Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell mitogen involved in normal and pathological angiogenesis. Our group recently reported that, among the several VEGF isoforms, VEGF189 (V189) is selectively induced in decidual endometrial cells during the mid-late phase of the menstrual cycle, together with polymorphonuclear neutrophil (PMN) influx. We thus compared the effects of various VEGF isoforms on PMN migration in vitro, and the mechanisms involved. In transmigration and under-agarose assays, V189 was both chemotactic and chemokinetic for PMN, while VEGF165 (V165) was only chemokinetic. The chemokinetic effect of V189 for PMN was blocked by neutralizing anti-VEGF antibodies, but not by neutralizing anti-KDR antibodies, suggesting that the Flt-1 VEGF receptor that is expressed in PMN mediates these effects. Flow cytometric analysis of several adhesion molecules at the PMN surface showed that all VEGF isoforms slightly upregulated beta1- and beta2-integrins and PECAM, and downregulated L-selectin; all these molecules are activation markers. The involvement of beta1-integrins was further supported by the ability of blocking antibodies to reduce VEGF-induced PMN migration. As human PMN can secrete several cytokines and growth factors, the selective secretion of VEGF isoforms was also further examined. RT-PCR analysis showed that V165 mRNA was more strongly expressed than V189 mRNA. Conversely, the major protein isoform secreted after optimal PMN degranulation was V189, which was located in both azurophilic and specific granules. PMN-derived VEGF can thus modulate PMN migration. This autocrine amplification mechanism would allow sustained VEGF release to occur at inflammatory sites, and may contribute to both normal and pathological angiogenesis.

Transcriptional regulation of vascular endothelial growth factor by estradiol and tamoxifen in breast cancer cells: a complex interplay between estrogen receptors alpha and beta.

Vascular endothelial growth factor (VEGF) is a potent angiogenic and prognostic factor for many tumors, including those of endocrine-responsive tissues such as the breast and uterus. Recent studies indicate that 17beta-estradiol (E(2)) modulates VEGF expression in breast and uterine cells, involving transcriptional activation through estrogen receptor (ER) alpha. However, molecular mechanisms of VEGF regulation mediated by the two ER subtypes and the potential role of ERbeta in the control of breast cancer angiogenesis have not yet been investigated. In transient transfection assays using the VEGF(-2275/+54) promoter-luciferase construct, E(2) (1 nM) increased transcription activity in MCF-7 cells (either untransfected or cotransfected with ERalpha) and it increased transcription activity in MDA-MB-231 cells cotransfected with ERalpha or ERbeta (1.8- and 2-fold induction, respectively). The positive effect was abolished when MCF-7 cells were treated with pure antiestrogen ICI 182,780 or the agonist/antagonist tamoxifen (1 micro M). To identify response elements involved in this transcriptional regulation, MCF-7 or MDA-MB-231 cells were transfected with several deletion constructs of the VEGF promoter. Deletion of 1.2-2.3 kb upstream to the transcription start in the VEGF promoter abrogated E(2)-dependent transcription in these cells. This region contains an imperfect estrogen-responsive element (ERE), ERE1520, and one activator protein 1 site. Transfection of MCF-7 cells (ERalpha) with the ERE1520-luciferase construct conferred transcriptional activity with 1 nM E(2) (1.9-fold induction). Also, the imperfect ERE formed a complex with ERalpha or ERbeta proteins in gel shift assay using MCF-7 or MDA-MB-231 nuclear extracts. In contrast to ERalpha, ERbeta could transactivate VEGF reporter construct in MDA-MB-231 cells, in the presence of E(2) or tamoxifen, suggesting different transactivational mechanisms between ERalpha and ERbeta in the presence of tamoxifen. Interestingly, E(2) inhibited VEGF transcription in MCF-7 cells transfected with ERbeta or MDA-MB-231 cells cotransfected with ERalpha and ERbeta, suggesting that heterodimerization of ERalpha/ERbeta has the ability to inhibit E(2)-induced VEGF expression in breast cancer cells. These results demonstrate that VEGF is a target gene for ERalpha and ERbeta in breast cancer cells; it remains to be determined whether ERalpha and ERbeta expression in breast biopsies correlates with VEGF expression and vascular density.

A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus.

A key mechanism underlying physiological angiogenesis of the human endometrium is its ability to regenerate the vascular capillary network and to perform vascular remodeling (i.e., development of spiral arteries). Vascular endothelial growth factor (VEGF) is associated with angiogenesis and capillary permeability in this tissue. VEGF is expressed as several spliced variants, its main human isoforms contain 121 and 165 aa; 17beta-estradiol (E(2)) increases endometrial VEGF, possibly in all isoforms. Here we show that progesterone (P) selectively increases the expression of the VEGF(189) (V(189)) isoform in the human uterus. V(189) is identified in the conditioned medium of stromal cells treated with E(2) + P; its presence in this in vitro model of decidual stromal cells is detected after 6-8 days, using ELISA, and after 8-10 days, using Western blot analysis with different antibodies, including one specific for V(189). The secretion pattern of V(189) parallels that of the decidual protein IGFBP-1. V(189) is secreted as a native isoform, as compared with the migration of recombinant V(189) by SDS/PAGE. In situ hybridization and immunocytochemistry(,) performed on the same biopsies, suggest that decidual cells express V(189) during the mid-late secretory phase of the menstrual cycle and early gestation. Finally, using an in vivo permeability assay, we show that native V(189) increases capillary permeability. These observations demonstrate that P regulates V(189) expression in decidual cells, which could have important implications for understanding uterine vascular remodeling and implantation, and may be relevant in a range of disease states such as edema and irregular bleeding.