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. 2007 Mar;210(3):807-18.
doi: 10.1002/jcp.20904.

Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1)

James Greenaway  1 , Jack Lawler, Roger Moorehead, Paul Bornstein, Jonathan Lamarre, Jim Petrik
Affiliations

Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1)

James Greenaway et al. J Cell Physiol. 2007 Mar.

Abstract

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 TNFalpha-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.

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Figures

Fig. 1
Fig. 1. Expression of CD31 and quantification of vessel density in ovaries from wild-type and TSP-1-nullmice
A: Ovaries from wild-type and TSP-1 null mice were immunostained for CD31 (brown stain). Ovaries of wild type mice demonstrated peri-follicular, thecal, and luteal immunopositive blood vessels. TSP-1-null mice exhibited increased expression of CD31, with the presence of large CD31-positive blood vessels. B: Western blot; there was a significant increase in CD31 protein in ovaries from TSP-1-null versus wild-type mice. C: Ovarian vessel density was calculated as the total area of CD31-positive blood vessels in wild-type and TSP-1-null mice. TSP-1 null ovaries had a significantly elevated blood vessel density, compared to wild-type controls. For the Western blot graph, * denotes a statistical difference in CD31 protein compared to wild-type controls (P < 0.05).
Fig. 2
Fig. 2. Expression of VEGF in ovaries from wild-type and TSP-1-null mice
A: Ovaries from TSP-1-null mice exhibited an increase in VEGF-positive cells (brown stain) that were localized to the corpus luteum, granulosa/theca cells of the follicle, and endothelial cells. B: There was a significant increase VEGF protein in TSP-1-null ovaries compared to wild-type ovaries, as measured by Western blotting. For the Western blot graph, * denotes a statistically significant difference in VEGF protein compared to wild-type controls (P < 0.05).
Fig. 3
Fig. 3. TSP-1 treatment of SIGC inhibits VEGF protein expression and induces apoptosis
A: Western blot analysis of VEGF protein from SIGC treated for 24 h with increasing amounts of purified TSP-1. Graph represents densitometric analysis of VEGF protein from three different in vitro experiments (mean ± SEM). B: Percent apoptosis in granulosa cells subjected to TNFα challenge in the absence or presence of variable concentrations of TSP-1. All cultures were performed in serum-free media. Lane 1: No treatment; Lane 2: 100 ng/ml TNFα; Lane 3: 100 ng/ml TNFα + 50 ng/ml VEGF + 10 ng/ml TSP-1; Lane 4: 100 ng/ml TNFα + 50 ng/ml VEGF + 50 ng/ml TSP-1; Lane 5: 100 ng/ml TNFα + 50 ng/ml VEGF + 100 ng/ml TSP-1; Lane 5: 100 ng/ml TNFα + 50 ng/ml VEGF + 1 µg/ml TSP-1. * denotes statistically different from serum free control (Lane 1) (P < 0.05) (n = 3 experiments).
Fig. 4
Fig. 4. Expression of proteins relevant to proliferation and apoptosis in response to VEGF, TSP-1, or combination treatment
A: Relief contrast overlay immunofluorescence images of untreated granulosa cells or after treatment with 50 ng/ml VEGF, 100 ng/ml TSP-1 or a combination of 50 ng/ml VEGF and 100 ng/ml TSP-1. B: Quantification of the percentage of immunopositive in vitro cells from (A). Graphs represent four replicate experiments (mean ± SEM). C: Representative (of four replicate) western blots of proteins isolated from granulosa cells treated with VEGF, TSP-1, or both. A decrease in cytoprotective VEGF, VEGR2, and bcl-2 is observed after treatment with 100 ng/ml TSP-1 compared to treatment with 50 ng/ml VEGF. Conversely, an increase in pro-apoptotic Fas occurs following treatment with TSP-1. * denotes statistically different from serum free controls (P > 0.05).
Fig. 5
Fig. 5. Binding and internalization of TSP-1/VEGF by the LRP receptor
A: Immunofluorescent co-localization of TSP-1 and VEGF in the absence or presence of the LRP-1 antagonist RAP. Cells were treated for 6 h with purified VEGF or TSP-1 labeled with AlexaFluor 488 dye (green stain). Co-staining for TSP-1 or VEGF was performed with an AlexaFluor 594 conjugated antibody (red stain). Reciprocal experiments are shown. A sample containing only AlexaFluor dye (no protein) is shown as negative control. B: 125I labeled VEGF is internalized by LRP-1-expressing Chinese hamster ovary (CHO) cells and native murine endothelial (mEC) and granulosa cells (SIGC), but not LRP-1-deficient CHO cells or by SIGC and mEC treated with 500 nM RAP, or by SIGC treated with TSP-1 siRNA. CHO, SIGC, and mEC were plated into 12-well dishes 24 h prior to the assay. 1 nM125I-Labeled VEGF was added to LRP-positive and -negative CHO cells, to SIGC and mEC in the presence or absence of 500 nM RAP, and to SIGC treated with TSP-1 siRNA. At the indicated times, the amount of internalized radioactivity was determined. Each data point represents the average of duplicate assays (n = 3).
Fig. 6
Fig. 6. TSP-1 knockdown results in increased VEGF expression and reduced apoptosis
A: Immunofluorescence for TSP-1 (green) and VEGF (red) SIGC treated with scrambled sequence (RNAi control) or TSP-1 siRNA. Images are acquired with identical acquisition settings. B: Western blot analysis of TSP-1 and VEGF in control and TSP-1 siRNA treated SIGC. C: Percent apoptosis in untreated, and cyokine-challenged SIGC in the presence or absence of controls or TSP-1 siRNA. All cultures were performed in serum free media. Lane 1: No treatment; Lane 2: 100 ng/ml TNFα; Lane 3: 100 ng/ml TNFα + 50 ng/ml VEGF + vehicle control; Lane 4: 100 ng/ml TNFα + 50 ng/ml VEGF + RNAi control; Lane 5: 100 ng/ml TNFα + 50 ng/ml VEGF + TSP-1 RNAi. * denotes statistically different from serum free control (Lane 1) (P < 0.05) (n = 3 experiments).
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