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The Ets family contains a growing number of transcriptional activators and inhibitors, which activity is regulated by phosphorylation and protein-protein interactions. Among these factors, Ets1, Erg1 and Fli1 are expressed in endothelial cells during angiogenesis in normal and pathological development. The expression of these transcription factors is regulated by angiogenic factors in cultured endothelial cells, as well as by various stresses occurring during angiogenesis. Transfection experiments and transgenic mice analysis revealed that Ets family members are involved in the transcriptional regulation of endothelial specific genes such as those encoding Tie1 and -2, VEGFR1 and -2 and VE-Cadherin. In vitro studies plead for a role of Ets family members in endothelial cell adhesion, spreading and motility. Gene inactivation experiments show that Ets1 is dispensable for embryonic development. The phenotype of knocked-out embryos indicates that Tel is required for maintenance of the developing vascular network in the yolk sac. Altogether, we suggest that Ets family members act both positively and negatively during the different steps of the angiogenic process. The regulation of the initiation of gene transcription arises from the combined activity of different transcriptional regulators. Therefore very few transcription factors are specific for a physiological process, or a given cell type. The transcriptional network that regulates blood vessel formation involves transcription factors which are expressed in a variety of situations. The Lung Kruppel Like Factor (LKLF) which is required for blood vessel stabilisation during murine development is also expressed in the primitive vertebrae and in the lung of the adult (C.T. Kuo, M.L. Veselits, K.P. Barton, M.M. Lu, C. Clendenin, J.M. Leiden, The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilisation during murine embryogenesis, Genes Dev. 11 (22) (1997) 2996-3006). Scl/Tal1 which is essential for angiogenic remodelling of the yolk sac capillary network (J.E. Visvader, Y. Fujiwara, S.H. Orkin, Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development, Genes Dev. 12 (4) (1998) 473-479), is involved in blood cell development and is also expressed in the developing brain. The EPAS transcription factor which was thought to be endothelial cell specific in the mouse embryo (H. Tian, S.L. McKnight, D.W. Russell, Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells, Genes Dev. 11 (1) (1997) 72-82) is also expressed in the liver, kidney and cells of the sympathetic nervous system of the chick embryo (J. Favier, H. Kempf, P. Corvol, J.M. Gasc, Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase, FEBS Lett. 462 (1-2) (1999) 19-24). Ets1, which expression was originally detected in lymphoid cells of adult tissues, has been the first transcription factor to be identified in endothelial cells during angiogenesis in the embryo (B. Vandenbunder, L. Pardanaud, T. Jaffredo, M.A. Mirabel, D. Stehelin, Complementary patterns of expression of c-etsl, c-myb and c-myc in the blood-forming system of the chick embryo, Development 107 (1989) 265-274 [5]) and in tumours (N. Wernert, M.B. Raes, P. Lassalle, M.P. Dehouck, B. Gosselin, B. Vandenbunder, D. Stehelin, The c-ets 1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularisation and other forms of angiogenesis in man, Am. J. Path. 140 (1992) 119-127 [6]). Since then, the Ets family has extended and this review will emphasise the relationships between these factors and angiogenesis.

Whole genome" expression profiles can be used as molecular signatures or portraits characteristic of a tumour or of the physiological state of a cell. This global description also provides a new approach for the study of the transcriptional regulation, and this review is dedicated to this fundamental aspect of the transcriptome. Gene expression studies will be first considered at the single cell level. In situ analyses reveal that transcription is a stochastic phenomenon, indicating that the transcriptome contains fluctuating regions. The transcriptional response of a cell population to an external stimulus has been used to evaluate the contribution of the various signalling pathways and transcription factors. One of the emerging themes is the co-ordinate expression of genes associated with the same physiological process. In the spirit of this analysis of the transcriptional regulatory networks, it is becoming possible to design artificial biological networks to implement desired functions, paving the way to new therapeutic approaches.

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

During morphogenesis of the vascular tree, the massive outgrowth of primitive capillaries is followed by the development and the maturation of some capillary branches whereas others regress. The direct observation and the manipulation of in vivo models, including a series of recent knock-out experiments, allow to delineate the mechanisms controlling this process, and to identify factors involved in the formation of a mature capillary, surrounded with a basal lamina and pericytes. The expression of several members of the Ets family of transcription factors, Ets1, Erg and Fli, correlates with the occurrence of invasive processes, such as angiogenesis during normal and pathological development. The description of the phenotype of cultured endothelial cells expressing the DNA binding domain of Ets1 suggests that members of the Ets family take part in the morphogenesis of the -vascular tree. Although transient transfection experiments allowed the identification of putative targets genes for Ets1 during angiogenesis, deciphering the Ets1 regulation networks remains a major goal for the future.

Various strategies led to the identification of transcription factors that take part to the control of different steps during the formation of new blood vessels: the description of the expression pattern of genes encoding these factors during embryonic development for ETS-1, ERG and FLI, SCL/TAL, GATA1 and 2, the description of the phenotype of embryos obtained after gene inactivation by homologous recombination for ARNT or LKLF, and the study of transcriptional regulation in cultured endothelial cells for EGR1 or HOX-D3. Altogether, these results showed that there is no transcription factor specific for endothelial cells or for one step in the formation of blood vessels. Rather, factors controlling gene expression induced by hypoxia, shear-stress or growth factors take part in the morphogenesis of the vascular tree. The study of these factors may allow to identify potential therapeutic targets for treatments aimed at inhibiting or stimulating the development of new blood vessels.

HIF-1 is a transcription factor which stimulates the expression of several genes induced by hypoxia, such as the gene encoding the vascular endothelial growth factor VEGF. Three papers report the effects of the inactivation of the HIF-1 alpha gene. The absence of HIF-1 alpha impairs morphogenesis of the vascular tree, both in the embryo and in teratocarcinomas obtained after injection of ES cells. HIF-1 alpha-/-embryos do not survive beyond midgestation. In contrast, one paper reports tumour growth inhibition, and another one its stimulation. These results suggest that the role of HIF-1 in the hypoxic response is dependent on the genetic background.

Angiopoietin-2 is a new member of the family of the ligands of Tek (Tie2), a transmembrane receptor with tyrosine kinase activity which is specifically expressed in endothelial cells during angiogenesis. Several lines of evidence indicate that angiopoietin-1 is involved in pericyte recruitment by endothelial cells and in the maintenance of cell-cell and cell matrix association in mature capillaries. Angiopoietin-2 behaves as an antagonist of angiopoietin-1. It is suggested that it looses capillary structure and renders endothelial cells to become responsive to angiogenic stimuli.

The c-ets-1 transcription factor has been involved in the in vitro transactivation of matrix-degrading protease genes that might play an important role in tumor invasion. Using in situ hybridization, we analyzed serial frozen sections for c-ets-1, collagenase 1, and urokinase-type plasminogen activator gene expression in 54 lung carcinomas including 34 non-neuroendocrine carcinomas (18 squamous carcinomas, 10 adenocarcinomas, 3 large cell carcinomas, and 3 basaloids) and 20 neuroendocrine carcinomas (7 small cell lung carcinomas, 4 large cell neuroendocrine carcinomas, 4 well differentiated neuroendocrine carcinomas, and 5 carcinoids). c-ets-1 gene was expressed in stromal cells in 44/54 lung carcinomas including one metastasizing carcinoid. c-ets-1 transcripts were also detected in cancer cells more frequently in neuroendocrine than in non-neuroendocrine carcinomas (P = 0.0059) and in stages III and IV and metastasis more frequently than in stages I and II ( P = 0.0065). Collagenase 1 gene was expressed in 16/34 non-neuroendocrine tumors and in 1/20 neuroendocrine tumors, either in stromal (12/17) or in cancer cells (6/17). Urokinase-type plasminogen activator mRNAs were expressed in 45/54 lung carcinomas in stromal and/or cancer cells. In non-neuroendocrine tumors, c-ets-1 and collagenase 1 gene expressions in stromal cells were correlated. These results demonstrate that the transcription factor c-ets-1, collagenase 1, and urokinase-type plasminogen activator are involved in lung cancer invasion and suggest that c-ets-1 protein might transactivate collagenase 1 gene during tumor invasion.

The formation of new blood vessels is an essential process in embryonic development and wound healing, for tumor growth and metastasis. In situ hybridization studies have revealed that the protooncogene c-est1 is expressed in endothelial cells at the beginning of blood vessel formation, in normal and pathological conditions. c-ets1 encodes a transcription factor, a protein which binds specifically to DNA and which regulates the transcription of genes containing these specific binding sequences in their promotors. Thus, in vitro experiments suggest that c-ets1 may activate the transcription of genes encoding collagenase 1, stromelysine 1 and urokinase plasminogen activator, proteases involved in extracellular matrix degradation. A working hypothesis is that c-ets1 takes part in regulating angiogenesis by controlling the transcription of these genes whose activity is necessary for the migration of endothelial cells from pre-existing capillaries. This hypothesis is discussed with respect to current experimental evidence and to the complexity of the regulatory network controlling gene transcription and extracellular matrix degradation.

The c-ets1 proteins are transcriptional activators expressed within endothelial cells during blood vessel development in chick embryos. The authors show by in situ hybridization that c-ets1 is transcribed in the endothelia during angiogenesis in human embryos, in granulation tissue, and especially during tumor vascularization. c-ets1 mRNAs were also detected in the fibrocytes of tumor stroma and in the spindle cells of Kaposi's sarcomas, regarded as cells of endothelial origin. It has been shown that the c-ets proteins activate transcription through a PEA3 motif that plays a role in the stimulation of transcription of urokinase-type plasminogen-activator (u-PA), stromelysin and collagenase genes. The authors demonstrate in vitro that the angiogenic factor TNF alpha increases transiently the amount of both c-ets1 and u-PA mRNA in confluent human umbilical vein endothelial cells. Therefore, the authors suggest that the c-ets1 proteins might regulate the transcription of the genes coding for matrix-degrading proteases, which are necessary for both angiogenesis and tumor invasion.