VEGF is a potent pro-angiogenic factor whose effects are opposed by a host of anti-angiogenic proteins, including thrombospondin-1 (TSP-1). We have previously shown that VEGF has important extravascular roles in the ovary and that VEGF and TSP-1 are inversely expressed throughout the ovarian cycle. To date, however, a causal interaction between TSP-1 and VEGF has not been identified. Here, we show that TSP-1 has a direct inhibitory effect on VEGF by binding the growth factor and internalizing it via LRP-1. Mice lacking TSP-1 are subfertile and exhibited ovarian hypervascularization and altered ovarian morphology. Treatment of ovarian cells with TSP-1 decreased VEGF levels and rendered the cells more susceptible to TNFα-induced apoptosis. Knockdown of TSP-1, through RNA interference, resulted in overexpression of VEGF and reduced cytokine-induced apoptosis.In conclusion, we demonstrate a direct inhibitory effect of TSP-1 on VEGF in the ovary. TSP-1's regulation of VEGF appears to be an important mediator of ovarian angiogenesis and follicle development.The growth of normal tissues and pathological structures such as tumors are dependent upon the formation of blood vessels for nutrient delivery and waste removal (Folkman, 1992). Growth of new vasculature is regulated by a balance between pro-and anti-angiogenic factors. A potent pro-angiogenic factor is vascular endothelial growth factor (VEGF), which is a heparin-binding glycoprotein secreted as a homodimer that stimulates endothelial cell proliferation and migration (Bernatchez et al., 2002;Castellon et al., 2002), promotes new vessel formation, increases vascular permeability (Ferrara, 2004), and acts as a survival factor for endothelial cells in vitro and in vivo (Gerber et al., 1998;Jia et al., 2004). In addition to its effects on endothelial cells, we recently reported that VEGF protects ovarian cells from apoptosis by signaling through VEGFR-2 expressed by these cells (Greenaway et al., 2004).The effects of pro-angiogenic factors are balanced by anti-angiogenic factors such as members of the thrombospondin (TSP) family, which consists of five proteins (TSP-1-5), of which TSP-1 and -2 share structural and functional homology (Bornstein, 1992; Adams and Lawler, 2004). TSP-1 is a secreted glycoprotein located in the extracellular matrix that has been shown to be a potent inhibitor of angiogenesis (Lawler, 2002; Armstrong and Bornstein, 2003;Wang et al., 2003;Cursiefen et al., 2004;Lawler and Detmar, 2004 (Detmar, 2000). In addition, a direct interaction of VEGF with TSP-1 and with a TSP-1 type I repeat domain of connective tissue growth factor has been reported (Gupta et al., 1999;Inoki et al., 2002). TSP-1 is also known to bind ligands and interact with the low-density lipoprotein receptorrelated protein (LRP-1), resulting in internalization and degradation of the protein (Mikhailenko et al., 1995;Emonard et al., 2004;Wang et al., 2004). LRP-1 is a member of the low-density lipoprotein receptor gene family, which also includes the LDL, VLDL, and apo...

Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the first time expression of Flk-1/KDR in ovarian granulosa cells and identify a novel extravascular role for VEGF and its receptor in ovarian function.

Angiogenesis does not normally occur in most adult tissues. However, in the ovary, there are cyclical vascular changes including angiogenesis that involve the interaction of numerous cytokines and growth factors. Angiogenic processes are regulated by a balance between pro- and antiangiogenic factors. The purpose of this study was to determine the expression of the antiangiogenic thrombospondin family and proangiogenic vascular endothelial growth factor (VEGF) in various sizes of healthy bovine follicles. Ovaries were collected from slaughterhouse animals and healthy follicles were sorted based on size (< 0.5 cm, small; 0.5-1.0 cm, medium; >1.0 cm, large). Thrombospondin (TSP) protein levels were significantly higher in small follicles. Immunohistochemistry confirmed the granulosa layer as the primary area within the follicle involved in TSP generation and that small follicles had the highest proportion of immunopositive cells. TSP-1 and -2 mRNA levels were significantly higher in small follicles than either medium or large follicles. TSP colocalized with CD36 on granulosa cells (GC) in the follicle and in cultured cells. In contrast with TSP, VEGF expression increased during growth and development of the follicle. FSH stimulated GC expression of TSP, while LH had no effect. In summary, TSP-1 and -2 were coordinately expressed in the extravascular compartment of the ovary during early follicle development. VEGF was inversely expressed, with expression increasing as follicles developed. Regulated expression and localization of these proteins suggests that they may be involved in regulating growth and development of the follicle in a novel fashion.

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Tumor development is a complex process that relies on interaction and communication between a number of cellular compartments. Much of the mass of a solid tumor is comprised of the stroma which is richly invested with extracellular matrix. Within this matrix are a host of matricellular proteins that regulate the expression and function of a myriad of proteins that regulate tumorigenic processes. One of the processes that is vital to tumor growth and progression is angiogenesis, or the formation of new blood vessels from preexisting vasculature. Within the extracellular matrix are structural proteins, a host of proteases, and resident pro- and antiangiogenic factors that control tumor angiogenesis in a tightly regulated fashion. This paper discusses the role that the extracellular matrix and ECM proteins play in the regulation of tumor angiogenesis.