Human endothelial cell cultures: phenotypic modulation by leukocyte interleukins

We report here that soluble factors from activated mononuclear leukocytes have a dramatic effect on cultured endothelial cells. While human umbilical vein endothelial cells grown under standard conditions show a polygonal, epithelial-like morphology, cells exposed to culture media conditioned by lectin-activated human mononuclear leukocytes become extremely elongated and/or send out numerous cytoplasmic processes, assuming a dendritic configuration. This effect cannot be mimicked by exogenous cyclic AMP, is reversible upon interruption of the treatment, and appears specific for endothelial cells, since it has not been observed so far with other cell types. The shape changes are accompanied by a reorganization of the endothelial cell cytoskeleton: actin microfilament bundles tend to be disposed in parallel arrays, while intermediate filaments and microtubules penetrate up to the extremity of the cytoplasmic processes. Colchicine prevents endothelial cell elongation but only slightly impairs the formation of lateral cell processes ("dendritic configuration"). Purified interleukins were tested for their ability to induce these changes of cell shape. Escherichia coli-recombinant human interleukin 2 had no effect, and gamma-interferon only a slight effect on endothelial cell morphology. Interleukin 1 induced moderate cell elongation, while combined treatment with both interleukin 1 and gamma-interferon resulted in shape changes indistinguishable from those elicited by supernatants of activated mononuclear leukocytes. The possible relevance of the observed endothelial cell changes to the reported angiogenic activity of mononuclear cell products is discussed.     

Exaggerated human vascular cell prostaglandin biosynthesis mediated by monocytes: role of monokines and interleukin 1

Incubation of cultured human umbilical vein endothelial cells with factors derived from human peripheral blood mononuclear cells (MNCF) or adherent monocytes (AMF) resulted in concentration-and time-dependent increases in prostacyclin and prostaglandin E2 (PGE2) production. MNCF and AMF also stimulated prostacyclin and PGE2 biosynthesis in cultured human arterial smooth muscle cells and human dermal fibroblasts. The effect of these monokines on endothelial cells and fibroblasts was mimicked by treatment with purified human interleukin 1 (IL 1). Mononuclear cell-conditioned medium subjected to gel filtration yielded fractions (Mr 12,000 to 18,000 daltons) which simultaneously contained endothelial cell and fibroblast prostaglandin-stimulating activity and IL 1 activity. Therefore, monokines, specifically IL 1, appear to serve as chemical mediators of the interaction between monocytes and vascular cells as would occur in blood vessel injury, inflammation, and atherosclerosis.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

Evidence for an endothelial cell-derived factor which stimulates the growth of human peripheral blood mononuclear cells

Evidence is provided which demonstrates that conditioned media of cultured endothelial cells derived from human umbilical veins contained a factor which stimulated peripheral blood mononuclear cell [3H]thymidine uptake. A dose-dependent response in peripheral blood mononuclear cell [3H]thymidine uptake was obtained when cells were incubated with increasing concentrations of supernatant of endothelial cell cultures. Studies on temporal kinetics demonstrated that stimulatory activity was evident when mononuclear cells had been incubated with endothelial cell supernatant for 120 hr or more. Preliminary characterization showed the growth immunoregulatory factor to have a molecular weight greater than 100,000 Da.     

Localization of neuropeptide Y and atrial natriuretic peptide in the endothelial cells of human umibilical blood vessels

The localization of neuropeptide Y (NPY) and atrial natriuretic peptide (ANP) in the endothelial cells of human umbilical blood vessels was studied using the pre-embedding peroxidase-antiperoxidase (PAP) technique for electron microscopy and avidin-biotin-complex (ABC) immunostaining for endothelial cells cultured from umbilical vein. Subpopulations of NPY- and ANP-immunoreactive endothelial cells were present in term umbilical vein and artery. The umbilical vein contained more positive cells than the artery. The percentage of NPY- and ANP-immunoreactive umbilical vein cells in culture was 32% and 44%, respectively, out of a total of 3013 cells examined. The possibility that these potent vasoactive substances located in the endothelial cells of the non-innervated umbilical vessels are involved in the local regulation of blood flow is discussed.     

The effect of second-line antirheumatic drugs on interleukin-8 mRNA synthesis and protein secretion in human endothelial cells.

Interactions between interleukin 8 (IL-8) and endothelial cells play an important role in the emigration of mononuclear cells from the blood into areas of inflammation. We examined the ability of specific second-line antirheumatic drugs to regulate (IL-8) gene expression and protein secretion in interleukin 1 (IL-1) stimulated human umbilical vein endothelial cells and peripheral blood mononuclear cells. The drugs sodium aurothiomalate, D-penicillamine and sulphasalazine were all able to modulate IL-8 mRNA synthesis in and protein secretion from endothelial cells. A bimodal effect was observed: at low concentrations IL-8 was suppressed, whereas higher concentrations resulted in an increased IL-8 production. In endothelial cells, treatment with hydrocortisone led to a linear suppression of IL-8 production in concentrations ranging from 0.5 micrograms/ml up to 500 micrograms/ml. Sulphapyridine, auranofin, hydroxychloroquine and methotrexate, had no effect on IL-8 secretion in endothelial cells. By contrast, 5-aminosalicylic acid induced a threefold increase in the IL-8 release. In peripheral blood mononuclear cells it was only possible to suppress the IL-8 production by hydrocortisone treatment. These results indicate that suppression of IL-8 production in endothelial cells could be an important factor in the mode of action for a number of second-line antirheumatic drugs.     

Detection of glycosaminoglycans on the surface of human umbilical vein endothelial cells using gold-conjugated poly-l -lysine with silver enhancement

Summary Endothelial glycosaminoglycans are important in a diverse range of vascular functions. In the course of a biochemical and histological study exploring the role of glycosaminoglycans in inflammation, we have investigated the use of gold-conjugated poly-l-lysine with silver enhancement to establish the nature and physical location of glycosaminoglycans on the surface of cultured human umbilical vein endothelial cells. Cationic gold was effective in locating anionic sites in both cultured endothelial cells and in paraffin-embedded renal tissue. By manipulating pH, and by using enzymes specific for degrading glycosaminoglycans, it was found that, at pH 1.2, staining was directed primarily at glycosaminoglycans. The surface of human umbilical vein endothelial cells was found to be extensively covered in heparan sulphate, the histological appearance of which was dependent upon the fixation procedure employed. Heparan sulphate was also seen to co-distribute with the extracellular matrix protein, fibronectin, when endothelial cultures were simultaneously stained with cationic gold and an antibody to cellular fibronectin.     

DDAVP-induced release of von Willebrand factor from endothelial cells in vitro: the effect of plasma and blood cells

The vasopressin analogue 1-deamino-8-D-arginine vasopressin (DDAVP) causes an immediate, transient rise in plasma levels of von Willebrand factor (vWF) after its administration. Although it is recognized that vascular endothelial cells play an essential role in this process, the molecular basis of the response is not understood. We have investigated the phenomenon using human umbilical vein endothelial cells as an in vitro model. When normal individuals were stimulated with DDAVP, plasma from blood samples collected subsequently caused the release of vWF from cultured endothelial cells over a 24 h period (22-46% increase over baseline), compared to control plasma (5-17%). DDAVP added directly to the endothelial cells produced no increase in vWF release. When whole blood was treated in vitro with DDAVP, and the plasma subsequently added to endothelial cells, a significant increase in vWF secretion was found. Peripheral blood mononuclear cells were then tested. In the presence of DDAVP, an increased response occurred. Further fractionation of these cells showed that monocytes were largely responsible, causing an increased vWF release of 162% at 2 h. These observations were reinforced by finding that the supernatants of monocytes incubated with DDAVP were also effective in causing increased vWF release (118% compared to 58% for the control sample). Our studies suggest that DDAVP plays an indirect role in causing the release of vWF from endothelial cells, and that peripheral blood monocytes may act as intermediary target cells, which then produce factor(s) acting directly on endothelial cells.     

An affordable method to obtain cultured endothelial cells from peripheral blood

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media‐2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell‐like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above‐mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Biomimetic hydrogels with VEGF induce angiogenic processes in both hUVEC and hMEC

Angiogenesis is the process by which new blood vessels arise from the pre-existing vasculature. Human endothelial cells are known to be involved in three key cellular processes during angiogenesis: increased cell proliferation, degradation of the extracellular matrix during cell migration, and the survival of apoptosis. The above processes depend upon the presence of growth factors, such as vascular endothelial growth factor isoform 165 (VEGF(165)) that is released from the extracellular matrix as it is being degraded or secreted from activated endothelial cells. Thus, the goal of the current study is to develop a system with a backbone of polyethylene glycol (PEG) and grafted angiogenic signals to compare the initial angiogenic response of human umbilical vein endothelial cells (hUVEC) or human microvascular endothelial cells (hMEC). Adhesion ligands (PEG-RGDS) for cell attachment and PEG-modified VEGF(165) (PEG-VEGF(165)) are grafted into the hydrogels to encourage the angiogenic response. Our data suggest that our biomimetic system is equally effective in stimulating proliferation, migration, and survival of apoptosis in hMEC as compared to the response to hUVEC.     

Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks

Endothelial progenitor cells (EPC) derived from the circulation may be used to enhance neovascularization. Since the combination of granulocyte colony-stimulating factor (GCSF) and CXCR4 antagonist AMD3100 efficiently mobilizes hematopoietic stem cells into peripheral circulation, it may increase the pool of endogenously circulating EPC. We tested this hypothesis by administering GCSF and AMD3100 to adult rabbits and rats, isolating mononuclear cells from peripheral blood by Ficoll density gradient centrifugation, and characterizing the blood-derived EPC based on morphology, immunophenotyping, gene expression and other functional analyses. These EPC showed clonal growth similar to that of human umbilical vein endothelial cells when cultured in complete EGM-2 medium on collagen I-precoated culture plates. The EPC exhibited a typical cobblestone-like morphology and were relatively homogeneous by the third passage. The cells expressed the typical endothelial marker CD31 based on flow cytometry and fluorescence microscopy, formed capillary-like structures when cultured in Matrigel, internalized DiI-acetylated low-density lipoprotein, bound Ulex europaeus agglutinin-1, and expressed CD31 and several other endothelial markers (VEGFR2, VE-cadherin, Tie-2, eNOS, vWF) at significantly higher levels than bone marrow-derived mesenchymal stem cells. These results suggest that the combination of GCSF and AMD3100 can efficiently release stem cells into peripheral circulation and generate EPC that show the desired morphological, immunophenotypic and functional characteristics. This minimally invasive approach may be useful for autologous cell transplantation for postnatal neovasculogenesis and tissue repair.     

The platelet glycoprotein IIb/IIIa-like protein in human endothelial cells promotes adhesion but not initial attachment to extracellular matrix

On platelets the membrane glycoprotein IIb/IIIa complex (GPIIb/IIIa) functions in adhesive interactions with fibrinogen, von Willebrand factor, and fibronectin. However, the function of GPIIb/IIIa-like proteins on endothelial cells, as well as the ligand(s) the complex binds, is unknown. Using a highly specific polyclonal antibody we have explored the function of GPIIb/IIIa-like proteins on human umbilical vein endothelial cells (HUVE). Analysis by immunoblotting shows that this antiserum recognizes the endothelial GPIIIa-like protein of the complex. The IgG fraction of the polyclonal antiserum and its Fab' fragments detach confluent and subconfluent HUVE from extracellular substrata. The effect of the anti-GPIIb/IIIa IgG is not toxic as the detached cells maintain their viability after trypsinization and replating. Anti-GPIIb/IIIa IgG does not inhibit HUVE binding to extracellular matrix or purified fibronectin in an attachment assay despite the presence of intact GPIIb/IIIa on HUVE detached from substrate by various methods. Apparently, the GPIIb/IIIa-like protein on HUVE is important in normal HUVE adhesion to the extracellular matrix, but it is not required in the initial attachment of HUVE to extracellular matrix.     

Vascular endothelial cells synthesize and secrete brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) is an abundant neurotrophin in brain and peripheral nerves, where it affects neural development, survival and repair after injury. BDNF has been detected in rat and human blood, but the source of circulating BDNF is not established. BDNF messenger and peptide were detected in cultured cells and in the culture medium of human umbilical vein endothelial cells. The expression of BDNF was up-regulated by elevation of intracellular cAMP and down-regulated by Ca(2+) ionophore, bovine brain extract and laminar fluid shear stress. These results suggest that vascular endothelial cells may contribute to circulating BDNF.     

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.     

The influence of exogenous fibronectin on blood granulocyte adherence to vascular endothelium in vitro

Fibronectin is a major cell surface and extracellular matrix glycoprotein. It binds to a variety of substrata and supports the attachment and spreading of a number of cell types. We have found that purified human plasma fibronectin can also support blood granulocyte adhesion to cultured human umbilical vein endothelial cells. This activity is protected by treatment of the fibronectin with a sulphhydryl-containing agent. The effect of granulocyte attachment was observed at fibronectin concentration of 100 ng/ml with maximum effect at a concentration of 10 μg/ml. The attached granulocytes retained a rounded appearance, compared with the flattening that occurs on attachment to plastic. Granulocytes attached poorly to cultured human vascular smooth muscle cells and no enhancement occurred when fibronectin was added. Immunofluorescence microscopy using monospecific rabbit anti-human fibronectin demonstrated that the sulphhydryl-treated fibronectin accumulated on the endothelial cell surface, forming aggregates on the apical surface by 3 h of continued incubation. Washed, cultured endothelial cells not exposed to fibronectin or exposed to untreated purified plasma fibronectin did not demonstrate an aggregation of cell-surface fibronectin.     

Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1).

Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.     

Determination of fifteen nucleotides in cultured human mononuclear blood and umbilical vein endothelial cells by solvent generated ion-pair chromatography

The paper describes the development of a method for the determination of 15 nucleotides in cultured mononuclear blood and umbilical vein endothelial cell lysates by solvent generated ion-pair chromatography. The phase system is generated via a mobile phase of 100 mM phosphoric acid adjusted to pH 6.2 with triethylamine. Nucleotides are eluted by applying a linear magnesium ion gradient. The method is robust, highly reproducible and easily adaptable to other cell lysates and allows the separation and quantitation of the nucleotides with detection limits in the range from 17 (ADP) to 126 (CDP) pmol in 20-microl aliquots.