Barbara C. Biedermann

Arterial Neovascularization and Inflammation in Vulnerable Patients Early and Late Signs of Symptomatic Atherosclerosis

Background—Atherosclerosis is complicated by cardiovascular events such as myocardial infarction, stroke, or peripheral arterial occlusive disease. Inflammation and pathological neovascularization are thought to precipitate plaque rupture or erosion, both causes of arterial thrombosis and cardiovascular events. We tested the hypothesis that arterial inflammation and angiogenic events are increased throughout the arterial tree in vulnerable patients, ie, in patients who suffered from cardiovascular events, compared with patients who never suffered from complications of atherosclerosis. Methods and Results—In a postmortem study, we quantified the inflammatory infiltrate and microvascular network in the arterial wall of iliac, carotid, and renal arteries. Tissue microarray technology was adapted to investigate full-thickness arterial sectors. We compared 22 patients with symptomatic atherosclerosis with 27 patients who never had suffered from any cardiovascular event. The absolute intimal macrophage content was 2- to 4-fold higher in vulnerable patients at all 3 arterial sites analyzed (P<0.05). Patients with symptomatic atherosclerosis had a denser network of vasa vasorum than patients with asymptomatic disease (33±2 versus 25±2 adventitial microvessels per 1 mm2; P=0.008). Hyperplasia of vasa vasorum was an early and macrophage infiltration was a late sign of symptomatic atherosclerosis. Conclusions—High intimal macrophage content and a hyperplastic network of vasa vasorum characterize vulnerable patients suffering from symptomatic atherosclerosis. These changes are uniformly present in different arterial beds and support the concept of symptomatic atherosclerosis as a panarterial disease.

Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease

BACKGROUND Vascular endothelial cells form the interface between recipient tissues and circulating alloreactive donor T cells after allogeneic stem cell transplantation. Vascular injury has been seen in patients with acute graft versus host disease (GVHD) in the skin. We aimed to see whether vascular injury mediated by cytotoxic T lymphocytes and microvessel loss arises in patients with chronic GVHD. METHODS We investigated eight patients with acute GVHD and ten with chronic GVHD for signs of endothelial injury and microvessel loss by measurement of von Willebrand factor (vWF) in plasma and blood vessel density in biopsy samples taken from lesional skin. Controls consisted of nine patients without GVHD who survived for longer than 100 days and nine healthy people. Inflammation and endothelial injury were assessed in selected samples by immunostaining for CD8 T cells, activated cytotoxic T lymphocytes, and vascular endothelial cells. FINDINGS We identified more extensive loss of microvessels in the skin of patients with GVHD (median 66 capillaries/mm(2); IQR 16-98) than of healthy controls (205 capillaries/mm(2); 157-226; p=0.005). Patients with GVHD had higher concentrations of vWF (238%; 168-288) than did those without GVHD (102%; 88-118; p=0.0005). Perivascular CD8 T cell infiltrates in skin correlated with vWF plasma concentrations in patients with GVHD (p=0.01), and activated cytotoxic T lymphocytes and endothelial injury were present in these same samples. INTERPRETATION Host endothelial cells are a target of alloreactive donor cytotoxic T lymphocytes. Substantial blood vessel loss may lead to impaired blood perfusion and tissue fibrosis, the hallmark lesion of chronic GVHD.

A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium.

Large-Scale Culture and Selective Maturation of Human Langerhans Cells from Granulocyte Colony-Stimulating Factor-Mobilized CD34 + Progenitors

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages. To address these issues, we have devised conditions for the culture of a single DC type, Langerhans cells (LCs), using CD34+ cells from G-CSF-mobilized patients. Homogenous populations of LCs, replete with abundant immunocytochemically demonstrable Birbeck granules, could be stably maintained as immature DCs for long periods in culture. Unlike other human DC preparations, the LCs remained fully differentiated after cytokine removal. Following exposure to TNF-α, LPS, or CD40 ligand, the LCs could be synchronously induced to mature. Depending on the agent used, distinct types of LCs emerged differing in their capacity for T cell stimulation, IL-12 production, intracellular localization of MHC products, and overall morphology. Most interestingly, the expression of different sets of Toll family receptors is induced or down-regulated according to the maturation stimulus provided. These results strongly suggest that different proinflammatory stimuli might drive distinct developmental events.

Cutaneous Graft-versus-Host Disease: A Guide for the Dermatologist

Graft-versus-host disease (GVHD) is defined by the aggregation of clinical and pathological manifestations in a recipient of allogeneic stem cells or bone marrow transplantation in which specific immunological as well as nonspecific phenomena lead to characteristic features. GVHD is one of the major complications after hematopoietic stem cell transplantations and responsible for posttherapeutic morbidity, mortality and decrease in quality of life of those patients. GVHD is critically induced and maintained by donor immunocompetent cells that particularly attack epithelia of fast proliferating tissues such as those from the liver, gastrointestinal tract and skin. On the basis of the time of presentation, cutaneous GVHD has been originally divided into an acute and chronic disease. The latter has traditionally been further subclassified into a more epithelial or lichenoid and a predominantly dermal or sclerodermoid form. With respect to the growing importance of this therapeutic procedure and increasing numbers of outpatients presenting with chronic GVHD, this article summarizes the updated knowledge on this disease focused for the dermatologist, and additionally it emphasizes the recent consensus documents on the various aspects of chronic GVHD of the National Institute of Health.

Invariant natural killer T cells : linking inflammation and neovascularization in human atherosclerosis

Atherosclerosis, a chronic inflammatory lipid storage disease of large arteries, is complicated by cardiovascular events usually precipitated by plaque rupture or erosion. Inflammation participates in lesion progression and plaque rupture. Identification of leukocyte populations involved in plaque destabilization is important for effective prevention of cardiovascular events. This study investigates CD1d‐expressing cells and invariant NKT cells (iNKT) in human arterial tissue, their correlation with disease severity and symptoms, and potential mechanisms for their involvement in plaque formation and/or destabilization. CD1d‐expressing cells were present in advanced plaques in patients who suffered from cardiovascular events in the past and were most abundant in plaques with ectopic neovascularization. Confocal microscopy detected iNKT cells in plaques, and plaque‐derived iNKT cell lines promptly produced proinflammatory cytokines when stimulated by CD1d‐expressing APC‐presenting α‐galactosylceramide lipid antigen. Furthermore, iNKT cells were diminished in the circulating blood of patients with symptomatic atherosclerosis. Activated iNKT cell‐derived culture supernatants showed angiogenic activity in a human microvascular endothelial cell line HMEC‐1‐spheroid model of in vitro angiogenesis and strongly activated human microvascular endothelial cell line HMEC‐1 migration. This functional activity was ascribed to IL‐8 released by iNKT cells upon lipid recognition. These findings introduce iNKT cells as novel cellular candidates promoting plaque neovascularization and destabilization in human atherosclerosis.

Requirements for CD8 T-cell migration into the human arterial wall.

Atherosclerotic lesions develop in the arterial intima. Among the leukocytes that accumulate in advanced atherosclerotic plaques, CD8 T cells play a quantitatively important role. They may be involved in disease progression and plaque destabilization, leading to plaque rupture or erosion. These events finally precipitate cardiovascular events. Therefore, we wished to determine the accessibility of the human arterial wall, particularly the arterial intima, for CD8-positive, cytotoxic T lymphocytes. We quantified the number of CD8-positive T cells in the arterial wall using human arterial tissue microarrays. The conditions for efficient cytotoxic T-lymphocyte migration into the arterial wall were determined in an in vitro tissue invasion assay. The invasion pattern of resting or activated cytotoxic T-lymphocyte clones was morphometrically analyzed by confocal microscopy. CD8 T cells represented up to 50% of the lymphocytes in advanced atherosclerotic lesions. Resting CD8-positive cytotoxic T lymphocytes were able to migrate into the arterial intima when it was affected by advanced lesions but not at the earliest stages of the disease. After T-cell receptor and/or proinflammatory cytokine activation, cytotoxic T lymphocytes migrated efficiently into the arterial intima, even in the healthy or mildly affected sites. This in vitro tissue invasion assay mimics conditions under which effector cytotoxic T lymphocytes migrate into the arterial wall to reach similar cell densities as observed in arterial tissue sections from autopsies. Interference with T-cell activation may be important to inhibit cytotoxic T-lymphocyte invasion into the unaffected, healthy artery but may not prevent cytotoxic T-lymphocyte invasion into arteries that are severely affected by atherosclerotic lesions.

Vascular Endothelial Cells Have Impaired Capacity to Present Immunodominant, Antigenic Peptides: A Mechanism of Cell Type-Specific Immune Escape

Vascular endothelial cells (EC) are an exposed target tissue in the course of CTL-mediated alloimmune diseases such as graft-vs-host disease (GVHD) or solid organ transplant rejection. The outcome of an interaction between CTL and target cells is determined by the amount of Ag presented and the costimulatory signals delivered by the target cells. We compared human EC with leukocytes and epithelial cells as targets for peptide-specific, MHC class I-restricted CTL clones. EC were poor targets for immunodominant CTL. Both endogenously processed antigenic proteins and exogenously added antigenic peptides are presented at 50- to 5000-fold lower levels on EC compared with any other target cell analyzed. This quantitative difference fully explained the poor CTL-mediated killing of EC. There was no evidence that lack of costimulation would contribute significantly to this cell type-specific difference in CTL activation. An HLA-A2-specific CTL clone that killed a broad selection of HLA A2-positive target cells equally well, killed EC less efficiently. Our data suggest that EC present a different Ag repertoire compared with other cell types. By this mechanism, these cells may escape an attack by effector CTL, which have been educated by professional APCs and are specific for immunodominant antigenic peptides.

Increased Apolipoprotein Deposits in Early Atherosclerotic Lesions Distinguish Symptomatic From Asymptomatic Patients

Objective—Apolipoprotein E (apoE) and apolipoprotein B100 (apoB) are both involved in receptor-mediated uptake of atherogenic lipoproteins by the liver. Inefficient hepatic clearance of these lipoproteins leads to symptomatic atherosclerosis. Using arterial tissue microarrays, we tested the hypothesis that apoE and apoB accumulation in the arterial wall discriminates between patients with symptomatic atherosclerosis and patients who never experienced cardiovascular events. Methods and Results—In a postmortem study involving 49 patients (22 patients with symptomatic atherosclerosis), we quantified apolipoprotein deposits in arterial rings obtained from the left main coronary, the common carotid, the common iliac, and the renal artery applying tissue microarray technology and semiquantitative immunohistochemistry. In early atherosclerotic lesions, even before atheroma appeared, symptomatic patients had significantly more arterial apoE and apoB deposits than patients without cardiovascular events (P<0.001). Among the symptomatic patients, those without diabetes had more intense apolipoprotein deposits than diabetics. Large amounts of apoE and apoB were found in advanced atherosclerotic lesions, regardless of the activity of the disease. Conclusions—Increased apolipoprotein deposits are an early sign of symptomatic atherosclerosis. They may reflect either enhanced retention of atherogenic lipoproteins or impaired local apolipoprotein degradation. The arterial lipoprotein turnover may be different in diabetic patients.

Antibody Phage Display Assisted Identification of Junction Plakoglobin as a Potential Biomarker for Atherosclerosis

To date, no plaque-derived blood biomarker is available to allow diagnosis, prognosis or monitoring of atherosclerotic vascular diseases. In this study, specimens of thrombendarterectomy material from carotid and iliac arteries were incubated in protein-free medium to obtain plaque and control secretomes for subsequent subtractive phage display. The selection of nine plaque secretome-specific antibodies and the analysis of their immunopurified antigens by mass spectrometry led to the identification of 22 proteins. One of them, junction plakoglobin (JUP-81) and its smaller isoforms (referred to as JUP-63, JUP-55 and JUP-30 by molecular weight) were confirmed by immunohistochemistry and immunoblotting with independent antibodies to be present in atherosclerotic plaques and their secretomes, coronary thrombi of patients with acute coronary syndrome (ACS) and macrophages differentiated from peripheral blood monocytes as well as macrophage-like cells differentiated from THP1 cells. Plasma of patients with stable coronary artery disease (CAD) (n = 15) and ACS (n = 11) contained JUP-81 at more than 2- and 14-fold higher median concentrations, respectively, than plasma of CAD-free individuals (n = 13). In conclusion, this proof of principle study identified and verified JUP isoforms as potential plasma biomarkers for atherosclerosis. Clinical validation studies are needed to determine its diagnostic efficacy and clinical utility as a biomarker for diagnosis, prognosis or monitoring of atherosclerotic vascular diseases.