Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. The importance of angiogenic factors such as VEGF, while clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Human hematopoietic tumor cell lines, representing multiple lineages and diseases, produce and secrete VEGF and express at least one of its two receptors. Exposure of human vascular endothelial cells to VEGF increased the expression of several hematopoietic growth factors known to be involved in myeloma including interleukin-6 (IL-6). Bone marrow samples from patients diagnosed with multiple myeloma were examined for expression of VEGF and its receptors. VEGF protein production was detected in malignant plasma cells from 78% of the myeloma patients studied. While expression of the Flt-1 and KDR receptors was not observed in the malignant plasma cells, both were markedly elevated in the normal marrow myeloid and monocytic cells surrounding the tumor. In bone marrow clot sections from normal allogeneic donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myelocytes, macrophages, and megakaryocytes. In vitro colony-forming assays using patient-derived material revealed that antibody neutralization of VEGF resulted in an inhibition of colony growth, whereas the addition of recombinant human VEGF stimulated colony formation. Neutralization of VEGF activity also suppressed the generation of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from bone marrow mononuclear cells. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through paracrine and perhaps autocrine mechanisms.     

"Vasocrine" formation of tumor cell-lined vascular spaces: implications for rational design of antiangiogenic therapies

Here we report that B16F10 murine melanoma cells mimic endothelial cell behavior and the angiogenic process in vitro and in vivo. Cord formation in vitro by tumor cells is stimulated by hypoxia and vascular endothelial growth factor (VEGF) and inhibited by antibodies against VEGF and the VEGF KDR receptor (VEGF receptor 2). We define regulation of tumor cell-derived vascular space formation by these vasoactive compounds as "vasocrine" stimulation. ICRF 159 (Razoxane; NSC 129943) prevents tumor cell but not endothelial cell cord formation in vitro, and the antiangiogenic drug TNP-470 (NSC 642492) inhibits endothelial but not tumor cell cord formation in vitro. Both drugs inhibit formation of blood-filled vascular spaces in vivo. These results bear on the anticipated action of ICRF 159 in human clinical trials and novel strategies for targeting tumor blood supplies.     

The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors

Angiogenesis, or the formation of new blood vessels from preexisting vasculature, plays a major role in tumor growth and metastasis formation. Therefore, inhibiting tumor angiogenesis may be a promising therapeutic strategy. Paracrine stimuli from tumor cells are the main promoters of angiogenesis. They activate endothelial cells to proliferate and migrate, subsequently resulting in new tube formation and blood flow. This complex process involves numerous biological activities. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic factor. Originally identified for its ability to induce vascular permeability and stimulate endothelial cell growth, VEGF is now known to be a key requirement for tumor growth. Currently, three high-affinity tyrosine kinase receptors for VEGF have been identified, of which VEGF receptor (VEGFR)-Flk-1/KDR (VEGFR-2) is exclusively expressed in vascular endothelial cells. Because the VEGFR-2 system is a dominant signal-transduction pathway in regulating tumor angiogenesis, specific inhibitors of this pathway inhibit metastases, microvessel formation, and tumor-cell proliferation. Induction of apoptosis in tumor cells and endothelial cells has also been observed. The clinical importance of VEGF for tumor growth is supported by the fact that most tumors produce VEGF and that the inhibition of VEGF-induced angiogenesis significantly inhibits tumor growth in vivo. In this review, we discuss the biologic role of VEGF and the therapeutic options for inhibiting VEGF in cancer patients.     

Regulation of VEGF/VPF expression in tumor cells: Consequences for tumor growth and metastasis

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF) is a multifunctional cytokine which potently stimulates angiogenesis in vivo. VEGF/VPF expression is elevated in pathological conditions including cancer, proliferative retinopathy, psoriasis and rheumatoid arthritis. The angiogenesis associated with human tumors is likely a central component in promoting tumor growth and metastatic potential. The regulation of VEGF/VPF expression during tumor progression may involve diverse mechanisms including activated oncogenes, mutant or deleted tumor suppressor genes, cytokine activation, hormonal modulators, and a particularly effective activator, hypoxia. Understanding the diverse mechanisms by which tumor cells overexpress VEGF/VPF, and which mechanisms are operating in specific tumor types is important for the design of effective anti-cancer therapies.Abbreviations bFGF basic fibroblast growth factor - EC endothelial cell - EGF epidermal growth factor - flk-1 fetal liver kinase-1 - flt-1 fms-like tyrosine kinase-1 - HIF hypoxia inducible factor - KDR kinase domain receptor - PDGF platelet-derived growth factor - PMA phorbol 12-myristate 13-acetate - TGF- transforming growth factor-beta - VEGF vascular endothelial growth factor - VPF vascular permeability factor     

In vivo activities of mutants of vascular endothelial growth factor (VEGF) with differential in vitro activities

VEGF mutants in which Cys51 or Cys60 are converted into a serine are poor inducers of proliferation in human umbilical vein endothelial cells, but they have wild-type activity in the Miles vascular permeability assay. To assess the contribution of proliferation vs. other VEGF activities such as vascular permeability, to tumor angiogenesis and growth, C127I cells, transfected with BPV-based expression plasmids carrying wild-type or mutated VEGF cDNAs, were injected subcutaneously in BALB/c nu/nu mice. From C127I cells expressing wtVEGF(165), intensely vascularized and invasive tumors developed within 2 to 3 weeks. From cells expressing VEGF-Cys51Ser or VEGF-Cys60Ser, tumors developed only after 2 to 3 months, comparable to the time of development of control tumors, i.e., tumors from cells transfected with empty vector. Despite the late take, the VEGF-Cys51Ser and VEGF-Cys60Ser tumors developed an extensive vascular bed with an architecture comparable to that of recombinant wtVEGF-producing tumors whereas control tumors had a considerably lower vascular density. No metastases were detected in mice carrying either wtVEGF or mutant VEGF expressing tumors. Thus, because proliferation-defective VEGF-mutants cannot induce angiogenesis, we conclude that the proliferation-inducing effect of VEGF is crucial for tumor angiogenesis and growth. The hypervasculature in the tumors expressing these VEGF-mutants suggests, however, that other VEGF-activities, such as the induction of vascular permeability, strongly affects vascular density and vascular structure. Furthermore, neither overexpression of VEGF or a high vascular density or hyperpermeability of tumor vasculature is necessarily followed by metastasis.     

VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice

The efficacy of a phosphorothioate antisense oligonucleotide (ASO) for KDR/Flk-1 (KDR/Flk-1-ASO), an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, was investigated on the peritoneal dissemination and angiogenesis of a human gastric cancer cell line in nude mice. Green fluorescent protein (GFP)-transduced NUGC-4 (NUGC-4-GFP) human gastric cancer cells were implanted into the peritoneal cavity of nude mice. KDR/Flk-1-ASO, -SO, or phosphate-buffered saline was administrated from days 7 to 14, 200 microg/mouse, once a day. The mice were sacrificed on day 28. Disseminated peritoneal tumor nodules expressing GFP were visualized by fluorescence microscopy. KDR/Flk-1-ASO significantly decreased the extent of peritoneal dissemination of the tumors. The number of cells undergoing apoptosis was significantly increased in the KDR/Flk-1-ASO-treated tumors. Microvessel density was significantly reduced in the KDR/Flk-1-ASO-treated tumor nodules. The KDR/Flk-1 antisense strategy, therefore, decreases tumor dissemination apparently by inhibiting angiogenesis.     

Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice

Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)-specific angiogenesis are all enhanced in beta(3)-integrin-null mice. Furthermore, endothelial cells isolated from beta(3)-null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that beta(3)-integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in beta(3)-null mice. Moreover, beta(3)-null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation, beta(3)-null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild-type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in beta(3)-null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in beta(3)-integrin-deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth observed in these mice in vivo.     

表达嵌合性T细胞受体的肿瘤浸润淋巴细胞对KDR表达阳性细胞的选择性杀伤作用

目的 研究表达嵌合性T细胞受体VEGF 121-hinger-FcRγ的肿瘤浸润淋巴细胞(TIL)对血管内皮生长因子(VEGF)受体KDR(kinase-domain insert containing receptor)表达阳性细胞的选择性杀伤作用。方法 应用分子克隆技术重组VEGF 121-hinger-FcRy(Vhγ),构建重组逆转录病毒质粒pMSCVneo-Vhγ,经PT67包装后筛选高滴度的病毒,体外感染分离于肝癌组织的TIL,获得MSCVneo-Vhγ-TIL。以空病毒感染TIL获得的MSCVneo-TIL作为对照。以MTT比色法分析不同的TIL细胞对4种靶细胞(不表达VEGF受体KDR的成纤维细胞系NIH3T3和肝癌细胞系HepG2,以及表达VEGF、受体KDR的人脐静脉血管内皮细胞系ECV304和恶性黑色素瘤细胞系A375)的杀伤活性。结果 未转染的TIL和MSCVneo-TIL对NIH3T3和ECV304无明显的杀伤作用,对HepG2和A375有一定杀伤作用,对4种靶细胞杀伤活性的差异无显著性;MSCVneo-Vhγ-TIL对NIH3T3无明显杀伤活性,对ECV304有明显杀伤活性,对HepG2的杀伤与TIL、MSCVneo-TIL相比差异无显著性,而对A375的杀伤明显增强。结论 嵌合性T细胞受体Vhγ经逆转录病毒载体介导,可在TIL细胞表面稳定表达,并获得选择性识别和溶解KDR阳性血管内皮细胞和肿瘤细胞的新效应功能。     

Interleukin-1beta regulates angiopoietin-1 expression in human endothelial cells

Angiopoietin (Ang)-1 is an important regulator of endothelial cell (EC) survival and stabilization. Ang-1 exerts its biological effects by binding to the EC-specific tyrosine kinase receptor Tie-2, and initiates intracellular signaling in ECs. However, regulatory mechanisms for endothelial Ang-1 expression have not been completely elucidated. In this study, we investigated the effects of angiogenic cytokines and growth factors on Ang-1 expression in human umbilical vein ECs (HUVECs). Northern blot analysis was performed after HUVECs were exposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, platelet-derived growth factor-BB, insulin-like growth factor-1, or vascular endothelial growth factor (VEGF). Both IL-1beta and tumor necrosis factor-alpha caused marked down-regulation of Ang-1 mRNA levels at 4 h with a further decrease observed at 24 h. Using signaling inhibitors, we identified the P38 pathway as the pathway that mediates IL-1beta down-regulation of Ang-1. Furthermore, treatment of cells with IL-1beta indirectly (via down-regulation of Ang-1) led to a decrease in Tie-2 autophosphorylation levels in HUVECs. We previously demonstrated that IL-1beta regulates VEGF expression in tumor cells. This observation was confirmed in ECs in the present study. Because pericytes play a role in regulating EC function, we also determined whether IL-1beta would also down-regulate Ang-1 in human vascular smooth muscle cells. Similar to our findings in HUVECs, we found that IL-1beta decreased Ang-1 expression in human vascular smooth muscle cells. Direct effects of IL-1beta on angiogenesis were investigated by use of an in vivo Gelfoam angiogenesis assay in which IL-1beta produced a significant increase in vessel counts (P = 0.0189). These results suggest that IL-1beta indirectly regulates angiogenesis by modulating the expression of Ang-1. IL-1beta may trigger a proangiogenic response by decreasing Ang-1 levels in ECs and pericytes and up-regulating VEGF in ECs and tumor cells.     

Vascular endothelial growth factor and glioma angiogenesis: Coordinate induction of VEGF receptors,distribution of VEGF protein and possible In vivo regulatory mechanisms

We have previously suggested that tumor angiogenesis in human gliomas is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and flt-I (VEGF-receptor I). VEGF, an endothelial-cell-specific mitogen, is abundantly expressed in glioma cells which reside along necrotic areas, whereas fit-I, a tyrosine-kinase receptor for VEGF, is expressed in tumor endothelial cells, but not in endothelial cells in normal adult brain. Recently, a second tyrosine-kinase receptor which binds VEGF with high affinity, designated KDR or flk-I, has been described. We performed in situ hybridization for VEGF mRNA, flt-I mRNA and KDR mRNA on serial sections of normal brain, low-grade and high-grade glioma specimens. We show that KDR mRNA is co-expressed with flt-I in vascular cells in glioblastoma but not in low-grade glioma. Since flt-I and KDR are not expressed in endothelial cells in the normal adult brain, the coordinate up-regulation of 2 receptors for VEGF appears to be a critical event which controls tumor angiogenesis. Immunocytochemistry with a monoclonal anti-VEGF antibody revealed significant amounts of VEGF protein in the same glioma cells that expressed VEGF mRNA. The largest amount of VEGF immunoreactivity, however, was detected on the vasculature of glioblastomas, the site where VEGF exerts its biological functions. These findings suggest that VEGF is produced and secreted by glioma cells and acts on tumor endothelial cells which express VEGF receptors. To further characterize VEGF-producer cells in vivo, we investigated cellular proliferation, immunoreactivity to the p53 tumor-suppressor gene product and epidermal-growth-factor-receptor(EGFR) expression on serial sections by immunocytochemistry. VEGF-producer cells did not show increased cellular proliferation, p53 immunoreactivity or EGFR immunoreactivity as compared with glioma cells which did not express VEGF. Our studies therefore do not demonstrate evidence for a growth advantage of VEGF-producer cells in vivo or VEGF induction by p53 mutation or EGFR over-expression. © 1994 Wiley-Liss, Inc.     

Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast.

The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis.     

Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis.

Increased vascular endothelial growth factor (VEGF) expression is associated with colon cancer metastases. We hypothesized that inhibition of VEGF receptor activity could inhibit colon cancer liver metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate metastases. Mice received daily i.p. injections of vehicle, tyrosine kinase inhibitor for Flk-1/KDR (SU5416) or tyrosine kinase inhibitor for VEGF, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). SU5416 and SU6668 respectively inhibited metastases (48.1% and 55.3%), microvessel formation (42.0% and 36.2%), and cell proliferation (24.4% and 27.3%) and increased tumor cell (by 2.6- and 4.3-fold) and endothelial cell (by 18.6- and 81.4-fold) apoptosis (P<0.001). VEGF receptor inhibitors increased endothelial cell apoptosis, suggesting that VEGF may serve as an endothelial survival factor.     

VEGF及其受体KDR在垂体腺瘤中的表达与肿瘤血管形成的关系

 目的　探讨血管内皮生长因子(VEGF) 及其受体( KDR) 的表达与垂体腺瘤血管生成的关系。方法　应用免疫组织化学S2P 法检测58 例人垂体腺瘤中的V E GF 及KD R 的表达,并对血管进行染色、计数。结果　其中54 例有VEGF 的表达(占93. 1 %) ,阳性表达主要位于肿瘤细胞胞膜及胞浆; KD R 在47 例中有阳性表达(占81. 0 %) ,阳性表达位于肿瘤血管内皮细胞、肿瘤细胞胞膜及胞浆。VEGF 和KD R 表达与垂体腺瘤的侵袭性密切相关;V E GF 和KD R 高表达组微血管密度明显高于低表达组( P <0. 01) 。结论　VEGF 以旁分泌、自分泌形式协同KDR 促进垂体腺瘤血管的生成,并与垂体腺瘤的侵袭密切相关。     

Inhibition of endothelial cell differentiation and proinflammatory cytokine production during angiogenesis by allyl isothiocyanate and phenyl isothiocyanate

Angiogenesis is a crucial step in the growth and metastasis of cancers. The activation of endothelial cells and their further behavior are very critical during angiogenesis. The authors analyze the effect of allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) on angiogenesis in an in vitro model using human umbilical vein endothelial cells (HUVECs). AITC and PITC significantly inhibited endothelial cell migration, invasion, and tube formation. (3)H-thymidine proliferation assay showed that AITC and PITC significantly inhibited the proliferation of HUVECs in vitro. The authors also studied the effect of AITC and PITC on the serum cytokine profiles of angiogenesis-induced animals and found that these compounds are highly potent in the downregulation of vascular endothelial growth factor (VEGF) and proinflammatory cytokines such as interleukin (IL)-1beta , IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha). Treatment with these compounds showed an elevation in the levels of IL-2 and tissue inhibitor of metalloproteinases (TIMP)-1, which are antiangiogenic factors. Moreover, studies using B16F-10 melanoma cells showed that both AITC and PITC significantly reduced VEGF mRNA expression. These findings suggest that AITC and PITC act as angiogenesis inhibitors through the downregulation of VEGF and proinflammatory cytokines such as IL-1beta, IL-6, GM-CSF, and TNF-alpha and upregulation of IL-2 and TIMP.     

抗KDR胞外Ⅲ区单抗抑制血管内皮细胞增殖的研究

目的研制能够封闭血管内皮生长因子(VEGF)受体KDR的单克隆抗体,探讨其体外抑制VEGF165诱导的生物学活性。方法以原核表达的KDR胞外Ⅲ区融合蛋白免疫Balb／c小鼠,采用传统杂交瘤技术制备抗KDR胞外Ⅲ区单克隆抗体,采用ELISA和FACS方法鉴定其抗原结合特异性,采用免疫沉淀和[^3H]-TdR掺入的方法分析该单克隆抗体阻断VEGF165刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,抑制VEGF165岱诱导内皮细胞增殖的活性。结果抗KDR胞外Ⅲ区单抗Ycom1D3不仅能与可溶性KDR结合,亦可与细胞表面表达的KDR结合,并可竞争性抑制VEGF165与可溶性KDR的相互作用,阻断VEGF165刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,显著抑制由VEGF165诱导脐静脉内皮细胞的增殖。结论抗KDR胞外Ⅲ区单抗Ycom1D3可通过封闭KDR而抑制VEGF活性,在肿瘤及其他血管新生疾病治疗中具有潜在的应用前景。     

VEGF及其受体KDR在宫颈癌的联合表达与局部肿瘤血管生成的关系

目的:探讨血管内皮生长因子及其受体KDR在早期宫颈癌的表达及其对宫颈癌肿瘤血管生成的作用。方法:采用免疫组织化学SP法检测18例宫颈上皮内瘤样病变(cervicalintraepithelialneoplasm,CIN)、75例早期宫颈癌(invasivecarcinomaofcervix,ICC)和15例正常宫颈上皮(normalcervicalepithelium,NCE)中VEGF和KDR的表达情况,并检测其中微血管密度(CD34标记)。结果:在ICC中,VEGF和KDR主要表达于癌细胞膜和(或)细胞浆;而CD34主要表达于癌巢间质血管内皮细胞。从NCE到CIN再到ICC,VEGF与KDR的阳性表达率以及MVD均显著升高(P<0.01)。在ICC中,VEGF、KDR阳性表达者其MVD分别显著高于VEGF、KDR阴性表达者(P<0.05)。VEGF在ICC的表达与KDR显著正相关(r=0.56,P<0.01),并且两者均与MVD显著正相关(前者r=0.60,P<0.01;后者r=0.33,P<0.01)。VEGF与KDR均阳性表达者,其微血管密度显著高于两者均阴性表达者(P<0.01)。结论:VEGF及其受体KDR表达在宫颈癌肿瘤血管生成中起上调作用,两者均过度表达,肿瘤血管生成显著增加。检测VEGF及其受体KDR的联合表达对进一步了解宫颈癌血管生成情况以及寻找抗肿瘤血管生成治疗新靶点有一定价值。     

Expression and localization of vascular endothelial growth factor receptors in human hepatocellular carcinoma and non-HCC tissues

Flt-1 (VEGF receptor-1) and KDR/Flk-1 (VEGF receptor-2) are the high-affinity receptors for the angiogenesis factor, vascular endothelial growth factor (VEGF). VEGF expression has been confirmed in human hepatocellular carcinoma (HCC), and VEGF is thought to be involved in the angiogenesis within HCC tissues. However, expressions of VEGF receptors in HCC have not been reported. We immunohistochemically examined expressions and localizations of Flt-1 and KDR in 28 surgically resected HCC tissues. In non-cancerous area, Flt-1 and KDR were mainly found in macrophages including Kupffer cells; both receptors were found in vascular endothelial cells in the portal veins and arteries within portal tracts; and KDR was also found in some sinusoidal endothelial cells. In cancerous area, Flt-1 and KDR were found in some macrophages, and also in the endothelial cells of intratumoral blood vessels. In 25 moderately and/or poorly differentiated HCCs, KDR expression in the blood space endothelial cells was clear and continuous in 20 cases, and focal in 5 cases. These results suggest that there would be an angiogenesis mechanism via VEGF/Flt-1 or VEGF/KDR in HCC, and the VEGF/KDR system would take a more important role.