Abundant data now demonstrate that the growth of new blood vessels, termed angiogenesis, plays both pathological and beneficial roles in human disease. Based on these data, a tremendous effort has been undertaken to understand the molecular mechanisms that drive blood vessel growth in adult tissues. Tie2 recently was identified as a receptor tyrosine kinase expressed principally on vascular endothelium. Disrupting Tie2 function in mice resulted in embryonic lethality with defects in embryonic vasculature, suggesting a role in blood vessel maturation and maintenance. Based on these studies, we undertook a series of studies to probe the function of Tie2 in adult vasculature that will form the focus of this chapter. Consistent with a role in blood vessel growth in adult vasculature, Tie2 was upregulated and activated in the endothelium of rat ovary and in healing rat skin wounds, both areas of active angiogenesis. Moreover, Tie2 was upregulated in the endothelium of vascular "hot spots" in human breast cancer specimens. Surprisingly, Tie2 also was expressed and activated in the endothelium of all normal rat tissues examined, suggesting a role in maintenance of adult vasculature. To determine the functional role of Tie2 in tumor vasculature, a soluble Tie2 extracellular domain (ExTek) was designed that blocked the activation of Tie2 by its activating ligand, angiopoietin 1 (Ang1). Administration of recombinant ExTek protein or an ExTek adenovirus inhibited tumor growth and metastasis in rodent tumor models, demonstrating a functional role for Tie2 in pathological angiogenesis in adult tissues. To begin to understand the endothelial signaling pathways and cellular responses that mediate Tie2 function, we identified signaling molecules that are recruited to the activated, autophosphorylated Tie2 kinase domain. Two of these molecules, SHP2 and GRB2, are part of the pathway upstream of mitogen-activated protein kinase (MAPK) activation, a pathway that may be responsible for morphogenetic effects of Tie2 on endothelial cells. Another signaling molecule, p85, is responsible for recruitment of phosphatidylinositol 3 kinase (PI3-K) and activation of the Akt/PI3-K pathway. Akt/PI3-K has emerged as a critical pathway downstream of Tie2 that is necessary for cell survival effects as well as for chemotaxis, activation of endothelial nitric oxide synthase, and perhaps for anti-inflammatory effects of Tie2 activation. Taken together, these studies and many others demonstrate that the Tie2 pathway has important functions in adult tissues, in both quiescent vasculature and during angiogenesis, and help to validate the Tie2 pathway as a therapeutic target.

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxiainducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-b-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3b. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.

Microvascular endothelial cells (RFCs) cultured in twodimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor ␤ 1 (TGF ␤ 1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF ␤ 1. TGF ␤ 1 promotes tube formation in 3D cultures. TGF ␤ 1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF ␤ type I and II receptors have been reported to regulate different activities induced by TGF ␤ 1, we compared the TGF ␤ receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF ␤ 1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF ␤ 1 while the type I receptor mediates the matrix response of RFCs to TGF ␤ 1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF ␤ receptors, altering the responsiveness of RFCs

Summary Endothelial receptor tyrosine kinases may play important roles in pathological vascular growth, particularly in tumours. In this study, immunohistochemistry was used to evaluate the expression of a novel endothelial receptor tyrosine kinase, Tie2/Tek, in the endothelium of vascular 'hotspots' in normal breast tissue (n = 10), benign breast lesions (n = 10) and in breast tumours (n = 123). Tie2 expression was detected in the endothelium of all breast tissues examined. However, the strongest expression of Tie-2 was seen in vascular 'hot spots' within the inflammatory infiltrate at the periphery of invasive tumours. Moreover, the proportion of Tie2-positive vessels (Tie2 counts/CD31 counts) was significantly higher in breast tumours than the proportion of Tie2-positive vessels in either normal breast tissue or benign breast lesions (P = 0.004 and 0.0001 respectively). These data are consistent with a role for Tie2 in tumour angiogenesis and demonstrate the potential use of Tie2 expression as a novel marker of the tumour vasculature.

Bovine aortic endothelial cells (BAECs) express both type I and type II receptors for transforming growth factor beta (TGF beta). These cells respond to TGF beta 1 but are relatively refractory to another isoform of TGF beta, termed TGF beta 2. TGF beta s are thought to signal through receptor complexes composed of type I and/or type II receptors, both of which appear to be functional serine-threonine kinases. The TGF beta type III receptor, on the other hand, does not seem to have any direct signaling capacity. We have now stably transfected BAECs with the type III receptor cDNA. These cells displayed surface expression of the type III receptor protein, as determined by cross-linking with iodinated TGF beta 1 and immunoprecipitation with antibodies to the type III receptor protein. Transfected BAECs exhibit increased responsiveness to TGF beta 2 by several different criteria including an increase in plasminogen activator inhibitor-1 protein and inhibition of migration and proliferation. Thus, the type III receptor protein may play a role in presenting TGF beta 2 to the type II receptor and increase responsiveness to TGF beta 2 to a level comparable to that of TGF beta 1.