Fluvastatin inhibits up-regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells.

BACKGROUND: Mechanisms of thrombosis induced by antiphospholipid (aPL) antibodies include up-regulation of tissue factor (TF) expression on endothelial cells (ECs). Statins have been shown to reduce levels of TF induced by tumor necrosis factor (TNF-alpha) and lipopolysaccharide (LPS) on ECs. In a recent study, fluvastatin inhibited thrombogenic and proinflammatory properties of aPL antibodies in in vivo models. The aim of this study was to determine whether fluvastatin has an effect on aPL-induced expression of TF on ECs. METHODS: IgGs were purified from four patients with APS (IgG-APS) and from control sera (IgG-NHS). Cultured human umbilical vein endothelial cells (HUVEC) were treated with IgG-APS or IgG-NHS or with medium alone or with phorbol myristate acetate (PMA), as a positive control. In some experiments, cells were pretreated with fluvastatin (2.5, 5 or 10 micro m) with and without mevalonate (100 micro m). TF expression on HUVECs was measured by ELISA. RESULTS: PMA and the four IgG-APS preparations increased the expression of TF on EC significantly (4.9-, 2.4-, 4.2-, 3.5- and 3.1-fold, respectively), in a dose-dependent fashion. Fluvastatin (10 micro m) inhibited the effects of PMA and the four IgG-APS on TF expression by 70, 47, 65, 22 and 68%, respectively, and this effect was dose-dependent. Mevalonate (100 micro m) completely abrogated the inhibitory effects of fluvastatin on TF expression induced by aPL. CONCLUSION: Because of the suggested pathogenic role of aPL on induction of TF on ECs, our data provide a rationale for using statins as a therapeutic tool in treatment of thrombosis in APS.

Regulation of tissue factor in microvascular dermal endothelial cells.

Inflammation is accompanied by activation of the coagulation cascade, manifested by thrombosis and fibrin generation. Whereas endothelial cells normally provide a nonthrombogenic surface, inflammatory mediators may induce the expression of tissue factor, rendering their surface thrombogenic. In order to define the mechanisms regulating the expression of tissue factor in the skin microvasculature, we examined tissue factor expression in human dermal microvascular endothelial cells. Quiescent human dermal microvascular endothelial cells did not constitutively express tissue factor protein, but were induced to express tissue factor by treatment with tumor necrosis factor-alpha in a time- and concentration-dependent fashion. Increased expression of tissue factor protein was accompanied by increases in steady-state mRNA levels. Tumor necrosis factor-alpha treatment resulted in increased expression of tissue factor heterogeneous nuclear RNA without changes in mRNA stability, suggesting that increased mRNA was mediated primarily via increased tissue factor gene transcription. In order to define the pathways regulating tissue factor induction, we examined the effects of MG-132, an inhibitor of nuclear factor-kappaB activation, PD98059, an inhibitor of MEK1 action, and SB203580, an inhibitor of activated p38 activity. MG132 only partially blocked tumor necrosis factor-alpha-induced tissue factor protein expression, despite an almost complete inhibition of tumor necrosis factor-alpha-induced E-selectin expression. In contrast, SB203580, almost completely inhibited tumor necrosis factor-alpha-induced tissue factor expression but inhibition of MEK1 by PD98059 had a minimal effect on tumor necrosis factor-alpha-mediated tissue factor induction in human dermal microvascular endothelial cells. Both SB203580 and MG132 treatment inhibited tumor necrosis factor-alpha-mediated increases in tissue factor mRNA and tissue factor gene transcription as measured by expression of tissue factor heterogeneous nuclear RNA. These data support a transcriptional role for both nuclear factor-kappaB and p38 mitogen-activated protein kinase, but not MEK1 in tissue factor gene expression in human dermal microvascular endothelial cells.

Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation.

OBJECTIVE: Upregulation of adhesion molecules on endothelial cells following irradiation has been shown, but the functional significance of this upregulation in various endothelial cell lines is not clear. We have developed an in vitro flow model to study the functional consequences of the radiation-induced upregulation of E-selectin and intercellular adhesion molecule (ICAM-1). METHODS: Human dermal microvascular endothelial cells (HDMEC), human umbilical vein endothelial cells (HUVEC), or transformed human microvascular endothelial cells (HMEC-1) were grown in 35-mm dishes and irradiated with a single dose of 10 Gy. HL-60 (human promyelocytic leukemia) cells were perfused over the irradiated endothelial cells in a parallel plate flow chamber at shear stress ranging from 0.5 to 2.0 dynes/cm2. Flow cytometry was used to quantify the expression of E-selectin and ICAM-1 on the various endothelial cells. RESULTS: Flow cytomeric analysis revealed an upregulation of ICAM-1 expression on all three cell types postirradiation (post-IR), and an upregulation of E-selectin expression only on HDMEC post-IR. E-selectin expression was detected on control HDMEC, but at a lower level than that detected on post-IR HDMEC. Flow assays revealed a significant increase in the number of rolling and firmly adherent HL-60 cells on post-IR HDMEC at shear stress < or =1.5 dynes/cm2; pretreatment of control and irradiated HDMEC with antibodies to E-selectin and ICAM-1 significantly diminished the number of rolling and firmly adherent HL-60 cells, respectively. No rolling or firm adhesion of HL-60 cells was observed on HUVEC or HMEC-1 monolayers post-IR. CONCLUSION: These findings suggest that ICAM-1 is upregulated on irradiated HDMEC, HUVEC, and HMEC-1. E-selectin is upregulated to a functional level only on irradiated HDMEC, and not on irradiated HUVEC or HMEC-1.

Pemphigus in the southeastern United States.

BACKGROUND: Our purpose was to review the course and management of pemphigus treated at a tertiary care center in the southeastern United States. METHODS: We describe 30 patients seen at the Emory Clinic from January 1992 to July 1999. RESULTS: Equal numbers of men and women from different ethnic backgrounds were affected. Pemphigus vulgaris was more common than pemphigus foliaceous. Pain, sore throat, and pruritus were the most common presenting symptoms. The mean diagnostic delay was 6 months in patients with pemphigus foliaceous and 4.5 months in patients with pemphigus vulgaris. Hospitalization was required in 47% of patients. Adjuvant therapy in addition to systemic orticosteroids was required in 93%. Herpes gingivostomatitis occurred in 33%. Clinical or total remission was obtained in 33%. CONCLUSIONS: Pemphigus occurs in multiple ethnic groups in the southeastern United States. Appropriate treatment is frequently delayed by lack of prompt diagnosis. The complications of pemphigus and its therapy were significant.

CD40 expression and function on human dermal microvascular endothelial cells: role in cutaneous inflammation.

CD40 is a member of the tumour necrosis factor (TNF) cell surface receptor family. It plays an important role in T-cell dependent B-cell functions and in dendritic cell development. It has also been identified as a cell surface receptor on endothelial cells. In this manuscript, we report that human dermal microvascular endothelial cells (HDMEC) express cell surface CD40 and that the expression of CD40 is increased by the proinflammatory cytokines TNF-alpha and interferon (IFN)-gamma. Additionally, we demonstrate that engagement of HDMEC CD40 with its recombinant CD40 ligand augments the induction of E-selectin, but not of intercellular or vascular cell adhesion molecules on the surface of HDMEC. Furthermore, we show that IFN gamma stimulation of HDMEC results in increased binding of Jurkat leucocytes to HDMEC by a CD40--CD154 dependent pathway. This study thus provides evidence that CD40 expression in HDMEC is regulated by proinflammatory cytokines, and that CD40 functions as an important mediator of cutaneous inflammation.

A review of cellular and molecular events of contact allergic dermatitis.

This biochemical review is presented for the first time in a dermatophytosis conference. The intent is to challenge biochemists to explain the formation of these lesions to clinicians.

Differential expression of active mitogen-activated protein kinase in cutaneous endothelial neoplasms: implications for biologic behavior and response to therapy.

BACKGROUND: Tumors of endothelium range from benign hemangiomas of infancy to highly malignant angiosarcomas of the elderly. Hemangiomas are the most common tumors in infants and may affect up to 10% of all children. The biologic behavior of these lesions ranges from self-resolving, in the case of hemangiomas and pyogenic granulomas, to lethal metastatic neoplasms in the case of angiosarcoma. Although the clinical outcomes of these diseases are easily distinguished, the biologic basis for these differences is not well understood. Activation of mitogen-activated protein kinase (MAPK) is an important signal transduction mechanism that may predict response of a tumor to chemotherapy. OBJECTIVE: Our purpose was to examine expression of phosphorylated (activated) MAPK in hemangiomas of infancy, pyogenic granulomas, hemangioendotheliomas, and angiosarcomas to determine whether phosphorylated MAPK was expressed in endothelial tumors. In addition, we examined endothelial tumors of infectious origin, Kaposi's sarcoma, and verruga peruana. METHODS: Skin sections from benign and malignant endothelial tumors, including hemangioma of infancy, angiosarcoma, and infectious endothelial lesions (Kaposi's sarcoma, verruga peruana) were stained with an antibody specific for phosphorylated MAPK. RESULTS: We demonstrated strong expression of phosphorylated MAPK in benign endothelial tumors, including capillary hemangioma of infancy and pyogenic granuloma, and greatly decreased expression in angiosarcoma. In addition, infectious endothelial tumors stained strongly with this antibody, similar to benign tumors. The presence of immunoreactive phosphorylated MAPK appears to be inversely correlated with degree of malignancy. CONCLUSION: We demonstrate that the use of antibodies specific for signal transduction pathways is feasible in paraffin-fixed tissue. Thus the activity of a given signal transduction pathway can be ascertained in a biopsy specimen. Immunohistochemistry for phosphorylated MAPK may help the pathologist distinguish benign from malignant endothelial processes and thus guide therapy.

Clinical review: thioureas and allergic contact dermatitis.

Thioureas are an uncommon underrecognized cause of allergic contact dermatitis (ACD). This article presents the findings in 3 individuals with ACD to thioureas and reviews the medical literature concerning thiourea-induced ACD. Thioureas are often the allergenic sources in ACD involving high-grade rubber products made of neoprene. Standard patch test series and rubber allergen patch test series usually do not contain thiourea allergens and will fail to diagnose these causes of ACD. Thioureas--most notably diethylthiourea, dibutylthiourea, and diphenylthiourea--should be considered in individuals with potential rubber allergy who fail to react to antigens in the standard allergen patch test tray.

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.

Induction of apoptosis in human dermal microvascular endothelial cells and infantile hemangiomas by interferon-alpha.

BACKGROUND: Hemangioma of infancy is an angiomatous disorder characterized by the proliferation of capillary endothelium. It has been shown that interferon-alpha (IFN-alpha) may induce involution of proliferating, life-threatening hemangiomas in children. This IFN-alpha-induced regression of hemangiomas is not accompanied by any T cell response nor by the occurrence of necrosis. METHODS: To determine whether IFN-alpha may induce apoptosis, we cultured human dermal microvascular endothelial cells (HDMEC) with IFN-alpha at concentrations of 100, 500, 1,000 and 2,000 U/ml for 24-72 h and detected apoptosis by terminal deoxynucleotidyl transferase mediated FITC-dUTP nick end labeling (TUNEL). Quantitative analysis was performed using the FACScan and morphological alterations were studied by confocal laser scanning microscopy. In addition to the in vitro study we also analyzed frozen skin sections from proliferating, spontaneously regressing, and IFN-alpha-treated hemangiomas by simultaneous determination of endothelial cells and apoptotic nuclei using an indirect immunofluorescence test in combination with TUNEL. RESULTS: Apoptosis was detected in up to 20% of the IFN-alpha-treated endothelial cells compared to the untreated controls. A maximum of apoptosis was observed after 48 h of stimulation with IFN-alpha in a dose-dependent manner. The analysis of hemangioma biopsies revealed apoptotic endothelial cells in IFN-alpha-treated as well as in spontaneously regressing hemangiomas, but not in proliferating ones. CONCLUSION: These data indicate that the therapeutic effect of IFN-alpha on hemangiomas is based on apoptosis induction of endothelial cells, which might also explain the clinically and histologically observed involution without any sign of inflammation or necrosis.

Neutralization of TNF by the antibody cA2 reveals differential regulation of adhesion molecule expression on TNF-activated endothelial cells.

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.

Culture and characterization of sinusoidal endothelial cells isolated from human liver.

Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.

Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We describe a novel method of measuring 2D binding characteristics of receptors and ligands that are attached to surfaces and whose bonds are subjected to forces. The method utilizes a common centrifugation assay to quantify adhesion. A model for the experiment has been formulated, solved exactly, and tested carefully. The model is stochastically based and couples the bond force to the binding affinity. The method was applied to examine tumor cell adherence to recombinant E-selectin. Satisfactory agreement was found between predictions and data. The estimated zero-force 2D Kd for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2), and the bond interaction range was subangstrom. Our results also suggest that the number of bonds mediating adhesion was small (<5).

The melanoma epidemic: more apparent than real?

To examine inconsistencies between the growth in incidence of melanoma and more modest changes in melanoma-related mortality, we reviewed the medical literature on the incidence of melanoma diagnosis and associated mortality and on the changes in diagnosis and mortality over time. Increases in melanoma surveillance activity have been associated with increases in the diagnosis of thin melanomas, but the incidence of advanced tumors has changed minimally. The large increases in diagnosis of melanoma without commensurate increases in advanced tumors and mortality are not compatible with presumed malignant behavior of thin melanomas. Increased intensity of melanoma surveillance may artificially increase the incidence of melanoma by harvesting histologically "malignant" but biologically benign tumors. Little available evidence suggests the presence of an actual increase in the frequency of biologically malignant tumors. Attempts to increase screening intensity for melanoma may result in further increases in diagnosis of melanomas. Nevertheless, limitations in histopathologic diagnostic techniques will continue to hinder efforts at early identification of those at risk for death from melanoma without diagnosing melanoma in large numbers of patients with biologically benign pigmented skin tumors.

Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation.

The endothelial cell-specific mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) represents a central regulator of cutaneous angiogenesis. Increased VPF/VEGF expression has recently been reported in psoriatic skin and healing wounds, both conditions in which transforming growth factor-alpha (TGF alpha) and its ligand, the epidermal growth factor receptor, are markedly up-regulated. Since TGF alpha strongly induces VPF/VEGF synthesis in keratinocytes, TGF alpha-mediated VPF/VEGF expression is likely to play a significant role in the initiation and maintenance of increased vascular hyperpermeability and hyperproliferation in skin biology. The objectives of the present studies were to determine the molecular mechanisms responsible for TGF alpha-induced transcriptional activation of the VPF/VEGF gene. We have identified a GC-rich TGF alpha-responsive region between -88 bp and -65 bp of the VPF/VEGF promoter that is necessary for constitutive and TGF alpha-inducible transcriptional activation. In electrophoretic mobility shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional TGF alpha-inducible protein complex that is distinct from Sp1 protein. Both AP-2 and Egr-1 transcription factors were detected as components of the TGF alpha-inducible protein complex in supershift EMSA studies. In co-transfection studies, an AP-2 but not an Egr-1 expression vector activated VPF/VEGF transcription, thus indicating that AP-2 protein is functionally important in TGF alpha-induced VPF/VEGF gene expression. By clarifying regulatory mechanisms that are critical for angiogenic processes in the skin, these studies may form the basis for new therapeutic strategies to modulate VPF/VEGF expression in cutaneous inflammation and wound healing.