Primary Human Umbilical Vein Endothelial Cells Research Articles

Anthocyanin attenuates CD40-mediated endothelial cell activation and apoptosis by inhibiting CD40-induced MAPK activation

CD40-mediated inflammatory signaling is a potent activator of endothelial cells (ECs) and effective in triggering the pathogenesis of atherosclerosis, a chronic inflammatory disease. Anthocyanin is considered to exert potent cardiovascular-protective effect partially through its anti-inflammatory property, however, the precise mechanism is still unknown. Here we chose cultured human umbilical vein endothelial cells (HUVECs) to explore the influence of anthocyanin on CD40-mediated endothelial activation and apoptosis and the underlying mechanism. Stimulation of human primary HUVECs by CD40 with its physiological ligand CD40L not only augmented MMP-1, -9 secretion and promoted MMP-1, -9 activities, but also induced endothelial cell apoptosis and death. Treatment of ECs with anthocyanins cyanidin-3- O-β-glucoside (Cy-3-g) and peonidin-3- O-β-glucoside (Pn-3-g) prevents CD40-induced endothelial activation by inhibiting production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In addition, exposure to anthocyanins inhibits CD40-induced endothelial apoptosis. Anthocyanins also decreased activation of JNK and p38 induced by CD40. Collectively, our findings suggested that the inhibition of JNK and p38 activation interrupts CD40 induced endothelial cell activation and apoptosis, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin and its athero-protective function.

Glucagon-like peptide-1 attenuates tumour necrosis factor-α-mediated induction of plasmogen activator inhibitor-1 expression

Glucagon-like peptide-1 (GLP-1) has been proposed as a target for treatment of type 2 diabetes. GLP-1 has also been demonstrated to improve endothelial cell dysfunction in diabetic patients. Elevated plasminogen activator inhibitor type-1 [corrected] (PAI-1) levels have been implicated in endothelial cell dysfunction. The effect of GLP-1 on PAI-1 expression in vascular endothelial cells has not been explored. In a spontaneously transformed human umbilical vein endothelial cell (HUVEC) line, C11-spontaneously transformed HUVEC (STH) and primary HUVEC cells, GLP-1 treatment, in the presence of a dipeptidyl peptidase IV inhibitor, attenuated induction of PAI-1 protein and mRNA expression by tumour necrosis factor-alpha (TNF-alpha). GLP-1 also inhibited the effect of TNF-alpha on a reporter gene construct harbouring the proximal PAI-1 promoter. In addition, GLP-1 attenuated TNF-alpha-mediated induction of Nur77 mRNA and TNF-alpha-mediated binding of nuclear proteins (NPs) to the PAI-1, Nur77, cis-acting response element nerve growth factor induced clone B response element (NBRE). GLP-1 treatment also inhibited TNF-alpha-mediated induction of Akt phosphorylation. Taken together, these observations suggest that GLP-1 inhibits TNF-alpha-mediated PAI-1 induction in vascular endothelial cells, and this effect may involve Akt-mediated signalling events and the modulation of Nur77 expression and NP binding to the PAI-1 NBRE.

Intracellular signaling pathways involved in cell growth inhibition of human umbilical vein endothelial cells by melatonin

Melatonin, an indolamine mainly produced in the pineal gland, has received a great deal of attention in the last decade because of its oncostatic effects, which are due to its immunomodulatory, antiproliferative, antioxidant and its possible antiangiogenesis properties. Herein, we document its antiproliferative action on human umbilical vein endothelial cells (HUVECs). Moreover, the possible cell signaling pathways when melatonin inhibited HUVEC proliferation were explored in this study. Primary HUVECs were isolated, cultured, purified and identified before the studies were performed. HUVECs were found to possess G-protein-coupled membrane receptors for melatonin (MT1 and MT2) and also nuclear melatonin receptors (RORalpha and RORbeta, especially RORbeta). No obvious expression of RORgamma was found. We investigated the membrane receptors and several intracellular signaling pathways including mitogen-activated protein kinases (MAPK)/extracellular signal-related kinases (ERK), phosphoinositol-3-kinase (PI3K)/Akt and protein kinases C (PKC) involved in antiproliferative action of melatonin on HUVECs. The blockade of these pathways using special inhibitors decreased cell growth. Furthermore, the constitutive activation of nuclear factor kappa B (NF-kappaB) contributed to the proliferation of HUVECs. High concentrations of melatonin inhibited both NF-kappaB expression and its binding ability to DNA, possibly through inactivation of ERK/Akt /PKC pathways. Taken together, high concentrations of melatonin markedly reduced HUVEC proliferation; the antiproliferative action of melatonin was closely correlated with following pathway: melatonin receptors/ERK/PI3K/Akt/PKC/ NF-kappaB.

Binding of ADAM28 to P-selectin Glycoprotein Ligand-1 Enhances P-selectin-mediated Leukocyte Adhesion to Endothelial Cells

ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions.

Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVEC)

Human-derived endothelial cells can now be routinely harvested from human umbilical veins. Studies with human umbilical vein endothelial cells (HUVEC) have been conducted with cells from passage 2 to 5. It is now also possible to cryopreserve primary and early-passaged HUVEC for future propagation and for forwarding to an end user by express courier. Stored HUVEC have been stably retrieved even after several years. These retrieval techniques have facilitated the deployment of HUVEC for many studies, including those for homeostasis, inflammatory disorders, atherosclerosis, cancer, and microbial adhesion and invasion. In this unit, we will delineate the procedure for harvesting, propagation, and storage of HUVEC.

Comparison of the pro-inflammatory potential of monocytes from healthy adults and those with peripheral arterial disease using an in vitro culture model

We adapted a monocyte:endothelial cell co-culture model to investigate the pro-inflammatory potential of monocytes from patients with peripheral arterial disease (PAD). Isolated monocytes were cultured with human umbilical vein endothelial cells (HUVEC) for 24 h, after which the ability of the HUVEC to recruit flowing neutrophils was tested. Development of a usable protocol required comparisons of primary HUVEC with cells that had been passaged and/or frozen and thawed, evaluation of optimal culture media and comparison of monocytes from freshly drawn and stored blood. We found, for instance, that expansion of HUVEC was assisted by inclusion of hydrocortisone, but this agent was withdrawn before the test phase because it reduced responses of HUVEC. Using the optimal practical protocol, we found great variation in the ability of monocytes from different donors to cause neutrophil adhesion. Slightly more (∼20%) monocytes from patients with PAD adhered to HUVEC than monocytes from healthy controls, and the monocytes from PAD patients induced ∼70% greater subsequent adhesion of neutrophils. Thus, we developed a functional model of inflammatory potential usable in clinically-related studies and found that patients with PAD had circulating monocytes with greater than normal ability to activate endothelial cells.

Eicosapentaenoic acid inhibits TNF-α-induced Lnk expression in human umbilical vein endothelial cells: involvement of the PI3K/Akt pathway

n−3 Polyunsaturated fatty acids (PUFAs) exert anti-inflammatory properties by influencing inflammatory cell activation processes. Lnk is an adaptor protein involving endothelial cell (EC) activation because it is induced by tumor necrosis factor-α (TNF-α). This study was conducted to evaluate the role of eicosapentaenoic acid (EPA), an n−3 PUFA, in the regulation of Lnk expression in human umbilical vein endothelial cells (HUVECs). Primary HUVECs were pretreated with EPA for 12 h at various concentrations (0–40 μM) and then exposed for another 12 h in the presence or absence of TNF-α (10 ng/ml). Lnk mRNA and protein were detected using reverse transcriptase polymerase chain reaction, immunoprecipitation and Western blot analysis. Results showed that pretreatment of HUVEC with EPA inhibited TNF-α-induced expression of Lnk in a dose-dependent manner. TNF-α-induced Lnk was also inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. Thus, we investigated the role of PI3K/Akt signaling pathway in this process. Phosphorylation of Akt was assessed by Western blot analysis. We found that EPA treatment decreased the amount of activated Akt. These results showed that EPA inhibited TNF-α-induced Lnk expression in HUVECs through the PI3K/Akt pathway. This may be a potential mechanism by which EPA protects ECs under inflammatory conditions.

Stereospecific and Redox-Sensitive Increase in Monocyte Adhesion to Endothelial Cells by Homocysteine

Previous studies have shown that elevated homocysteine (Hcy) levels promote the development of atherosclerotic lesions in atherosclerosis-prone animal models. There is evidence that oxidant stress contributes to Hcy's deleterious effects on the vasculature. The accumulation and adhesion of monocytes to the vascular endothelium is a critical event in the development of atherosclerosis. We investigated the effects of Hcy on the interaction between human endothelial cells (EC) (EC line EA.hy 926 and primary human umbilical vein EC [HUVEC]) and the monocytic cell line THP-1, and the impact of vascular oxidant stress and redox-sensitive signaling pathways on these events. L-Hcy, but not D-Hcy, increases the production of reactive oxygen species inside EC, enhances nuclear factor(NF)-kappaB activation, and stimulates intercellular adhesion molecule-1 (ICAM-1) RNA transcription and cell surface expression. This leads to a time- and dose-dependent increase in monocyte adhesion to ECs. Pretreatment of ECs with superoxide scavengers (MnTBAP and Tiron) or with an inhibitor of NF-kappaB activation abolished Hcy-induced monocyte adhesion, ICAM-1 expression, and nuclear translocation of NF-kappaB. These findings suggest that reactive oxygen species produced under hyperhomocysteinemic conditions may induce a proinflammatory situation in the vessel wall that initiates and promotes atherosclerotic lesion development.

Multimodular Analysis of Angiogenic Biomarkers in the Primary Treatment of Multiple Myeloma: Correlative Studies of the BiRD Trial.

Biomarkers that predict the angiogenic propensity of multiple myeloma and the impact of therapy on angiogenesis remain unknown. Nevertheless, many putative anti-angiogenic agents are currently being evaluated in clinical trials. We describe the results of a multimodular analysis of angiogenic biomarkers including a novel in vitro functional assay, the human umbilical vein endothelial cell (HUVEC)-based angiogenic scale (HBAS), to assess the overall angiogenic activity in the plasma of patients with multiple myeloma. Plasma fractionated from the peripheral blood of myeloma patients was added to primary HUVEC and cultured under serum-free conditions for 24 hours. Angiogenic morphology was then examined and scored using a six-point angiogenic scale. Analysis of a cohort of ten consecutive patients with multiple myeloma undergoing the BiRD protocol (Biaxin, Revlimid and dexamethasone) showed that the majority had higher HBAS scores at baseline than age-matched normal subjects. Plasma samples from stage IIIA disease consistently scored higher than those from stage II disease on the functional angiogenic scale (mean score of 2.8 versus 1.0, p<0.005), suggesting a more pro-angiogenic state for further advanced disease. The HBAS also correlated with increased levels of plasma and platelet vascular endothelial growth factor-A (VEGF-A) as quantified by ELISA. Interestingly, VEGF-A levels in platelet lysate were significantly higher than those in plasma and more closely paralleled the functional HBAS scores. In addition, two-color flow cytometric analysis of cellular biomarkers demonstrated an angiogenic switch defined by increased mobilization of circulating CD133+VEGFR2+ endothelial progenitor cells in patients with active disease as compared to normal subjects (2.2-fold increase; p<0.005). Treatment with BiRD effectively attenuated all tested angiogenic parameters and correlated with a positive clinical response. Collective assessment of angiogenic biomarkers may be a promising resource for predicting clinical outcomes and for validating the potential impact of anti-angiogenic therapy in multiple myeloma.

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination. The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-alpha (tumor necrosis factor-alpha), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

Internal Translation Initiation Mediated by the Angiogenic Factor Tie2

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5'-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5'-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5'-UTR and how its production is secured under unfavorable environmental conditions.

A microchip for electroporation of primary endothelial cells

Abstract Electroporation (EP) is a technique with which DNA molecules can be delivered into cells suspended in a chamber using high electric field pulses. We have utilized the MEMS technology to fabricate the EP microchip. In this device, the interdigitated electrodes should provide higher electric field on the chip surface and cells could be manipulated in situ without detachment if adherent cells were used for EP. Moreover, this EP microchip overcame the use of high voltage and the excess heat generated during electroporation, which are the main drawbacks of the current electroporation technology. One primary cell type, human umbilical vein endothelial cells (HUVEC), and two different cell lines, Huh-7 cell (human hepatocellular carcinoma cell line) and 293T cell (human embryonic kidney cell line) were used to test this device. Experimental results show that this EP microchip could successfully deliver green fluorescent protein (GFP) genes into these cells. In the case of primary HUVEC, the survival rates were greater than 31%, and the transfection rates were about 13–36% based on the number of surviving cells under the applied voltage ranging from 2 to 8 V. In summary, this EP microchip should provide many potential applications for gene therapy, particularly those using primary cells for gene delivery.

Platelet adhesion to dengue-2 virus-infected endothelial cells.

Platelets in circulation normally do not adhere to resting endothelial cells. However, in response to vascular injury they adhere to stimulated endothelium and thereby play an essential role in hemostasis and thrombosis. Infection with dengue-2 virus can cause illness accompanied by thrombocytopenia and hemorrhage. Increased adherence of platelets to stimulated endothelial cells could contribute to the thrombocytopenia. In this study, adherence of radioisotopically labeled platelets to 1) unstimulated, 2) lipopolysaccharide (LPS)-stimulated, and 3) dengue-2 virus-infected human umbilical vein endothelial cells (HUVEC) was measured in an in vitro assay. Primary HUVEC were cultured in 96-well tissue culture plates in the presence or absence of LPS or dengue-2 virus. These cells were co-incubated with 3H-adenine-labeled fresh platelets for 30 min after which the cells were assayed for adherent platelets. Within 30 min there was maximum adherence of platelets to confluent LPS-stimulated HUVEC (36 +/- 4% over controls; P = 0.005). In comparison, there was a significant increase in adherence to dengue-2 infected HUVEC (78 +/- 7%; P < or = 0.001). Additionally, platelet adherence was visualized using fluorescent microscopy. Dengue-2 infection stimulated the HUVEC as monitored by expression of E-selectin. Platelets that adhered to dengue-2 or LPS-stimulated HUVEC were activated as visualized by dual fluorescent probes. These data demonstrate that human platelets adhere to dengue-2 virus-stimulated HUVEC and this interaction could contribute to the thrombocytopenia observed during infection.

REVERSIBLY IMMORTALIZED HUMAN ENDOTHELIAL SCAVENGER CELLS TO DEVELOP A BIOARTIFICIAL ORGAN

Purpose: Endothelial cells are responsible for microcirculation and scavenger functions in various organs and seems to be one of the important components for tissue engineering. The present study describes for the first time a strategy of expansion of primary endothelial cell population by transfer of the simian virus 40 large T antigen (SV40T) gene that can be subsequently excised by Cre/loxP site-specific recombination. Methods: To transfer the SV40T gene into human umbilical vein endothelial cells (HUVECs), we constructed a retroviral vector SSR#69 expressing SV40T flanked by a pair of loxP. An adenoviral vector producing Cre recombinase (Ad-Cre) was used for recombination. The gene expression of endothelial markers, uptake of acetylated LDL, and efficacy of Cre/loxP-mediated recombination were evaluated in SV40T-transduced HUVECs. Results: One of SSR#69-transformed HUVECs, HNNT-1, grew economically in culture and showed gene expression of factor VIII, VEGF receptors (flt-1 and KDR/flk-1), CD34, and HGF. The cells demonstrated the uptake-of acetylated LDL. SV40T was efficiently removed following infection of Ad-Cre, while retaining the characteristics of primary HUVECs. Conclusion: We present here for the first time a method to control the expansion of primary human endothelial cells as a source for developing a novel hybrid bioartificial organ.

RNA Damage and Inhibition of Neoplastic Endothelial Cell Growth: Effects of Human and Amphibian Ribonucleases

Angiogenesis defines the many steps involved in the growth and migration of endothelial cell-derived blood vessels. This process is necessary for the growth and metastasis of tumors, and considerable effort is being expended to find inhibitors of tumor angiogenesis. This usually involves screening of potential anti-angiogenic compounds on endothelial cells. To this end, two candidate anti-angiogenic RNA-damaging agents, onconase and (-4)rhEDN, were screened for their effects on endothelial cell proliferation using three distinct types of endothelial cells in culture: HPV-16 E6/E7-immortalized human umbilical vein endothelial cells (HUVECs), a Kras-transformed HPV-16 E6/E7 HUVEC (Rhim et al., Carcinogenesis 4, 673-681, 1998), and primary HUVECs. Onconase similarly inhibited proliferation in all three cell lines (IC(50) = 0.3-1.0 microM) while (-4)rhEDN was more effective on immortalized HUVEC cell lines (IC(50) = 0.02-0.06 microM) than on primary HUVECs (IC(50) > 0.1 microM). Differential sensitivity to these agents implies that more than one endothelial cell type must be used in proliferation assays to screen for novel anti-angiogenic compounds.

Effects of C5a and FMLP on interleukin-8 production and proliferation of human umbilical vein endothelial cells.

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1alpha, TNFalpha, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1alpha and TNFalpha significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1alpha, TNFalpha, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a 3H-thymidine incorporation assay. In contrast with IL-1alpha and TNFalpha, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.

Human Umbilical Vein Endothelial Cells Differ from Other Endothelial Cells in Failing to Express ABO Blood Group Antigens

Most in vitro investigations of endothelial cell function have been based on the behaviour of human umbilical vein endothelial cells (HUVEC) in primary tissue culture. However, it is becoming apparent that there is a marked degree of heterogeneity among endothelial cells derived from different vascular beds. We have studied primary HUVEC from 45 umbilical cords. Contrary to previously published reports, we were unable to detect ABO antigens on the surface of cultured HUVEC from group A or B cords. This was not due to an absence of precursor H substance which was uniformly expressed on HUVEC in primary tissue culture. Further investigation revealed an absence of A enzyme activity in A type HUVEC and a level of mRNA only just detectable by RT-PCR. The absence of functional A enzyme activity was confirmed by demonstrating the absence of A substance on von Willebrand factor secreted by A type cells. HUVEC appear to be the only vascular endothelial cells that do not express ABO blood group antigens. We speculate that this may help protect the cord from maternal antibodies during gestation. The lack of ABO blood group antigens on HUVEC may significantly affect their surface function, and therefore care should be taken when extrapolating conclusions from results obtained with these cells (the most widely used human endothelial cell model in vitro) to the properties of adult endothelial cells.

A comparison of primary endothelial cells and endothelial cell lines for studies of immune interactions

The purpose of this study was to assess the suitability of using endothelial cell (EC) lines for studies of endothelial/immune interactions. The immortal human EC lines HMEC-1, ECV304 and EaHy926 were compared to human umbilical vein endothelial cells (HUVEC) for constitutive and induced expression of surface antigens known to be involved in interactions with T cells. These cell lines were also compared to HUVEC in transendothelial migration assays. Flow cytometry was used to measure cell surface expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, major histocompatibility complex (MHC) class I and MHC class II, CD40, CD95 (fas) and lymphocyte function associated antigen-3 (LFA-3) before and after treatment with the cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Polymerase chain reaction (PCR) was used to detect expression of the MHC class II transactivator. Significant differences were found in the ability to respond to cytokines between HUVEC and the cell lines, the greatest differences being induction of VCAM-1 and E-selectin in response to TNF-α and induction of MHC class II antigens in response to IFN-γ. Thus unlike HUVEC, induction of VCAM-1 and E-selectin was not detectable on EaHy926 and ECV304 and barely detectable on HMEC-1. MHC class II antigens were not induced on ECV304 in response to IFN-γ and nor was the class II transactivator (CIITA). Unlike HUVEC and the other cell lines, ECV304 were constitutively negative for PECAM-1. Constitutive and induced expression of MHC class I, ICAM-1, LFA/3, CD40 and fas were most conserved between the cell lines and showed little difference to HUVEC. The migration of peripheral blood mononuclear cells (PBMC) through all cell lines was significantly reduced compared to through HUVEC, suggesting that there is a functional difference between the cell lines with regard to interactions with lymphocytes. In conclusion this study has demonstrated significant differences in the ability of endothelial cell lines to respond to cytokines compared to primary HUVEC cultures. In particular ECV304 compares very poorly with HUVEC. Whether these differences are caused by immortalization procedures or reflect heterogeneity of EC arising from different vascular beds is discussed.

Different growth behaviour of human umbilical vein endothelial cells and an endothelial cell line seeded on various polymer surfaces

Major obstacles for successful application of endothelial cell seeding in synthetic vascular grafts include the source of autologous endothelial cells, the efficiency of cell seeding and detachment of adherent endothelial cells from the graft surface after restoration of circulation. Human umbilical vein endothelial cells (HUVECs) are frequently used to investigate the in vitro adhesion and proliferation of endothelial cells on polymer surfaces. In order to minimize the biological variation of HUVECs isolated from different umbilical veins, it would be advantageous to use an endothelial cell line in in vitro proliferation experiments. Aim of the present study was to compare the proliferation of primary HUVECs and the endothelial cell line EC-RF24 on several polymer surfaces coated with various concentrations of the adhesive protein fibronectin. EC-RF24 cells grow to a higher density than primary HUVECs. Moreover, the EC-RF24 cell line is able to proliferate on surfaces with sub-optimal adhesive properties. Therefore, it is concluded that the EC-RF24 cell line is less suitable for evaluation of the in vitro proliferation of endothelial cells on polymer surfaces.

Human umbilical vein endothelial cells lack expression of the estrogen receptor.

Estrogens may influence the expression of various cytokines, adhesion molecules, von Willebrand factor and prostacyclin produced by endothelial cells. However, reports concerning expression of the estrogen receptor in endothelial cells are controversial. Primary human umbilical vein endothelial cells (HUV-EC), the non continuous human umbilical vein endothelial cell line HUV-EC-C (ATCC CRL 1730) and endothelial cells from 10 frozen umbilical cords were analyzed for the expression of the estrogen receptor. Immunological studies using estrogen receptor specific antibodies failed to detect the expression of the receptor in all human umbilical vein endothelial cells tested. No estrogen receptor transcripts were found in primary HUV-EC or HUV-EC-C by reverse transcriptase-polymerase chain reaction. Weak hybridization signals were detected when the PCR amplicons were hybridized with estrogen receptor cDNA sequences as a probe. In vitro protein-DNA interaction studies revealed no complexes between a fully consensus estrogen response element and HUV-EC-C extracts. Finally, transient transfection studies in HUV-EC-C could not demonstrate 17beta-estradiol-induced transcription of the beta-galactosidase reporter gene linked to a consensus estrogen response element. These observations suggest that human umbilical vein endothelial cells lack the estrogen receptor.