Cell surface overexpression of alphavbeta5 integrin impedes alphavbeta3-mediated migration of the human ovarian adenocarcinoma cell line IGROV1

Human ovarian surface epithelium and epithelial tumors express integrin alphavbeta5, which can interact with vitronectin. In addition, in vitro acquisition of cisplatin resistance by alphavbeta3-expressing IGROV1 cells is accompanied by cell-surface expression of integrin alphavbeta5. To further explore the role of alphavbeta5 in ovarian carcinoma cells, IGROV1 cells were stably transfected with a human beta5 integrin cDNA construct, and three beta5 transfectant clones were selected for the expression of alphavbeta5 integrin at their cell surface. Despite a delayed entry in the exponential phase of growth, beta5-transfectant cells kept a proliferation ability similar to that of parental cells, while their growth rate was hindered in the presence of an anti-alphavbeta5 blocking antibody. Only simultaneous blockade of alphavbeta3 and alphavbeta5 by specific antibodies impeded the adhesion to vitronectin of beta5 transfectants and of the beta5-expressing cisplatin-resistant variant IGROV1-R10, suggesting that the two heterodimers cooperated in the regulation of this process. Cell surface expression of alphavbeta5 resulted in an attenuation of alphavbeta3-mediated migration on vitronectin. Alphavbeta5 participated to migration events in the absence of exogenous growth factors only in one transfectant clone and in IGROV1-R10 cells. Finally, the response to cisplatin was not significantly modified in beta5 transfectants when compared to IGROV1 parental cells.     

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.     

Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.     

Mesothelial vitronectin stimulates migration of ovarian cancer cells

Ovarian carcinomas, the most fatal gynaecological malignancies, are associated with poor prognosis predominantly because of a high recurrence rate. Ovarian cancer cells spread widely throughout the abdominal cavity leading to peritoneal metastasis. The influence of the mesothelial microenvironment on the biological mechanisms leading to cancer cell colonization of the mesothelium is poorly understood. This study aims to investigate whether mesothelial secretions affect the migration of ovarian cancer cells and focuses on the role of the adhesive molecule Vn (vitronectin) and its integrin receptors. An in vitro co‐culture model indicated that clusters of IGROV1 and SKOV3 cells adhere to MeT‐5A mesothelial cells preferentially at intercellular sites, invade the mesothelial monolayer and alter the integrity of the mesothelium. In addition, mesothelial CM (cell‐conditioned medium) induces migration of IGROV1 and SKOV3 cells in Boyden chambers and wound healing assays. Furthermore, blocking molecules directed against vitronectin or its αv integrin receptor decrease mesothelial‐CM‐induced migration by approximately 40% and 60–70% for IGROV1 and SKOV3 ovarian cancer cells, respectively, in Boyden chamber assays. Wound healing assays that allow cell migration to be measured over 24 h periods demonstrated that blocking molecules prevent the migration of IGROV1 and SKOV3 cells. Vitronectin is present in CM MeT‐5A (mesothelial conditioned medium) and in metastatic peritoneal tissue sections. The expression of vitronectin at the periphery of mesothelial cells and within ovarian cancer cell clusters suggests a potential role for this molecule during intraperitoneal implantation of ovarian cancer cells. Vitronectin could represent a target for the development of anti‐adhesive strategies to impede ovarian cancer dissemination.     

Adhesive force of human hepatoma HepG2 cells to endothelial cells and expression of E-selectin

Expression of adhesion molecules may play an important role in the interaction of tumor cells with vascular endothelial cells during tumor invasion and metastasis. In this study, the adhesive force of human hepatoma HepG2 cells to human umbilical vein endothelial cells (HUVECs) was investigated using a micropipette aspiration technique. Expression of an adhesion molecule, E-selectin, was also observed by immunofluorescence microscopy. In particular, the adhesive force after stimulation of HUVECs with recombinant human interleukin-1beta (rhIL-1beta) was examined. The results demonstrated that the adhesive force of HepG2 cells to stimulated HUVECs is significantly higher than that of unstimulated control cells, and that immunofluorescence of E-selectin in stimulated HUVECs showed a higher fluorescent intensity compared to control cells. Moreover, addition of monoclonal anti-human E-selectin decreased the adhesive force of HepG2 cells to stimulated HUVECs by 50%. These results suggest that endothelial E-selectin may be a main mediator of carcinoma metastasis of malignant tumor through blood circulation, possibly increasing the adhesive force of human hepatoma HepG2 cells to HUVECs in the early stage of metastases.     

RGD-peptide presents anti-adhesive effect, but not direct pro-apoptotic effect on endothelial progenitor cells

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization in tumor or ischemic tissues by multi-step events, including adhesion, migration, chemoattraction, and differentiation to endothelial cells. Anti-angiogenic RGD-peptides have been shown to directly induce apoptosis in human umbilical vein endothelial cells (HUVECs) and T cells. Here, we examined the effects of RGD-peptides on EPCs in terms of adhesive differentiation and apoptosis. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, RGD-peptide treatment decreased dose-dependently the number of adherent cells double positive for DiI-ac-LDL uptake and UEA-1 binding. The cells treated with RGD peptide were also stained less strongly by vWF or KDR antibody by immunofluorescence staining. Immobilization of the RGD-peptide promoted cell adhesion, but resulted in a deficiency in the development of ability of ac-LDL uptake and UEA-1 binding, showing an antagonistic effect. Accordingly, ex vivo-cultivated EPCs expressed integrin alpha5, alphav, beta1, beta3, and beta5, and antibodies to integrins alpha5, alphav, and beta1 decreased the number of adherent cells. However, viability of total MNCs containing early EPCs was not affected by RGD-peptide. In addition, neither an increase in apoptotic cell death nor a direct activation of caspase-3 by RGD-peptide was detected in ex vivo-cultivated EPCs, unlike in HUVECs. Interestingly, RGD-peptide rather enhanced Bcl-2 expression in ex vivo-cultivated EPCs and the EPCs themselves with a high Bcl-2/Bax ratio are comparatively resistant to apoptosis. Therefore, these results suggest that RGD-peptides may inhibit EPC differentiation by anti-adhesive effect, but not by a direct pro-apoptotic effect.     

Colon cancer cell adhesion to endothelial E-selectin inhibits detachment of endothelial cells through activation of beta(1)-integrin

Previous studies have implicated a role for E-selectin in carcinoma cell adhesion to vascular endothelium. We examined the role of colon cancer cell adhesion to vascular endothelium via E-selectin using adenoviral vector-mediated transfection in human umbilical vein endothelial cells (HUVECs). We found that the amount of HUVEC detachment from the gelatin matrix 24 h after LS-180 cell adhesion was inhibited only when the HUVECs were transduced with wild-type E-selectin, but not with a cytoplasmic domain truncated mutant E-selectin or the control Lac-Z vector. We also found that the adhesion of LS-180 cells to wild-type E-selectin transduced HUVEC-induced activation of beta(1)-integrin receptors without affecting MMP activity. These results indicate that colon cancer cell adhesion via E-selectin inhibits HUVEC detachment from the monolayer, at least in part by modulating beta(1)-integrin activity in HUVECs. In addition, they indicate the importance of the cytoplasmic domain of E-selectin with this phenomenon.     

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2 h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells.     

The role of integrins in the maintenance of endothelial monolayer integrity

This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells.

In postcapillary venules, marginating neutrophils (PMNs) are often seen rolling along the vessel wall prior to stopping and emigrating. There is substantial evidence in vitro and in vivo that the adhesion receptors E- and L-selectin participate in this phenomenon on cytokine-stimulated endothelium, and recent evidence has shown that a closely related adhesion receptor, P-selectin, is capable of mediating neutrophil rolling on an artificial membrane. Here we demonstrate and characterize PMN rolling on monolayers of human umbilical vein endothelial cells (HUVECs) stimulated with histamine to induce surface expression of P-selectin. Peak association of PMNs with the HUVECs occurs 10 min after histamine stimulation, and at a postcapillary venular wall shear stress of 2.0 dyn/cm2 the rolling velocity is 14 microns/s. Approximately 95% of the PMNs roll on the endothelial cells, 5% adhere firmly, and none migrate beneath the endothelial monolayer. Monoclonal antibody (MAb) G1, which binds P-selectin and blocks its adhesive function, completely prevents association of the PMNs with histamine-stimulated HUVEC, whereas the nonblocking anti-P-selectin MAb S12 does not. Treatment of PMNs with the anti-L-selectin MAb DREG56 reduces PMN adherence by approximately 50%. Anti-CD54 MAb R6.5 and anti-CD18 MAb R15.7 have little effect on the number of PMNs rolling on the HUVECs but completely prevent PMNs from stopping and significantly increase rolling velocity. Nonblocking control MAbs for R6.5 (CL203) and R15.7 (CL18/1D1) lack these effects. Rolling adhesion of PMNs on histamine-stimulated HUVECs therefore appears to be completely dependent on endothelial cell P-selectin, with a minor adhesion-stabilizing contribution from intercellular adhesion molecule 1 and beta 2 integrins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking alphavbeta5 integrin

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.     

Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin alpha(v)beta(3), has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of alpha(v)beta(3) in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin alpha(v)beta(3) on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. alpha(v)beta(3) was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-alpha(v)beta(3) monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin alpha(v)beta(3), only L1 serves as a potential ligand for alpha(v)beta(3) during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and alpha(v)beta(3) antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin alpha(v)beta(3) on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.     

Divergent responses of different endothelial cell types to infection with Candida albicans and Staphylococcus aureus

Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.     

Integrin alpha x stimulates cancer angiogenesis through PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression in blood vessel endothelial cells

Integrin alpha x (ITGAX), a member of the integrin family, usually serves as a receptor of the extracellular matrix. Recently, accumulating evidence suggests that ITGAX may be involved in angiogenesis in dendritic cells. Herein, we report a direct role of ITGAX in angiogenesis during tumor development. Overexpression of ITGAX in human umbilical vein endothelial cells (HUVECs) enhanced their proliferation, migration, and tube formation and promoted xenograft ovarian tumor angiogenesis and growth. Further study showed that overexpression of ITGAX activated the PI3k/Akt pathway, leading to the enhanced expression of c-Myc, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor 2 (VEGFR2), whereas, the treatment of cells with PI3K inhibitor diminished these effects. Besides, c-Myc was observed to bind to the VEGF-A promoter. By Co-Immunoprecipitation (Co-IP) assay, we manifested the interaction between ITGAX and VEGFR2 or the phosphorylated VEGFR2. Immunostaining of human ovarian cancer specimens suggested that endothelial cells of micro–blood vessels displayed strong expression of VEGF-A, c-Myc, VEGFR2, and the PI3K signaling molecules. Also, overexpression of ITGAX in HUVECs could stimulate the spheroid formation of ovarian cancer cells. Our study uncovered that ITGAX stimulates angiogenesis through the PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression during cancer development.     

Alendronate suppresses tumor angiogenesis by inhibiting Rho activation of endothelial cells

We previously reported that alendronate inhibits intraperitoneal dissemination in an in vivo ovarian cancer model. Recently, nitrogen-containing bisphosphonates have been reported to have antiangiogenic activities. In this study, alendronate inhibited human umbilical vein endothelial cell (HUVEC) migration and capillary-like structure formation in vitro. These inhibitory effects were associated with reduced Rho activation and suppression of the formation of actin stress fibers and focal adhesions in HUVECs. Furthermore, the inhibition by alendronate was reversed by geranylgeraniol, which abrogated the inhibition of Rho geranylgeranylation. Next, we examined the effect of alendronate on angiogenesis in disseminated ovarian tumors of athymic immunodeficient mice. Alendronate treatment reduced the intra-tumor neoangiogenesis compared with that in the non-treated mice, although tumor-derived VEGF expression was not altered. In conclusion, the in vivo anti-tumor effect of alendronate might be derived, at least in part, from its direct antiangiogenic effects on intra-tumor endothelial cells by inhibiting Rho geranylgeranylation.     

Hypoxic trophoblast‐derived sFlt‐1 may contribute to endothelial dysfunction: implication for the mechanism of trophoblast—endothelial dysfunction in preeclampsia

The maternal systemic disorder of widespread endothelial dysfunction is a primary focus in understanding the development of preeclampsia. sFlt‐1 (soluble fms‐like tyrosine kinase receptor 1), an endogenous inhibitor of VEGF (vascular endothelial growth factor), may play important roles in endothelial dysfunction. The present study aimed to determine whether hypoxic trophoblast‐derived sFlt‐1 could lead to endothelial dysfunction by establishing a cocultured model of anoxic TEV‐1s (human first‐trimester extravillous trophoblasts) and HUVECs (human umbilical vein endothelial cells). The results showed that the hypoxic treatment significantly promoted sFlt‐1 mRNA and protein expression in TEV‐1s in a time‐dependent manner compared with the effect in HUVECs. When HUVECs were cocultured with anoxic TEV‐1s, the endothelial function, which was characterized by NO (nitric oxide) synthesis and monolayer barrier function of HUVECs, were notably decreased, accompanied by increasing sFlt‐1 and decreasing VEGF in cell‐conditioned medium. Moreover, the observed endothelial dysfunction described above was consistent with the dysfunction observed in VEGF siRNA‐treated cultures. The findings presented herein imply that chronically hypoxic trophoblasts may release sufficient sFlt‐1 to cause endothelial dysfunction by depriving cells of VEGF activity.     

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross‐talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co‐culture between osteoblastic and endothelial cells. Through a well defined direct contact co‐culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular‐like structures. VEGF₁₆₅ gene expression was detected in the HOPs, was up‐regulated in the co‐cultured HOPs and both Flt‐1 and KDR gene expression increased in co‐cultured HUVECs. However, the cell rearrangement observed in co‐culture was promoted by a combination of soluble chemoattractive factors and not by VEGF₁₆₅ alone. Despite having no observable effect on endothelial cell tubular‐like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co‐culture‐stimulated osteoblastic phenotype. This co‐culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering. J. Cell. Biochem. 106: 390–398, 2009. © 2009 Wiley‐Liss, Inc.