Possible role of WNT10B in increased proliferation and tubule formation of human umbilical vein endothelial cell cultures treated with hypoxic conditioned medium from human adipocytes

SOX6 plays important roles in cell proliferation, differentiation, and cell fate determination. It has been confirmed that SOX6 is a tumor suppressor and downregulated in various cancers, including esophageal squamous cell carcinoma, hepatocellular carcinoma, and chronic myeloid leukemia. Netrin-1 is highly expressed in various human cancers and acts as an anti-apoptotic and proangiogenic factor to drive tumorigenesis. The role of SOX6 and netrin-1 in regulating the growth of ovarian tumor cells still remains unclear. Real-time polymerase chain reaction and western blot were used to determine the SOX6 messenger RNA and protein levels, respectively, in ovarian cancer cells and tumor tissues. Stable transfection of SOX6 was conducted to overexpress SOX6 in PA-1 and SW626 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Invasion of ovarian cancer cells and migration of human umbilical vein endothelial cells were confirmed by Transwell assays. To overexpress netrin-1, ovarian cancer cells with SOX6 restoration was transduced with netrin-1 lentiviral particles. PA-1 xenografts in a nude mice model were used to conduct in vivo evaluation of the role of SOX6 and its relationship with netrin-1 in tumor growth and angiogenesis. In this study, we found significantly reduced SOX6 levels in PA-1, SW626, SK-OV-3, and CaoV-3 ovarian cancer cell lines and human tumor tissues in comparison with normal human ovarian epithelial cells or matched non-tumor tissues. SOX6 overexpression by stable transfection dramatically inhibited proliferation and invasion of PA-1 and SW626 cells. Also, conditioned medium from PA-1 and SW626 cells with SOX6 restoration exhibited reduced ability to induce human umbilical vein endothelial cells migration and tube formation compared with conditioned medium from the cells with transfection control. Furthermore, an inverse relationship between SOX6 and netrin-1 expression was observed in PA-1 and SW626 cells. Overexpression of netrin-1 in ovarian cancer cells with forced SOX6 expression remarkably abrogated the inhibitory effect of SOX6 on proliferation, invasion of the cells, and tumor xenograft growth and vascularity in vivo. Human umbilical vein endothelial cell migration and tube formation were enhanced in the conditioned medium from the ovarian cancer cells transduced with netrin-1 lentivirus particles. Our observations revealed that SOX6 is a tumor suppressor in ovarian cancer cells, and SOX6 exerts an inhibitory effect on the proliferation, invasion, and tumor cell-induced angiogenesis of ovarian cancer cells, whereas nerin-1 plays an opposite role and its expression is inversely correlated with SOX6. Moreover, our findings suggest a new role of SOX6 and netrin-1 for understanding the progression of ovarian cancer and have the potential for the development of new diagnosis and treatment strategies for ovarian cancer.

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MODULATION of PROSTACYCLIN PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS WITH ECGF/HEPARIN MEDIUM : ROLE OF CELLULAR DENSITY AT CONFLUENCE

10.1055/s-0038-1643376 ◽

1987 ◽

Author(s):

O BOUTHERIN-FALSON ◽

N BLAES

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Umbilical Vein ◽

Calf Serum ◽

Lower Amount ◽

Endothelial Cell Proliferation ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in vascular endothelial cells. In addition to the role of exogenous agents, its production could be modulated by culture conditions : proliferative state, medium renewal, subcultivation... The use of endothelial cell growth factor (ECGF) associated with heparin has been shown to improve human endothelial cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) grown in that medium produce less prostacyclin than without growth factor.HUVEC were cultured in RPMI-199 1:1 + 20% fetal calf serum, added or not with ECGF (Bovine hypothalamus extract BTI Cambridge, 24 ug/ml) and heparin (from porcine intestinal mucosa, Signa, 90 ug/ml). After 4 days in culture, medium was removed and replaced by Tyrode Hepes buffer and basal production was measured after 20 min. Cells were then submitted to 5 min thrombin to assess PGI2 production in stimulated conditions. PGI2 production was estimated by specific radioimmunoassay for 6 keto PGFjalpha. For each point, cell number in the culture was counted after Trypsin EDTA treatment. In the present study, cells grown in ECGF-heparin medium produce lower amount of PGI2, compared to heparin or control medium. This result was observed in both basal and stimulated conditions. For each medium (ECGF-heparin, heparin, control), correlations between PGI2 production per cell and log cell density were shown to be significantly negative.These observations suggest that ECGF effect on PGI2 production could be a consequence of its growth factor activity, notably by the fact that it leads to an endothelial monolayer made of more numerous cells. Since it is now suggested by a number of clinical observations that PGI2 is rather produced in pathological conditions, culture models showing a weak production of PGI2 appear in that connection doser to the physiological conditions.

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Adenosine stimulates proliferation of human endothelial cells in culture

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.265.1.h131 ◽

1993 ◽

Vol 265 (1) ◽

pp. H131-H138 ◽

Cited By ~ 9

Author(s):

M. F. Ethier ◽

V. Chander ◽

J. G. Dobson

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Growth ◽

Cell Cultures ◽

Umbilical Vein ◽

Fibroblast Cell ◽

Human Endothelial Cells ◽

Cell Counts ◽

Endothelial Cell Cultures ◽

Endothelial Cell Growth

The effect of adenosine on proliferation of human endothelial cells was investigated by adding adenosine to the medium of cultures derived from human umbilical veins. Cell counts on cultures grown in 10 microM adenosine for 4–7 days were 41–53% greater than counts from control cultures. In contrast, 10 microM adenosine had no effect on growth of a human fibroblast cell strain (IMR-90). Neither inosine nor 2',5'-dideoxyadenosine influenced endothelial cell growth at concentrations of 0.1 or 10 microM. Addition of adenosine deaminase abolished the proliferative effect of added adenosine and inhibited proliferation by 16% in control cultures, suggesting that endogenous adenosine may enhance proliferation in culture. The adenosine receptor antagonist, 8-phenyltheophylline, at 0.1 and 1.0 microM blocked the enhanced proliferation caused by 10 microM adenosine. Addition of 10 microM adenosine enhanced DNA synthesis in endothelial cell cultures as indicated by an increased incorporation of [3H]thymidine into acid-insoluble cell material. The results indicate that addition of physiological concentrations of adenosine to human umbilical vein endothelial cell cultures stimulates proliferation, possibly via a surface receptor, and suggest that adenosine may be a factor for human endothelial cell growth and possibly angiogenesis.

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Influence of Umbilical Cord Blood-Derived Endothelial Precursor Cells on a Human Umbilical Vein Endothelial Cell Co-Culture In Vitro Angiogenesis Assay.

Blood ◽

10.1182/blood.v106.11.4218.4218 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4218-4218

Author(s):

Nicholas J. Greco ◽

Brandon Eilertson ◽

Jason J. Banks ◽

Paul Scheid ◽

Marcie Finney ◽

...

Keyword(s):

Endothelial Cell ◽

Growth Factor ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Umbilical Vein ◽

Tubule Formation ◽

Human Umbilical Vein ◽

In Vitro Angiogenesis

Abstract To assess in vitro angiogenesis, cellular co-culture assays have been utilized to study adherence, spreading, differentiation and proliferation, and migration of endothelial cells. Formation of tubule or capillary-like networks is influenced by the presence of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) but other factors provided by cell sources and/or direct contact with multiple cell types may facilitate this formation. The hypothesis of this study is that umbilical cord blood (UCB)-derived endothelial precursor cells (EPCs) may influence the formation of human umbilical vein endothelial cell (HUVEC) tubule structures during angiogenesis. Methods: UCB-derived EPCs were isolated from CD133negative cells after a 7-day culture on human fibronectin in EGM-2 media. Tubule formation was evaluated (passage 1–2, 20 x 103 or 2 x 103 cells) by adding HUVECs without or with EPCs to cultures of human bone marrow-derived mesenchymal stromal cells (MSCs) under normoxic (20%) conditions (37°C, 5% CO2, containing VEGF, epidermal growth factor, FGF, insulin-like growth factor, heparin, hydrocortisone, and ascorbic acid in EGM-2 medium) for a 2-week period. HUVECs were added to cultures without or with labeling with Vybrant® CM-DiI which allows the temporal observation of tubule formation progress and cellular incorporation. Final tubule formation was confirmed using a primary anti-CD31 (PECAM) antibody followed by a FITC-conjugated secondary antibody for signal amplification. Results: After 2–4 days, linear aggregates of labeled HUVECs (2-D arrangement) were observed. After 14 days, there was remodeling of HUVECs into the development of a 3D network of linear and branched tubule structures. EPCs facilitated the formation of tubules affecting both the extent of tubule formation and also enhanced proliferation of HUVEC cells. A minority (< 5%) of EPCs were incorporated into developing tubules (estimated using CM-Dil-labeled EPCs). To quantify tubule formation, digital pictures of representative areas of culture wells (2–4/well) were acquired. Using Image Pro Plus software, tubules were quantified using multi-parameter analysis with respect to length, area, and perimeter. The presence of EPCs (equal to the number of added HUVECs) significantly enhanced all parameters. In comparison to control samples, the presence of EPCs increased the area, perimeter and size by 15.2-fold, 3.4-fold, and 3.2-fold, respectively. Confocal microscopy revealed that the co-cultures formed anatamoses, indicating the formation of a connected network. Conclusions: Taken together, these results suggest that the presence of cord blood-derived EPCs facilitate tubule formation and development via a heterotypic cell-cell interaction without integrating into the angiogenic structures. Further studies will evaluate the secretion of cytokines and growth factors.

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