Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be mediated by TNF-R1

Thrombospondin-1 (TSP-1) treatment of dermal microvascular endothelial cells (MvEC) has been shown to upregulate Fas ligand (FasL) and to induce apoptosis by a mechanism that requires caspase-8 activity. We have examined the potential anti-angiogenic effects of TSP-1 on primary human brain MvEC. The addition of TSP-1 to primary human brain MvEC cultured as monolayers on type 1 collagen, induced cell death and apoptosis (evidenced by caspase-3 cleavage) in a dose- (5-30 nM) and time-dependent (maximal at 17 h) manner. TSP-1 treatment for 17 h induced caspase-3 cleavage that required caspase-8 activity and the tumor necrosis factor receptor 1 (TNF-R1). We did not find a requirement for Fas, or the tumor necrosis-related apoptosis-inducing ligand receptors (TRAIL-R) 1 and 2. We confirmed the findings using caspase inhibitors, blocking antibodies and small interfering RNA (siRNA). Further analysis indicated that the TSP-1 induction of caspase-3 cleavage of primary human brain MvEC adherent to collagen required the synthesis of new message and protein, and that TSP-1 induced the expression of TNFalpha mRNA and protein. Consistent with these findings, when the primary human brain MvEC were propagated on collagen gels mAb anti-TNF-R1 reversed the inhibitory effect, in part, of TSP-1 on tube formation and branching. These data identify a novel mechanism whereby TSP-1 can inhibit angiogenesis-through induction of apoptosis in a process mediated by TNF-R1.     

Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein that influences cellular phenotype and the structure of the extracellular matrix. These effects are important components of the tissue remodeling that is associated with angiogenesis and neoplasia. The genetic mutations in oncogenes and tumor suppressor genes that occur within tumor cells are frequently associated with decreased expression of TSP-1. However, the TSP-1 that is produced by stromal fibroblasts, endothelial cells and immune cells suppresses tumor progression. TSP-1 inhibits angiogenesis through direct effects on endothelial cell migration and survival and through indirect effects on growth factor mobilization. TSP-1 that is present in the tumor microenvironment also acts to suppress tumor cell growth through activation of transforming growth factor β in those tumor cells that are responsive to TGFβ. In this review, the molecular basis for the role of TSP-1 in the inhibition of tumor growth and angiogenesis is summarized.     

In vitro assessment of iron effect on porcine ovarian granulosa cells: secretory activity, markers of proliferation and apoptosis

It would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at doses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, proliferation and apoptosis.     

Response of bovine endothelial cells to FGF-2 and VEGF is dependent on their site of origin: Relevance to the regulation of angiogenesis

Angiogenesis, the formation of new capillary blood vessels, occurs almost exclusively in the microcirculation. This process is controlled by the interaction between factors with positive and negative regulatory activity. In this study, we have compared the effect of two well described positive regulators, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) on bovine adrenal cortex-derived microvascular endothelial (BME) and bovine aortic endothelial (BAE) cells. The parameters we assessed included (a) cellular reorganization and lumen formation following exposure of the apical cell surface to a three-dimensional collagen gel; (b) organization of the actin cytoskeleton; (c) expression of thrombospondin-1 (TSP-1), an endogenous negative regulator of angiogenesis; and (d) extracellular proteolytic activity mediated by the plasminogen activator (PA)/plasmin system. We found that (a) collagen gel overlay induces rapid reorganization and lumen formation in BME but not BAE cells; (b) FGF-2 but not VEGF induced dramatic reorganization of actin microfilaments in BME cells, with neither cytokine affecting BAE cells; (c) FGF-2 decreased TSP-1 protein and mRNA expression in BME cells, an effect which was specific for FGF-2 and BME cells, since TSP-1 protein levels were unaffected by VEGF in BME cells, or by FGF-2 or VEGF in BAE cells; (d) FGF-2 induced urokinase-type PA (uPA) in BME and BAE cells, while VEGF induced uPA and tissue-type PA in BME cells with no effect on BAE cells. Taken together, these findings reveal endothelial cell-type specific responses to FGF-2 and VEGF, and point to the greater specificity of these cytokines for endothelial cells of the microvasculature than for large vessel (aortic) endothelial cells. Furthermore, when viewed in the context of our previous observation on the synergistic interaction between VEGF and FGF-2, our present findings provide evidence for complementary mechanisms which, when acting in concert, might account for the synergistic effect.     

Tumor progression: the effects of thrombospondin-1 and -2

The thrombospondins (TSPs) are a family of proteins that regulate tissue genesis and remodeling. In many tumors, down-regulation of TSPs accompanies activation of oncogenes or inactivation of tumor suppresser genes and appears to be a prerequisite for the aquisition of a pro-angiogenic phenotype. The normal suppression of angiogenesis by TSP-1 and -2 involves multiple mechanisms including direct interaction with vascular endothelial cell growth factor (VEGF), inhibition of matrix metalloproteinase 9 (MMP9) activation, inhibition of endothelial cell migration and induction of endothelial cell apoptosis. The importance of down-regulation of TSPs for tumor progression is further established by the fact that several different approaches that are designed to increase the levels of TSP-1 or -2 in tumor tissue inhibit tumor growth. These approaches include cell-based gene therapy, low dose chemotherapeutics and systemic delivery of recombinant proteins or synthetic peptides that include type 1 repeat (TSR) sequences. Initial studies indicate that these reagents, in combination with established approaches for the treatment of cancer, will offer more efficacious therapies.     

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones that overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knock down NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared with V189shCtl, under stress conditions. Unexpectedly, NRP1 knockdown had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity toward NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.Key words: VEGF isoforms, survival, apoptosis, NRP-1, breast cancer cells     

Differential involvement of TGF-beta1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1–10 µg/ml) and inhibition at high concentrations (25–100 µg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5–25 µg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to αvβ3 integrin. TGF-β1 (0.1–200ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-β1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-β1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-β1, whereas the effects on fibroblast and tumour cell migration are TGF-β1-independent.     

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.     

The role of thrombospondin-1 in tumor progression

The role of thrombospondin-1 (TSP-1) in tumor progression is both complex and controversial. It is clear from the literature that the function of TSP-1 in malignancy depends on the presence of other factors and the level of TSP-1 expression in the tumor tissue. High levels of TSP-1 secreted by tumors, which were engineered to overexpress TSP-1, inhibit tumor growth, while anti-sense inhibition of TSP-1 production in certain tumors also inhibits growth. Clearly, the presence of other factors in these experimental systems must be important. The role of TSP-1 in angiogenesis also depends on the levels of TSP-1, the presence and level of angiogenic stimulators such as basic fibroblast growth factor (bFGF), and the localization of TSP-1 in the tissue. Matrix-bound TSP-1 promotes capillary tube formation in the rat aorta model of angiogenesis, while TSP-1 inhibits bFGF- induced angiogenesis in the rat cornea model. The inhibitory effect also depends on the proteolytic state of TSP-1 since the amino terminus promotes angiogenesis in the cornea model, while the remaining 140-kDa fragment inhibits bFGF-induced angiogenesis. Both the stimulatory and inhibitory effects of TSP-1 are likely due to upregulation of matrix-degrading enzymes and their inhibitors. These enzymes are critical for maintaining optimal matrix turnover during angiogenesis. These varied TSP-1-dependent mechanisms offer new targets for the development of anti-angiogenic therapeutics for the treatment of a variety of cancers, as well as other pathologies involving inappropriate angiogenesis such as diabetic retinopathy.     

Thrombospondin-1 Modulates Angiogenesisin Vitroby Up-Regulation of Matrix Metalloproteinase-9 in Endothelial Cells

Evidence suggests that thrombospondin-1 (TSP-1), a 450-kDa glycoprotein in platelets and extracellular matrix, is involved in angiogenesis. However, the mechanisms by which TSP-1 regulates angiogenesis are unknown, and the exact role of TSP-1 in angiogenesis has been controversial: both stimulatory and inhibitory effects of TSP-1 have been reported. In this study, we evaluated the effect of TSP-1 on the capacity of bovine aortic endothelial (BAE) cells to both invade and form microvessel-like tubes in collagen gels. BAE cell tube formation was enhanced by exogenous TSP-1 at relatively low concentrations (1–10 μg/ml) but inhibited at higher concentrations of TSP-1 (>15 μg/ml). In addition, we correlated this biphasic effect on tube formation with the capacity of TSP-1 to stimulate the activity of a matrix metalloproteinase-9 (MMP-9) in BAE cell collagen gel cultures. The TSP-1-mediated stimulation of MMP-9 activity was specific and dose- and time-dependent. Furthermore, TSP-1-stimulated BAE cell invasion and tube formation were reversed by antibodies against both TSP-1 and MMP-9, suggesting that TSP-1 modulates endothelial cell invasion and morphogenesisin vitroby a mechanism involving the regulation of MMP-9 activity. These findings support the conclusion that TSP-1 is a multifunctional modulator of angiogenesis and are consistent with the dynamic presence of TSP-1 in remodeling tissues in which matrix degradation is required.     

Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development

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.     

Thombospondin-1 disrupts estrogen-induced endothelial cell proliferation and migration and its expression is suppressed by estradiol

The natural hormone 17beta-estradiol (17beta-E2) is known to induce tumor angiogenesis in various target organs by activating positive regulators of angiogenesis. In this study, we show for the first time that in human umbilical vein endothelial cells (HUVECs), 17beta-E2 transiently down-regulates the expression and secretion of a potent negative regulator of angiogenesis, thrombospondin-1 (TSP-1). This inhibitory effect of 17beta-E2 is mediated through nongenomic estrogen receptor (ER)/mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) 1/2 and c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathways, because this effect can be abolished by a pure ER antagonist (ICI 182,780) and inhibitors of downstream signaling proteins of MAPK signaling cascades, including MAPK kinase 1/2 and ERK1/2 inhibitor and JNK/SAPK inhibitor. To understand the functional role(s) of TSP-1 during estradiol-induced angiogenesis, we examined the growth and migration of endothelial cells in different experimental environments. Using a recombinant protein, we show that increments of TSP-1 protein concentration in culture medium significantly reduce the migration and proliferation of HUVECs stimulated by 17beta-E2. Together, these studies suggest that TSP-1 can be considered an important negative factor in understanding the increased angiogenesis in response to estrogens.     

Chronic Delivery of a Thrombospondin-1 Mimetic Decreases Skeletal Muscle Capillarity in Mice

Angiogenesis is an essential process for normal skeletal muscle function. There is a growing body of evidence suggesting that thrombospondin-1 (TSP-1), a potent antiangiogenic protein in tumorigenesis, is an important regulator of both physiological and pathological skeletal muscle angiogenesis. We tested the hypothesis that chronic exposure to a TSP-1 mimetic (ABT-510), which targets the CD36 TSP-1 receptor, would decrease skeletal muscle capillarity as well as alter the balance between positive and negative angiogenic proteins under basal conditions. Osmotic minipumps with either ABT-510 or vehicle (5% dextrose) were implanted subcutaneously in the subscapular region of C57/BL6 mice for 14 days. When compared to the vehicle treated mice, the ABT-510 group had a 20% decrease in capillarity in the superficial region of the gastrocnemius (GA), 11% decrease in the plantaris (PLT), and a 35% decrease in the soleus (SOL). ABT-510 also decreased muscle protein expression of vascular endothelial growth factor (VEGF) in both the GA (−140%) and SOL (−62%); however there was no change in VEGF in the PLT. Serum VEGF was not altered in ABT-510 treated animals. Endogenous TSP-1 protein expression in all muscles remained unaltered. Tunnel staining revealed no difference in muscle apoptosis between ABT-510 and vehicle treated groups. These data provide evidence that the anti-angiogenic effects of TSP-1 are mediated, at least in part, via the CD36 receptor. It also suggests that under physiologic conditions the TSP-1/CD36 axis plays a role in regulating basal skeletal muscle microvessel density.     

The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF--but not TSP-1--stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.     

Transforming growth factor‐beta 1 (TGF‐β1) induces angiogenesis through vascular endothelial growth factor (VEGF)‐mediated apoptosis

VEGF and TGF‐β1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF‐β1 induces apoptosis. We have previously shown that VEGF/VEGF receptor‐2 (VEGFR2) signaling mediates TGF‐β1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF‐mediated apoptosis is required for TGF‐β1 induction of angiogenesis. In vitro the apoptotic effect of TGF‐β1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF‐β1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord‐like structures by TGF‐β1 with an effect comparable to that of z‐VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF‐β1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF‐β1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF‐β1 induction of both apoptosis and angiogenesis, an effect similar to that of z‐VAD. Thus, TGF‐β1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF‐mediated apoptosis as a potential target to control angiogenesis. J. Cell. Physiol. 219: 449–458, 2009. © 2009 Wiley‐Liss, Inc.     

HIF-1在卵巢黄体发育过程中对血管新生的调控作用

在哺乳动物中,卵巢黄体（corpus luteum,CL）是由破裂排卵后的卵泡所形成的,也是血管增生比较激烈的地方。尤其是在卵巢黄体早期发育阶段,这种快速形成的致密毛细血管网可以确保产生激素的细胞获得氧气、营养和合成激素等所必要的前体,同时释放大量的激素用于早期妊娠的建立和维持。目前的研究已经表明,血管内皮生长因子（vascular endothel ial growth factor,VEGF）作为重要的促血管生成因子,在卵巢黄体发育过程中对血管增生具有至关重要的调节作用,而VEGF作为转录因子HIF－1的下游靶基因,受缺氧诱导因子HIF－1信号通路的调控。该文一方面对卵巢黄体发育过程中VEGF依赖性血管增生的调控机制进行概述,另一方面就转录因子H1F－1对VEGF的转录激活调控机制进行系统阐述,从而揭示HIF－1对卵巢黄体发育过程dgVEGF依赖性血管新生的调控作用,为进一步研究哺乳动物卵巢黄体发育过程中血管增生的分子调控机制提供坚实的理论基础。