markers including CD1a and S-100 (for identification of DCs), CD68 (macrophages), CD3 (T-cells), von Willebrand factor (endothelial cells), and smooth muscle alpha-actin (smooth muscle cells). In vitro, DCs were differentiated from human peripheral blood monocytes using GM-CSF and IL-4. Peripheral blood monocytes were differentiated to macrophages using M-CSF. The expression of C1q in monocytes and in vitro monocyte-derived DCs and macrophages was determined by RT-PCR, Western blotting, immunofluorescence microscopy and flow cytometry. RESULTS In all the arterial specimens studied, DCs expressing C1q were detected. C1q was also found in macrophages, macrophage foam cells and in neovascular endothelial cells in atherosclerotic lesions, but no C1q expression was detected in T-cells and smooth muscle cells. In vitro analysis demonstrated that monocyte-derived DCs and macrophages express C1q but no C1q was detected in monocytes. CONCLUSION C1q is expressed by DCs residing in the arterial wall as well as by monocyte-derived DCs in vitro. Expression of C1q occurs during differentiation of monocytes to DCs and macrophages and might be important in binding and trapping immune complexes in atherosclerotic lesions.

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties.

Abstract Plasminogen activator inhibitor-1 (PAI-1) and twochain high molecular weight kininogen (HKa) exert antiadhesive properties in vitronectindependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilicalvein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin VFACS assay, similar to αvintegrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the antiadhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectindependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of antiadhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.