Anti-endothelial Antibodies Research Articles

Prevalence and biological effects of anti-trophoblast and anti-endothelial cell antibodies in patients with recurrent spontaneous abortions.

Trophoblasts and endothelial cells represent a potential target for antibodies in women with recurrent spontaneous abortions. These antibodies have been shown to be associated with anti-phospholipid antibodies. Are they also present in women with unexplained pregnancy losses in the absence of anti-phospholipid antibodies? The anti-trophoblast antibodies were tested by an immunofluorescence assay on cells purified from pooled first-trimester placentae, whereas the anti-endothelial cell antibodies were measured by enzyme-linked immunoadsorbent assay (ELISA) on cells isolated from the umbilical vein and were cultured to confluence. The cytotoxicity of trophoblasts was evaluated in a homologous system. The expression of adhesion molecules on endothelial cells was quantitated by ELISA using specific monoclonal antibodies, and the expression of tissue factor was quantitated by a chromogenic assay measuring the formation of factor Xa. Complement-fixing antibodies to trophoblast represent a better marker to discriminate patients with recurrent spontaneous abortions from controls and are cytotoxic for the target cells. Anti-endothelial antibodies are also present in these patients and exhibit pro-inflammatory and pro-coagulant activities.

Molecular Cloning and Expression of 56-58 KD Antigen Associated with Transplant Coronary Artery Disease

High circulating levels of anti-endothelial antibodies have been reported in patients with accelerated transplant coronary artery disease (TxCAD); however, the nature or characteristics of the antigen to which these antibodies bindin vivoremain unclear. To identify the antigen, a human endothelial cell cDNA expression library was constructed in λZAP and immuno-screened with serum containing a high titre of anti-endothelial antibodies. Nucleotide sequence of cloned cDNA inserts and GenBank database searches revealed a 94% identity in a 35 bp overlap to the human vimentin gene. The cloned antigen fusion protein co-migrated with human vimentin of molecular mass 56-58 KD in SDS-PAGE and exhibited immunoreactivity towards monoclonal and polyclonal anti-human vimentin antibodies. These results suggest that the 56-58 KD antigen associated with accelerated TxCAD is vimentin (or a vimentin-like protein), a cytoskeletal protein present in cells of the blood vessel walls.

A study of coagulation and anti-endothelial antibodies in idiopathic livedo reticularis.

Livedo reticularis is associated with collagen vascular diseases and other vaso-occlusive disorders in a substantial number of cases. In the remaining cases the cause of livedo reticularis is still unknown. (i.e., idiopathic). We sought to determine a possible causal relationship between idiopathic livedo reticularis and autoimmune factors associated with the coagulation system, including antiendothelial cell antibodies. Nine patients with idiopathic livedo reticularis were studied. All patients were found to have normal platelet count, fibrinogen levels, and prothrombin and activated partial thromboplastin times, as well as negative results for Venereal Disease Research Laboratory and D-timer tests. Anticoagulant activity was detected in 2 patients: one had positive results of thromboplastin titration index and Russell's viper venom test, as well as increased levels of anticardiolipin antibodies and anti-endothelial cell antibodies; the other has positive thromboplastin titration index, mildly increased levels of anti-endothelial cell antibodies, and markedly increased levels of antinuclear antibodies. A third patient had mildly increased levels of anti-endothelial cell antibodies alone, and a fourth patient had mildly increased levels of antinuclear antibodies only. The clinical outcome was uneventful in all of the patients during an 18-month follow-up period. These findings suggest involvement of autoimmune factors associated with the coagulation system in some patients with idiopathic livedo reticularis, whose clinical significance remains to be determined.

Anti-cell surface endothelial antibodies in sera from cardiac and kidney transplant recipients: association with chronic rejection

The aetiologies of accelerated or transplant-associated coronary artery disease (TxCAD) following cardiac transplantation and chronic rejection following renal transplantation remain ill-defined. Previous studies have used Western blotting to demonstrate an association between the formation of anti-endothelial (anti-EC) antibodies and TxCAD after heart transplantation. However, Western blotting favours detection of cytosolic proteins. The objectives of this study were to determine whether flow cytometry, a method which detects antigens on the cell surface, could be used to detect anti-EC antibodies and also whether the observations would extend to renal transplant patients with chronic rejection. Flow cytometry was used to look for antibodies reactive with the surface antigens of macrovascular and microvascular endothelial cell lines in sera from 44 cardiac and 35 renal transplant recipients before and after transplantation. In addition, sera from normals ( n = 20), patients with nontransplant CAD ( n = 50) and patients with unrelated diseases ( n = 40) were investigated. Of 23 cardiac recipients who had developed TxCAD at one or two years post-transplant, 61% had IgM and 13% had IgG anti-EC antibodies post-transplantation. In contrast, in 21 cardiac recipients who had not developed TxCAD 14% had IgM and 14% IgG anti-EC antibodies. There was little evidence for the presence of anti-EC antibodies in cardiac recipients before transplantation. Of 26 renal transplant recipients whose transplants failed due to chronic rejection, 42% and IgG and 19% IgM anti-EC antibodies post-transplantation. of nine renal recipients whose grafts were either functioning normally or who had acutely rejected, none had IgG or IgM anti-EC antibodies either pre- or post-transplantation. The anti-EC antibodies were not found in normals and were rare (less than 4%) in the other disease groups; they do not appear to be autoantibodies. In conclusion, these results suggest that FACS assay could be an informative and rapid test to provide more information on chronic rejection following cardiac and renal transplantation.

A Novel Technique for Culture of Human Dermal Microvascular Endothelial Cells under either Serum-Free or Serum-Supplemented Conditions: Isolation by Panning and Stimulation with Vascular Endothelial Growth Factor

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr,since mRNA for kdrwas detected using RT–PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.

Anti-endothelial cell IgG antibodies from patients with Wegener's granulomatosis bind to human endothelial cells in vitro and induce adhesion molecule expression and cytokine secretion.

To elucidate the role of anti-endothelial cell antibodies (AECA) in vascular inflammation in patients with Wegener's granulomatosis (WG). IgG fractions from 3 AECA-positive WG patients, IgG from 3 AECA-negative WG patients, and IgG from healthy donors were tested for their ability to: a) bind to endothelial cells and to display complement-dependent or antibody-dependent cellular cytotoxicity, b) modulate cell membrane expression of adhesion molecules, as evaluated by cytofluorometry and by immunoenzymatic assay, and c) induce the secretion of interleukin-1 beta (IL-1 beta), IL-6, IL-8, and monocyte chemotactic protein 1 (MCP-1). We found that AECA IgG from WG patients do not display any significant cytotoxicity but are able to up-regulate the expression of E-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 and to induce the secretion of IL-1 beta, IL-6, IL-8, and MCP-1. AECA in patients with WG could play a potential pathogenetic role by activating endothelial cells, and thus facilitating leukocyte recruitment and adhesion to endothelial surfaces, rather than by displaying a cytotoxic activity.

Endothelial cell activation in cutaneous vasculitis

Markers of endothelial cell activation were measured in 28 patients presenting with various forms of limited or focal type cutaneous vasculitis. Plasma levels of tissue plasminogen activator antigen (t-PA:Ag), plasminogen activator inhibitor type 1 antigen (PAI-1:Ag) and PAI-1 activity, fibrin plate, von Willebrand factor antigen (vWF:Ag), tissue factor (TF) and soluble thrombomodulin (sTM) were measured. In comparison with the control group (n = 20) there was a significant increase in t-PA:Ag, vWF:Ag and TF (P < 0.05, Mann-Whitney U-test) in the cutaneous vasculitis group. This study confirms that measurable degrees of endothelial activation occur in cutaneous vasculitis. Cutaneous vasculitis includes a diverse group of clinical conditions, which are associated with inflammatory changes in cutaneous blood vessels with local fibrin deposition. The aetiology and pathogenesis of the majority of these entities remain unknown. Causative mediators are thought to include immune complexes, anti-endothelial cell antibodies, cytotoxic lymphocytes and viruses. Histologically, immune complexes and complement are frequently detected on the vessel wall, and serologically anti-endothelial antibodies are often detected in patients with vasculitis and in systemic lupus erythematosus (SLE) which correlate with the severity of cutaneous vasculitis, arthritis and nephritis. Lymphocyte-mediated toxicity to endothelial cells has been reported in a small number of patients with giant cell arteritis and Takayasu's arteritis. The vascular endothelium plays a central part in the control of haemostasis. Under physiological conditions endothelial cells present an anticoagulant surface to blood constituents, partially due to surface expression of heparan sulphate and thrombomodulin (TM). Heparan sulphate binds antithrombin III (ATIII), thereby accelerating inactivation of intrinsic coagulation enzymes. Thrombomodulin is an endothelial cell surface glycoprotein which promotes anticoagulation by forming a complex with thrombin which then activates protein C. Activated protein C together with a cofactor, protein S, inactivates FVa and FVIIIa. von Willebrand factor (vWF) is synthesized by endothelial cells, stored in Weibel-Palade bodies and released into the circulation upon endothelial stimulation. vWF mediates the binding of platelets to the subendothelium and is the carrier molecule for FVIIIC. The endothelium controls fibrinolysis by producing t-PA and its inhibitor PAI-1. Inflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF) activate endothelial cells, causing a shift from an antithrombotic to prothrombotic state, including expression of tissue factor, increased synthesis of PAI-1 and decreased expression of TM. Fibrin deposition and intravascular thrombosis are seen in cutaneous vasculitis syndromes, suggesting local endothelial cell activation. The aim of this pilot study was to assess whether perturbation of the endothelium in cutaneous vasculitis could be detected in the patients' plasma samples. If so, further studies to assess any correlation in levels of these markers with disease activity might prove useful in the future.

Plasma Endothelin Correlates With Antiendothelial Antibodies in Patients With Mixed Connective Tissue Disease

Elevated circulating levels of the vasoactive peptide endothelin-1 have been reported in various cardiovascular disorders. Because these conditions are frequently associated with endothelial dysfunction and damage and the vasoconstrictor effect of endothelin-1 is believed to be produced at the local vascular level, it is uncertain whether circulating endothelin-1 is a causal factor in enhanced vascular tone or instead a marker of endothelial injury. We tested whether elevated immunoreactive endothelin-1 could be detected by radioimmunoassay in plasma and whether endothelin-1 levels correlated with antiendothelial autoantibodies in patients with mixed connective tissue disease. Venous blood samples were collected from 21 patients in the morning after an overnight fast and before medication. The plasma immunoreactive endothelin-1 level was 2.7 +/- 0.5 pg/mL (range, 1.1 to 5.2 pg/ml; n = 9) and 7.3 +/- 1.5 pg/mL (range, 2.8 to 20.7 pg/mL; n = 12) in patients who had no antiendothelial antibodies and in patients with antiendothelial antibodies, respectively. These latter values were significantly (P < .001) increased compared with 10 age-matched healthy volunteers (2.0 +/- 0.3 pg/mL; range, 0.5 to 3.0 pg/mL). Plasma endothelin-1 level strongly correlated with antiendothelial antibodies (rs = .836, n = 21, P < .001), whereas there was no correlation between age, systolic and diastolic blood pressures, antinuclear antibodies, and duration of the disease and endothelin-1 values. The incidence of Raynaud's phenomenon and angina did not differ significantly in patients with low and high endothelin-1 levels. This study showed that mixed connective tissue disease is associated with elevated plasma immunoreactive endothelin-1 and that endothelin-1 levels significantly correlate with antiendothelial autoantibodies. These findings suggest that increases in plasma endothelin-1 concentration may be secondary to vascular injury and do not necessarily represent enhanced susceptibility to vasoconstriction.

Vasculitis in Children

Common vasculitic disorders in children include those associated with infections (e.g., Rickettsiae, subacute bacterial endocarditis), Schonlein-Henoch purpura, and Kawasaki disease. Recent advances have occurred in understanding the pathogenesis of vasculitides. In this review, the reader will be exposed to some of the developments in adhesion molecules, antineutrophil cytoplasmic antibodies, antiendothelial antibodies, and antiphospholipid antibodies. Classification criteria and diagnostic strategies are also summarized.

Mosaic of anti-endothelial antibodies. Review of the first international workshop on anti-endothelial antibodies: clinical and pathological significance Milan, 9 November 1994.

Mosaic of anti-endothelial antibodies. Review of the first international workshop on anti-endothelial antibodies: clinical and pathological significance Milan, 9 November 1994.

726-5 Autoantibodies to Stress Proteins in Dilated Cardiomyopathy and Biopsy Proven Myocarditis

Stress proteins are synthesized either constitutively or in response to heat shock or other metabolic insults. Stress proteins are immunodominant antigens of mycobacteria and other nonviral microorganisms and have been highly conserved during evolution. Stress proteins have therefore been thought to be involved in the pathogenesis of various autoimmune diseases. As myocarditis and dilated cardiomyopathy (DCM) are both thought to be initiated by autoimmune mecha nisms, we looked for autoa ntibodies to stress proteins. Through their exposed situation in the vasculature, endothelial cells may participate in the initiation of these diseases. We therefore used both purified stress proteins and stressed endothelial cells as antigenic substrate. Sera were obtained from healthy controls and patients with dilated cardiomyopathy and biopsy proven myocarditis. Endothelial cells were isolated from umbilical veins by collagenase digestion, cultivated to the 2nd or 3rd passage, and then subjected to a heat shock (45°C, 15 min). The cells were allowed to recover for 16 h at 37°C, scraped off the culture vessel, and lysed in an urea buffer. The cell lysate or purified hsp 90 and 27 was then used in a 1D and 2D-immunoblot. Diagnosis N Antibodies against purified hsp 27 hsp 90 cell lysate DCM 113 15/13% 15/13% 41/36% Myocarditis 62 9/14.5% 4/6.5% 38/61% Controls 96 9/9% 7/7% 23/24% In the sera of patients with DCM and myocarditis antibodies to endothelial proteins were found, indicating that antiendothelial antibodies may be involved in the pathogenesis of these diseases. In contrast, autoantibodies to stress proteins were not found in significantly higher number in the sera of patients as compared to healthy controls. A role of autoantibodies to hsp 27 and hsp 90 in the pathogenesis of these diseases can thus not be assumed.

Characterization of the Endothelial Surface Proteins Recognized by Anti-Endothelial Antibodies in Primary and Secondary Autoimmune Vasculitis

The antigenic structures recognized by anti-endothelial cell antibodies (AECA) in sera from 10 Wegener's granulomatosis (WG) and 12 systemic lupus erythematosus (SLE) patients with signs of vasculitis were characterized by immunoprecipitation of selectively radiolabeled surface membrane proteins from human umbilical vein endothelial cells. Electrophoretic analysis of the immunoprecipitated proteins revealed reactivities against endothelial antigens ranging in size from 200 to 25 kDa. AECA antigens were not cell specific, since the same sera also reacted, at least in part, with radiolabeled human fibroblast surface proteins. The majority of WG patients displayed a constant precipitation pattern of five proteins (180, 155, 125, 68, and 25 kDa). On the contrary, AECA from SLE sera reacted with a more heterogeneous series of endothelial proteins. A group of four proteins, however, was also found in the majority of SLE sera: 200, 180, 155, and 25 kDa. In addition, some endothelial antigens were immunoprecipitated only by WG (125 kDa) or by SLE sera (200 kDa), suggesting a different endothelial reactivity in different vasculitic processes. The reaction did not involve intracellular proteins as demonstrated by the lack of reactivity of SLE sera negative for AECA but positive for anti-cytoplasmic or anti-nuclear antibodies. These data confirming that AECA recognize surface endothelial determinants further support a potential pathogenetic role for these antibodies in autoimmune vasculitis.

Anti-endothelial antibodies in Takayasu arteritis.

Anti-endothelial antibodies in Takayasu arteritis.

INTRAPARENCHYMAL., IMMUNOCYTOCHEMICAL DETECTION OF RAT BLOOD-BRAIN BARRIER ANTIGEN

A rat-specific monoclonal antibody exclusively recognizing, the blood-brain and blood-nerve barrier protein has been demonstrated in rat brain from postnatal day 3 (Sternberger and Sternberger, 1987). In the present study, we have employed the anti-endothelial barrier antibody to assess the morphological alterations and the intrnparenehymal distribution of barrier competent microvessels in the developing rat brain. At postnatal day 6, the antibody binding endotbelia appeared as single round cells with central nuelei and granulated cytoplasm, within which the reaction product was evenly dispersed. These cells appeared over the pial surface of the temporal neocortex. At day 8 postnatally, the endothelial cells were arranged in vascular columns of 2 to 4 contiguous cells within the cerebral cortex and the hippocampus. These reactive cells assumed an oval shape with elongated nuclei. The reaction product was present throughout the cytoplasm although more concentrated on the lumenal side of the cell. From postnatal day 10 to 19 months, there was a rapid increase in individual cellular and overall vascular staining intensity and length of stained collaterals arising from reactive parent microvessels. The cerebral cortex is more highly vascularized by barrier competent microvessels than white matter, while vessels in the hippocampus show higher reaction intensity but less vascular network than the rest of the neocortex, the white matter is less vascularized by blood-brain in barrier than either the hippocampus or the cerebral cortex.

Xenotransplantation rejection is antibody-mediated in both sensitized and nonsensitized recipients.

This study analyzes the mechanisms involved in xenotransplantation rejection between closely related species. Hamster hearts were transplanted heterotopically into both normal rats and rats previously sensitized by the transfusion of donor blood. Sequential ultrastructural and immunohistochemical analyses were performed on the grafts, spleens, and sera. The data obtained support the view that induced antibodies directed against the xenograft endothelium play a very important role in producing graft damage. Moreover, the demonstration of antibodies against myocyte determinants suggests that it is possible, in this particular model, that the antiendothelial antibodies are not the only ones involved in the injury process.

Antiendothelial cell antibodies in systemic lupus erythematosus: enhanced antibody binding to interleukin-1-stimulated endothelium.

IgG antiendothelial antibodies (IgG AEA), as measured by enzyme-linked immunosorbent assay, could be detected in serum samples of 38 out of 41 patients with systemic lupus erythematosus. Incubation of endothelial cells (EC) with interleukin-1 alpha (IL-1 alpha), in contrast to incubation with interferon gamma or tumor necrosis factor alpha, resulted in an enhanced IgG AEA binding. Immunoblotting revealed reactivity of AEA against a variety of EC antigens. The upregulation of IgG-AEA-binding reactivity to IL-1 alpha-stimulated EC was due to binding to antigens that were already expressed by unstimulated EC. The IgG-binding reactivity to both IL-1 alpha-stimulated and unstimulated EC was significantly higher in the serum of patients with joint or skin abnormalities as compared with patients without these manifestations. These data suggest that upregulated binding of IgG to EC induced by IL-1 alpha may play a role in immune vascular damage.

Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.

The endothelium of the coronary vascular system has been described in the literature as originating from different sources, varying from aortic endothelium for the main coronary stems, endocardium for the intramyocardial network, and sinus venosus lining for the venous part of the coronary system. Using an antibody against quail endothelial cells (alpha-MB1), we investigated the development of the coronary vascular system in the quail (Hamburger and Hamilton stages 15 to 35) and in a series of 36 quail-chicken chimeras. In the chimeras, pieces of quail epicardial primordium and/or liver tissue were transplanted into the pericardial cavity of a chicken host. The results showed that the coronary vascular endothelial distribution closely followed the formation of the epicardial covering of the heart. However, pure epicardial primordium transplants did not lead to endothelial cell formation, whereas a liver graft with or without an epicardial contribution did have this capacity. The first endothelial cells were seen to reach the heart at the sinus venosus region, subsequently spreading through the inner curvature to the atrioventricular sulcus and the outflow tract and, last of all, over the ventricular surfaces. At these sites, the precursor cells and small vessels were seen to invade the sinus venosus wall, the ventricular and atrial myocardium, and the mesenchymal border of the aortic orifice. Connections with the endocardium of the heart tube were only observed in the right ventricular outflow region. Initially, the connections with the aortic endothelium were multiple, but later in development only two of these connections persisted to form the proximal part of the two main coronary arteries. Connections to the pulmonary orifice were never observed. Our transplantation data showed that the entire coronary endothelial vasculature originated from an extracardiac source. Moreover, using the developing subepicardial layer as a matrix, we showed that the endothelial cells reached the heart from the liver region. Ingrowth into the various cardiac segments was also observed. Implications for the relation to specific congenital cardiac malformations are discussed.

Serum autoantibodies in patients with Alzheimer's disease and vascular dementia and in nondemented control subjects.

In this study we sought to evaluate the clinical significance of serum autoantibodies to dementing processes. We assessed 40 age-matched subjects: 10 patients with probable Alzheimer's disease, 10 with possible Alzheimer's disease with cerebrovascular disease, 10 with vascular dementia, and 10 nondemented control subjects. Serum from each subject was tested for the presence of antithyroglobulin antibody, thyroid antimicrosomal antibody, gastric anti-parietal cell antibody, anti-smooth muscle antibody, antinuclear antibody, rheumatoid factor, antineuronal antibody, and anticardiolipin antibody. In addition, we investigated the sera of these patients for the presence of an antivascular antibody directed against the vascular basement membrane proteoglycan antigen and for circulating immune complexes. Autoantibodies were present in 100% of the patients with possible Alzheimer's disease with cerebrovascular disease, 80% of those with vascular dementia, 40% of those with probable Alzheimer's disease, and 30% of the nondemented control subjects. The highest number of autoantibodies was observed in patients with vascular dementia and possible Alzheimer's disease with cerebrovascular disease. Antinuclear antibody was present in 60% of vascular dementia patients and antineuronal antibody in 50% of these patients. However, no individual autoantibody could differentiate Alzheimer's disease from cerebrovascular disorders. Immune complexes were detected in the serum of 20-30% of each patient group. Neither the patient nor the control sera was found to contain antiendothelial antibody. Despite the relatively small number of individuals examined in each category, the elevated number of autoantibodies associated with possible Alzheimer's disease with cerebrovascular disease and vascular dementia indicates a possible link between the presence of autoantibodies and cerebrovascular disorders in dementia.

Anti-endothelial antibodies and coronary artery disease after cardiac transplantation.

Accelerated coronary artery disease is the most serious complication after cardiac transplantation. The disease has a multifactorial aetiology, with little agreement about the relative importance of the various risk factors. We have investigated the frequency of anti-endothelial antibodies against human umbilical vein endothelial cells by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting. Peptide-specific anti-endothelial antibodies were found in 15/21 heart transplant recipients with accelerated coronary artery disease, and 1/20 transplant patients who had not developed the disease. Positive immunofluorescence of patients' serum on frozen sections of coronary vessels confirmed the endothelial specificity of antibodies. These results provide evidence of an immune aetiology for transplant-associated coronary artery disease and could have important implications for its diagnosis and therapy.

The role of antiendothelial antibodies in myocarditis and its sequelae

The role of antiendothelial antibodies in myocarditis and its sequelae