Thrombospondin 1 as a scavenger for matrix-associated fibroblast growth factor 2

The antiangiogenic factor thrombospondin 1 (TSP-1) binds with high affinity to several heparin-binding angiogenic factors, including fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), and hepatocyte growth factor/scatter factor (HGF/SF). The aim of this study was to investigate whether TSP-1 affects FGF-2 association with the extracellular matrix (ECM) and its bioavailability. TSP-1 prevented the binding of free FGF-2 to endothelial cell ECM. It also promoted the mobilization of matrix-bound FGF-2, generating a TSP-1/FGF-2 complex. The region of TSP-1 responsible for these activities was located within the 140-kDa antiangiogenic and FGF-2 binding fragment, whereas the 25-kDa heparin-binding fragment was inactive. Matrix-released FGF-2/TSP-1 complex had a reduced ability to bind to and induce proliferation of endothelial cells. TSP-1 depleted the ECM laid by FGF-2-overproducing tumor cells of its FGF-2-dependent mitogenic activity for endothelial cells. Besides FGF-2, TSP-1 also inhibited VEGF and HGF/SF binding to the ECM and mobilized them from the ECM. Our study shows that TSP-1 acts as a scavenger for matrix-associated angiogenic factors, affecting their location, bioavailability, and function.     

Levels of angiogenic factors in patients with multiple myeloma correlate with treatment response

Angiogenesis plays a significant role in the pathogenesis of multiple myeloma (MM). We have measured concentrations of angiogenesis activators, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and hepatocyte growth factor (HGF), and inhibitors, including endostatin, thrombospondin-1 (TSP-1), and angiostatin in the peripheral and bone marrow blood of MM patients at diagnosis and after high-dose chemotherapy. We have analyzed 96 patients with secretory MM. Serial measurements of angiogenesis factors/inhibitors were analyzed in the plasma by subgroups based on the best treatment response. Concentrations of angiogenic factors were determined in the peripheral blood and bone marrow plasma. There were significant decreases of VEGF and HGF levels and a significant increase in TSP-1 concentrations in the bone marrow plasma of patients who achieved complete or very good partial response in contrast to those who had partial or no response. VEGF and HGF levels decrease but those of TSP-1 increase after successful treatment for MM, indicating a reduction in the rate of angiogenesis.     

Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation.

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.     

Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors

Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth. Our experiments demonstrate that normal physiological levels of these inhibitors serve to retard the growth of tumors, and that their absence leads to enhanced angiogenesis and a 2- to 3-fold increase in tumor growth. The tumor-suppressive action of TSP-1, endostatin, and tumstatin correlates with expression of CD36 receptor, alpha5beta1 integrin, and alphavbeta3 integrin on proliferating endothelial cells, respectively. Moreover, tumors grow 2-fold faster in the tumstatin/TSP-1 double-knockout mice, compared with either the tumstatin- or the TSP-1-deficient mice, strongly suggesting that ceiling rate of cancer growth is not completely dependent on the genetic defects of cancer cells but also depends on the host-derived tumor microenvironment. Additionally, tumor growth in transgenic mice overproducing endostatin specifically in the endothelial cells (a 1.6-fold increase in the circulating levels; mimicking Down's syndrome condition) is 3-fold slower than the tumor growth in wild-type mice. Collectively, our data suggest that physiological levels of endogenous inhibitors of angiogenesis can serve as endothelium-specific tumor suppressors.     

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.     

离散因子/肝细胞生长因子cDNA转染对肝癌SMMC7721细胞生长和转移的影响

目的 通过离散因子／肝细胞生长因子（scatter factor/Hepatocyte factor,SF/HGF）cDNA转染肝癌SMMC7721细胞来探讨SF／HGF对肝癌生长和转移的影响。方法 用脂质体法进行基因转染,以ELISA和western blot检测SF／HGF及其受体c-met的表达,通过生长曲线、划良实验比较转染前后细胞的增长状况及细胞的运动能力。以转染前后细胞分别接种裸鼠,观察移植的生长及转移情况。结果 转染后细胞表达SF／HGF的量为694pg/ml,其受体c-met量未见明显变化;转染后细胞增殖明显加快,运动能力增强,并伴明显的形态学变化。初步动物实验显示转染后细胞生长较快,瘤组织中有瘤栓形成,并在肺内发现转移灶。结论 SF／HGF在肝癌细胞中的高表达能促进肝癌细胞生长、侵袭及转移。     

Cell surface proteoglycan syndecan-1 mediates hepatocyte growth factor binding and promotes Met signaling in multiple myeloma

Heparan sulfate proteoglycans (HSPGs) play a crucial role in growth regulation by assembling signaling complexes and presenting growth factors to their cognate receptors. Within the immune system, expression of the HSPG syndecan-1 (CD138) is characteristic of terminally differentiated B cells, ie, plasma cells, and their malignant counterpart, multiple myeloma (MM). This study explored the hypothesis that syndecan-1 might promote growth factor signaling and tumor growth in MM. For this purpose, the interaction was studied between syndecan-1 and hepatocyte growth factor (HGF), a putative paracrine and autocrine regulator of MM growth. The study demonstrates that syndecan-1 is capable of binding HGF and that this growth factor is indeed a potent stimulator of MM survival and proliferation. Importantly, the interaction of HGF with heparan sulfate moieties on syndecan-1 strongly promotes HGF-mediated signaling, resulting in enhanced activation of Met, the receptor tyrosine kinase for HGF. Moreover, HGF binding to syndecan-1 promotes activation of the phosphatidylinositol 3-kinase/protein kinase B and RAS/mitogen-activated protein kinase pathways, signaling routes that have been implicated in the regulation of cell survival and proliferation, respectively. These results identify syndecan-1 as a functional coreceptor for HGF that promotes HGF/Met signaling in MM cells, thus suggesting a novel function for syndecan-1 in MM tumorigenesis.     

Hepatocyte growth factor stimulates nitric oxide production through endothelial nitric oxide synthase activation by the phosphoinositide 3-kinase/Akt pathway and possibly by mitogen-activated protein kinase kinase in vascular endothelial cells.

Hepatocyte growth factor (HGF) has recently been the focus of attention due to its angiogenic effects, which are similar to those of vascular endothelial growth factor (VEGF); because of these effects, HGF is considered to be a novel therapeutic agent against vascular disorders, including atherosclerotic angiopathies. Although nitric oxide (NO), which is derived from vascular endothelial cells (ECs), is also involved in angiogenesis, little is known regarding the interactions between HGF and NO. We therefore examined the effects of HGF on NO production as well as endothelial NO synthase (eNOS) phosphorylation, and investigated their mechanisms. In bovine aortic ECs, HGF induced a rapid (5 min) increase of NO production measured by diaminofluorescein-2 diacetate. Moreover, HGF rapidly (2.5 min) stimulated eNOS phosphorylation (Ser-1179) as determined by Western immunoblot analyses. Both of these effects were almost completely suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and were partially suppressed by the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126. HGF also stimulated Akt phosphorylation (Ser-473), which was completely suppressed by LY294002 and was partially suppressed by U0126. Moreover, HGF stimulated extracellular signal-regulated kinase 1/2 phosphorylation (Thr-202/Tyr-204), which was completely suppressed by U0126 and was partially suppressed by LY294002. Taken together, these results indicate that HGF not only phosphorylates eNOS through the PI3K/Akt pathway, but also partially through the MAPK pathway, and that these two pathways may interact. Compared with VEGF, HGF was more potent in both NO production and eNOS phosphorylation. Our study thus demonstrates a novel activity of HGF-the stimulation of NO production-which occurs via eNOS phosphorylation that may in turn be mediated by cross-talk between the PI3K/Akt and MAPK pathways.     

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.     

Hemoglobin induces the production and release of matrix metalloproteinase-9 from human malignant cells.

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in both angiogenesis and tumor invasion. Vascular endothelial growth factor (VEGF) has been shown to up-regulate the expression of MMP-9 in vascular smooth muscle cells. We recently reported that hemoglobin (Hb) enhances the expression of tissue factor (TF) and VEGF on TF-positive human malignant cells. Therefore, to explore the relationship between tumor cell angiogenic protein VEGF and MMP-9, we studied the effect of Hb on MMP-9 production in human A375 malignant melanoma and J82 bladder carcinoma (TF+) cells and in KG1 myeloid leukemia (TF-) cells. Malignant cells were incubated with varying concentrations (0-1.0 mg/ml) of Hb and analyzed for released MMP-9 by gelatin zymography, dot immunoblotting, enzyme-linked immunosorbent assay, and Western blotting. Hb (0.50 mg/ml) induced an almost two-fold increase of MMP-9 in both A375 malignant melanoma (398 +/- 62 versus 233 +/- 61.0 ng/ml, P = 0.027) and J82 bladder carcinoma cells (1.55 +/- 0.12 versus 0.80 +/- 0.004 ng/ml, P = 0.004), compared with cells incubated without Hb. This release of MMP-9 was significantly inhibited by cycloheximide (95%) and by the specific inhibitors of protein tyrosine kinase, genistein (70 +/- 3.0%, P = 0.00027 and 67 +/- 1.0%, P = 0.00005) and mitogen-activated protein (MAP)-kinase, PD98059 (56 +/- 2.0%, P = 0.0001 and 62 +/- 1.0%, P = 0.00003) in A375 and J82 cells, respectively. In contrast, Hb (2.0 mg/ml) did not increase MMP-9 in KG1 cells. We conclude that Hb-induced synthesis of active MMP-9 in TF-bearing malignant cells is due to de novo synthesis of newly formed protein and is mediated by protein tyrosine kinase and by mitogen-activated protein kinase pathways.     

刚地弓形虫对荷瘤小鼠肿瘤组织微血管生成的抑制作用

摘 要：目的 观察刚地弓形虫感染对BALB/c小鼠ct26细胞皮下移植瘤血管生成的抑制作用及其作用机理。方法 建立刚地弓形虫感染的小鼠结肠癌ct26皮下移植瘤模型,观察弓形虫感染对小鼠的生存延长率、对肿瘤的质量抑制率,并采用免疫组化方法检测弓形虫感染对肿瘤组织MVD、VEGF、TSP-1表达的影响。结果 弓形虫感染的小鼠生存时间比对照组长（P<0.05）,生存延长率为57.45%,肿瘤质量抑制率为47.23%;弓形虫感染的小鼠肿瘤组织中MVD及VEGF表达水平低于对照组（P=0.005,P=0.03）,TSP-1表达高于对照组（P=0.005）。结论 弓形虫感染能抑制荷瘤小鼠肿瘤组织微血管生成;下调VEGF及上调TSP-1与弓形虫感染抗肿瘤血管生成有关。