Retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells.

The regulated expression of cell adhesion molecules (CAM) on endothelial cells is central to the pathogenesis of various inflammatory processes. Retinoic acid and synthetic derivatives have been demonstrated to exert antiinflammatory effects in cutaneous diseases. To determine modes of retinoid action in the modulation of inflammatory responses, we explored effects of all-trans-retinoic acid (t-RA) on the TNFalpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in cultured human dermal microvascular endothelial cells. Pretreatment with t-RA specifically prevented TNFalpha-induced VCAM-1 expression, but not ICAM-1 and E-selectin induction. t-RA significantly reduced VCAM-1-dependent T cell binding to TNFalpha-treated human dermal microvascular endothelial cells as well. This differential modulation of TNFalpha-induced CAM expression by t-RA was reflected at steady state mRNA levels and in nuclear run-on studies. In transcriptional activation studies, the TNFalpha-mediated activation of the human VCAM-1 promoter was inhibited after t-RA treatment, while the ICAM-1 promoter activation was unaffected, indicating that the selective inhibition of CAM expression is regulated in part at the level of gene transcription. Furthermore, the transcriptional inhibition by t-RA appears to be mediated by its effects upon the activation of NF-kappaB-dependent complex formation. Analysis of protein-DNA binding assays revealed marked inhibition of specific NF-kappaB-dependent binding to the tandem NF-KB sites of the VCAM-1 promoter, but not to the functional NF-kappaB motif of the ICAM-1 promoter. The specific inhibition of cytokine-mediated VCAM-1 gene expression in vitro may provide a potential basis by which retinoids exert their biological effects at sites of inflammation in vivo.

Human microvascular endothelial cells adhere to thrombospondin-1 via an RGD/CSVTCG domain independent mechanism.

Thrombospondin-1 (TSP-1), 450-kDa glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, plays an important role in angiogenesis, inflammation, and vascular occlusive skin diseases. Many of the physiologic and pathologic activities of TSP-1 are dependent upon its interactions with endothelial cells. To better understand the basis of these activities, we examined the mechanisms mediating the binding of human dermal microvascular endothelial cells (HDMEC) to immobilized TSP-1. HDMEC bound to but did not spread on TSP-1 in a concentration-dependent manner. Monoclonal antibodies (MoAbs) which recognize two purported TSP-1 binding proteins, CS36 and the alphav integrin chain, or TSP-1-derived peptides CGRGDS and CSVTCG, alone or in combination with heparin, did not inhibit HDMEC adhesion to immobilized TSP-1. Furthermore, CSVTCG-ovalbumin conjugates failed to support HDMEC adhesion. Although RGD-containing peptides immobilized on plastic wells supported HDMEC binding, they also induced cell spreading not characteristic of cell binding to TSP-1 and binding was inhibited by free RGD peptide. Two MoAbs against different domains of TSP-1 (A 4.1 and C 6.1) failed to block HDMEC binding to TSP-1, but both MoAbs inhibited G361 human melanoma cell binding to TSP-1 by 60%. Acid treatment of TSP-1 almost completely abrogated its ability to support HDMEC binding, while acid treatment inhibited G361 binding by 50%. However, either antibody completely abrogated G-361 cell binding to acid-treated TSP-1. These data demonstrate that HDMEC bind to immobilized TSP-1 in an RGD- and CSVTCG-independent manner via an acid labile epitope(s) which recognized via a receptor or receptors distinct from CD36 or alphavbeta3 integrin receptor.

Differential regulation of vascular cell adhesion molecule-1 gene transcription by tumor necrosis factor alpha and interleukin-1 alpha in dermal microvascular endothelial cells.

As part of the inflammatory response, the localization of leukocytes depends to an important degree on cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells (EC). We have previously shown that VCAM-1 expression is induced on human umbilical vein EC (HUVEC) by both tumor necrosis factor alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha), whereas on human dermal microvascular EC (HDMEC) only TNF alpha results in VCAM-1 expression. To explore molecular mechanisms responsible for these contrasting patterns of VCAM-1 induction in HUVEC versus HDMEC, we performed transcriptional activation studies with VCAM-1-based reporter constructs and in vitro binding assays using two adjacent NF-kappa B binding sequences of the VCAM-1 promoter as a DNA probe. Previous studies have established that these NF-kappa B motifs are required for cytokine-induced VCAM-1 transcription, and may further mediate cell-specific VCAM-1 gene activation by cytokines. The findings reported here demonstrate a significant HDMEC-specific attenuation of VCAM-1 gene transcription in response to IL-1 alpha, but not TNF alpha. An upstream VCAM-1 gene regulatory region distinct from the NF-kappa B sites appears to function as an IL-1 alpha-mediated transcriptional repressor within HDMEC. This repressor region conveys IL-1 alpha-dependent, but not TNF alpha-dependent, inhibition of transcription driven by a heterologous cytokine response element and promoter.

Nodular scleroderma.

We describe a 40-year-old woman with systemic scleroderma who had hundreds of firm nodules that developed on the trunk and upper extremities during several months. We briefly review previously reported cases of this rare variant of scleroderma.

Polarized expression and basic fibroblast growth factor-induced down-regulation of the alpha 6 beta 4 integrin complex on human microvascular endothelial cells.

Endothelial cells rest on a basement membrane that anchors them to the vessel wall. The alpha 6 beta 4 integrin complex has been described on epithelial cells, frequently localizes to basement-membrane structures, and appears to play a role in binding epithelial cells to laminin. We have determined that human microvascular endothelial cells express the beta 4 integrin chain in vivo and that it preferentially localizes to the endothelial basement membrane. Human microvascular endothelial cells and human umbilical vein endothelial cells also express cell-surface beta 4 in vitro. In addition, the expression of beta 4 appears to be polarized to the undersurface of endothelial cell monolayers in vitro, mimicking its in vivo localization. Stimulation of microvascular endothelial cells with basic fibroblast growth factor or phorbol 12-myristate 13-acetate, agents previously shown to induce endothelial cell migration in vitro, resulted in a marked decrease in cell-surface expression of the beta 4 integrin chain, associated with a decrease in beta 4 mRNA. These data demonstrate that human endothelial cells express the beta 4 integrin chain in vivo and in vitro, the expression of this integrin chain is polarized, and its expression is regulated on microvascular endothelial cells by factors important in wound healing and vascular regeneration.

Basic fibroblast growth factor increases expression of the alpha v beta 3 integrin complex on human microvascular endothelial cells.

Modulation of the expression of the alpha v beta 3 complex on human dermal microvascular endothelial cells (HDMEC) may be crucial in wound healing and angiogenesis. Therefore, we examined the influence of basic fibroblast growth factor (bFGF), transforming growth factor beta, and interferon-gamma (IFN-gamma) on the expression of this complex. Stimulation of HDMEC with bFGF increased cell surface expression of both alpha v and beta 3 in a dose- and time-dependent manner associated with the development of a spindled, elongated cell morphology. Northern blot analysis of HDMEC stimulated with bFGF demonstrated a marked increase in beta 3 but not alpha v mRNA expression. Incubation of HDMEC with transforming growth factor-beta or interferon-gamma alone resulted in modest decreases in cell surface alpha v beta 3, and co-incubation of HDMEC with bFGF and transforming growth factor-beta or interferon-gamma inhibited bFGF-induced changes in cell morphology, increases in cell surface alpha v beta 3 expression, and increases in beta 3 mRNA. These data demonstrate that both growth factors and pro-inflammatory cytokines alter the expression of alpha v beta 3 on microvascular endothelial cells and that these alterations correlate with changes in cell morphology.

Selective upregulation of intercellular adhesion molecule (ICAM-1) by ultraviolet B in human dermal microvascular endothelial cells.

Although a portion of ultraviolet light (UV) penetrates into the dermis, histologic changes that occur within the dermal microvasculature have largely been attributed to the elaboration of biologic substances, such as interleukin 1 (IL-1), from other constitutive cells of the skin, as opposed to a direct effect of UV on the endothelial cell. As a potential model for understanding early molecular events occurring in UV-induced cutaneous inflammation, we have examined the direct effects of UVB, as well as cytokine-positive controls, upon human dermal microvascular endothelial cells (HDMEC) cell adhesion molecule (CAM) gene expression. Cultured HDMEC were exposed to varying dosages of UVB, and examined for cell surface and mRNA expression of the CAMs intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (formerly ELAM-1). Following UVB exposure, dose-dependent increases in baseline cell surface expression of ICAM-1 were demonstrated by fluorescence-activated cell sorter analysis with concomitant increases in ICAM-1 mRNA, as shown by Northern blot analysis; there was no induction of either E-selectin or VCAM-1. The UVB-induced ICAM-1 upregulation could not be blocked by antibodies to IL-1 or tumor necrosis factor alpha (TNF-alpha). In fact, ICAM-1 gene regulatory region based CAT reporter gene plasmids, including constructs containing IL-1- and TNF-alpha-responsive elements, did not display increased CAT expression after transfection into HDMEC followed by UVB exposure, though control cytokine-treated transfectants did. Thus, UVB selectively upregulates ICAM-1, but not E-selectin or VCAM-1, mRNA and cell surface expression in HDMEC, and this upregulation is not dependent upon the autologous secretion and activity of either IL-1 or TNF-alpha.

Characterization of expression and modulation of cell adhesion molecules on an immortalized human dermal microvascular endothelial cell line (HMEC-1).

We have recently reported the creation of the first immortalized cell line derived from human dermal microvascular endothelial cells (HMEC-1). In preliminary studies this line was found to closely resemble microvascular endothelial cells in regard to many phenotypic characteristics. Because two key functional features of endothelial cells are their ability to bind to peripheral blood leukocytes and extracellular matrix proteins via cell adhesion molecules, we have now characterized HMEC-1 in terms of expression and regulation of cell adhesion molecules of the integrin, immunoglobulin gene superfamily, and selectin families. HMEC-1 can either constitutively express or can be induced to express key integrins, including alpha-1, -2, -3, -4, -5, -6, and -V, as well as beta-1, -3, -4, and -5. They also express or are capable of expressing immunoglobulin gene superfamily molecules, such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and a member of the selectin family, E-selectin. A number of important cell adhesion molecules that are either constitutively expressed or that must be induced are regulated in a time- and dose-dependent fashion by selected cytokines. Experiments comparing the phenotypic characteristics of HMEC-1 with human dermal microvascular endothelial cells or human umbilical vein endothelial cells reveal HMEC-1 to have features of both small- and large-vessel endothelial cells.

A factor in human plasma permits persistent expression of E-selectin by human endothelial cells.

E-selectin is an inducible endothelial cell adhesion protein that is a critical element in the binding of leukocytes to activated endothelial cells. It is induced by a variety of pro-inflammatory soluble substances including interleukin-1 (IL-1), tumor necrosis factor (TNF), or bacterial lipopolysaccharide (LPS). In vitro studies of a large vessel endothelial cells demonstrate that stimulation with TNF or IL-1 leads to a rapid, but transient, induction of E-selectin expression that disappears within 24 hours. However, in vivo studies have shown that microvascular endothelial cells persistently express E-selectin in chronic inflammatory states, particularly in the skin where it serves as a homing receptor for memory T cells. Stimulation of dermal-derived microvascular endothelial cells (HDMECs) with single doses of IL-1 alpha, TNF alpha, or LPS resulted in transient but slightly more persistent expression of E-selectin than seen after stimulation of large vessel derived umbilical vein endothelial cells (HUVECs). However, stimulation of either HDMECs or HUVECs with repetitive doses of IL-1 alpha, TNF alpha, or LPS in the presence of human serum or plasma resulted in persistent E-selectin expression in vitro. The persistent E-selectin cell surface expression was associated with persistent E-selectin mRNA expression and correlated with E-selectin-mediated HL-60 binding to endothelial cell monolayers. The effect of human plasma or serum was dose dependent, and fractionation of human plasma by gel filtration demonstrated that the E-selectin persistence activity resolved into high and low molecular peaks. These data demonstrate that human endothelial cells are capable of persistent E-selectin expression in vitro and that factors in human serum or plasma are critical in preventing cytokine refractoriness and loss of E-selectin expression. This study provides a basis to resolve the apparent discrepancies between previous in vivo and in vitro dynamics of E-selectin expression.

Monoclonal antibody modulates human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (fMLP).

Utilizing standard hybridoma techniques and a neutrophil chemotaxis assay for screening we produced a mouse monoclonal IgM antibody, 59/4, selected for specific inhibition of human neutrophil chemotaxis to the N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP). Antibody 59/4 inhibited neutrophil chemotaxis to fMLP, but not human C5a or leukotriene B4. The antibody exhibited specific homogeneous binding to PMNs, heterogeneous binding to monocytes, and did not bind to lymphocytes in a pattern similar to the profile of N-formyl peptide binding in flow cytometric analysis. The antibody did not inhibit the binding of fluorescein-conjugated fMLPK or fML(3H)P ligands to neutrophils in flow cytometric or competitive binding assays. Other neutrophil functions including myeloperoxidase release and rosette formation with immunoglobulin or immunoglobulin C3b-coated sheep erythrocytes were not affected in the presence of antibody 59/4. These results suggest that 59/4 specifically inhibits chemotaxis to fMLP.

A 5' portion of the ICAM-1 gene confers tissue-specific differential expression levels and cytokine responsiveness.

Intercellular adhesion molecule-1 (ICAM-1), a cell-adhesion molecule critically involved in leukocyte trafficking and adherence, displays tissue-specific and cytokine-specific expression profiles. Although human dermal microvascular endothelial cells (HDMEC) constitutively express ICAM-1, keratinocytes (HK) do not. Interleukin-1 (IL-1) upregulates ICAM-1 expression in HDMEC, but fails to do so in either HK or A431, a human squamous carcinoma cell line, even though both have IL-1 receptors and express ICAM-1 on exposure to other cytokines. We have previously characterized a human ICAM-1 genomic clone that contains the 5' flanking transcriptional regulatory region. To test the hypothesis that tissue- and cytokine-specific ICAM-1 gene expression results from the interaction of constitutive and inducible tissue-specific trans-acting factors with distinct cis-elements of the ICAM-1 gene, various ICAM-1-based reporter gene (CAT) plasmids were constructed. Transcriptional activity of these various constructs was assessed after transient transfection into HDMEC and A431. A critical ICAM-1 region was identified that conferred enhanced expression of CAT in HDMEC and suppressed expression of CAT in A431. This same region further enhanced CAT expression in transfected HDMEC treated with IL-1 alpha, yet no such enhancement was seen with IL-1 treatment of identically transfected A431. However, treatment of A431 transfectants with IFN gamma did result in enhanced CAT expression, demonstrating reversal of A431 cell context suppression of the ICAM-1-based reporter gene construct. These data implicate the existence of both tissue- and cytokine-specific responsive elements in the 5' flanking region of the ICAM-1 gene and demonstrate that regulatory effects directed by such elements are dependent upon their cellular context. Moreover, they provide the basis for identification of specific cis-acting genetic elements, the trans-acting factors with which they interact, and the molecular mechanisms by which they regulate transcription of the ICAM-1 gene.

Antigen presentation by a continuous human microvascular endothelial cell line, HMEC-1, to human T cells.

Endothelial cells line the vessels and lymphatics of the body, acting as a barrier between the blood and extravascular tissue. These cells are, therefore, in a prime position to play a role in lymphocyte activation. Indeed, it has been shown that primary endothelial cells in culture are capable of presenting particulate and soluble antigens to T cells and that this response is not dependent on macrophages. Recently, we developed an immortalized line of human microvascular endothelial cells, CDC/EU.HMEC-1 (HMEC-1). This endothelial line has the advantage not only of being devoid of contaminating cells but also of being a continuous cell line and therefore not subject to a restricted number of useful passages. The focus of this study was to determine whether HMEC-1 cells (like primary endothelial cells) could present antigen to T cells in the absence of macrophages. We demonstrate that a cloned and purified endothelial cell line can independently provide all the necessary signals for antigen-specific T-cell activation.

Use of a human microvascular endothelial cell line as a model system to evaluate cholesterol uptake.

CDC/EU.HMEC-1 (HMEC-1) cells provide a reliable source of human microvascular endothelial cells free of mycoplasma and viral infection. This cell line has potential for use in the further study of the endothelial cell modification of low-density lipoproteins and for anticholesterol drug evaluation assays. HMEC-1 cells will fill a gap that is present for in vitro investigations of cholesterol metabolism in conjunction with previously established hepatic, monocytic, or macrophage cell lines. This paper presents a simple assay that demonstrates a linear uptake of tritiated cholesterol by he HMEC-1 cells and shows that the cellular cholesterol load can be regulated using anticholesterol drugs.

Role of microvascular endothelial cells in inflammation.

Endothelial cells are critical elements in the evolution of all types of cutaneous inflammation. They participate through the synthesis and secretion of pro-inflammatory cytokines, including interleukin 1 (IL-1), IL-6, and IL-8, as well as M-CSF, G-CSF, GM-CSF, gro alpha, and MCP. They also express a series of cell-surface proteins and glycoproteins known as cell adhesion molecules that allow circulating leukocytes to bind to endothelial cells and allow endothelial cells to bind to matrix proteins. The regulated expression of these molecules, including those in the integrin, immunoglobulin gene, and selection families, allows for the precise trafficking of circulating leukocytes to sites of inflammation, injury, or immunologic stimulation in the skin. Furthermore, emerging evidence clearly indicates that selected differences exist between endothelial cells of the microvasculature and those that line large blood vessels. These include differences in secreted products, differences in the expression of cell adhesion molecules, and differences in cytokine-induced regulation of commonly expressed cell adhesion molecules, among others. Thus, a precise delineation of the biology of cutaneous microvascular endothelial cells is important to our understanding of cutaneous inflammation.

Expression and modulation of the vitronectin receptor on human dermal microvascular endothelial cells.

Microvascular endothelial cells express a variety of cell-surface integrins in vivo and in vitro with varying affinities for matrix proteins. The vitronectin receptor (VnR), a complex of the alpha v and beta 3 integrin chains, is capable of binding to a variety of matrix proteins that are deposited in injured tissues, including vitronectin, fibrinogen, and thrombin. Staining of frozen sections of human skin with antibodies recognizing the VnR and examination by immunofluorescence microscopy demonstrates staining in a vascular pattern suggesting in vivo expression of the vitronectin receptor on endothelial cells. Examination of pure cultures of human dermal microvascular endothelial cells (HDMEC) by flow-cytometric analysis and enzyme-linked immunosorbent assay confirmed that HDMEC also express cell surface VnR complex in vitro. Stimulation of human dermal microvascular endothelial cells in vitro with agents that stimulate protein kinase C resulted in dose- and time-dependent increases in expression of alpha v and beta 3 integrin chains. Additionally, stimulation with basic fibroblast growth factor induced similar increases, but stimulation with transforming growth factor-beta or interleukin-1 alpha failed to increase VnR expression. Increases in cell-surface VnR expression also correlated with an increased ability of microvascular endothelial cells to bind to vitronectin, but not fibronectin-coated surfaces. Although increases in cell-surface expression of beta 3 paralleled increases in expression of cell-surface alpha v, regulation of mRNA expression was distinct for each chain. These data suggests that microvascular endothelial cells express the VnR complex in vivo, that the cell-surface expression of this integrin on dermal microvascular endothelial cells can be regulated, and that this regulation may be important in cell adherence, cell migration, and wound healing.

HMEC-1: establishment of an immortalized human microvascular endothelial cell line.

The study of human microvascular endothelial cells has been limited, because these cells are difficult to isolate in pure culture, are fastidious in their in vitro growth requirements, and have a very limited lifespan. In order to overcome these difficulties, we have transfected human dermal microvascular endothelial cells (HMEC) with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and succeeded in immortalizing them. These cells, termed CDC/EU.HMEC-1 (HMEC-1), have been passaged 95 times to date and show no signs of senescence, whereas normal microvascular endothelial cells undergo senescence at passages 8-10. HMEC-1 exhibit typical cobblestone morphology when grown in monolayer culture, express and secrete von Willebrand's Factor, take up acteylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel. HMEC-1 grow to densities three to seven times higher than microvascular endothelial cells and require much less stringent growth medium. HMEC-1 will grow in the absence of human serum, whereas microvascular endothelial cells require culture medium supplemented with 30% human serum. These cells express other cell-surface molecules typically associated with endothelial cells, including CD31 and CD36 and epitopes identified by monoclonal antibodies EN4 and PAL-E. They also express the cell adhesion molecules ICAM-1 and CD44 and following stimulation with interferon-gamma express major histocompatibility complex class II antigens. HMEC-1 specifically bind lymphocytes in cell adhesion assays. Thus HMEC-1 is the first immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells.

Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells.

Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.