Expression and regulation of the novel vascular endothelial growth factor receptor neuropilin-1 by epidermal growth factor in human pancreatic carcinoma

BACKGROUND: It was recently shown that neuropilin-1 (NRP-1), which was described originally as a receptor for the semaphorins/collapsins (ligands involved in neuronal guidance), is a coreceptor for vascular endothelial growth factor (VEGF) and increases the affinity of specific isoforms of VEGF to its receptor, VEGF-R2. METHODS: The authors investigated the expression and regulation of NRP-1 in human pancreatic adenocarcinoma specimens and cell lines. RESULTS: Immunohistochemical analysis revealed that NRP-1 was expressed in 12 of 12 human pancreatic adenocarcinoma specimens but was absent in nonmalignant pancreatic tissue. Northern blot analysis revealed NRP-1 mRNA expression in 8 of 11 human pancreatic adenocarcinoma cell lines. NRP-1 mRNA expression was increased by epidermal growth factor (EGF) but not by tumor necrosis factor alpha in several of the human pancreatic adenocarcinoma cell lines studied. Treating human Panc-48 adenocarcinoma cells with EGF activated Akt and Erk but not P-38. Blockade of the phosphatidylinositol-3 kinase (PI-3K)/Akt, mitogen-activated protein kinase (MAPK)/Erk, or P-38 pathways abrogated EGF-induced NRP-1 expression. Finally, EGF receptor blockade in vivo led to a decrease in NRP-1 expression in an orthotopic model of human pancreatic carcinoma. CONCLUSIONS: NRP-1 is expressed in most human pancreatic adenocarcinomas and cell lines but not in nonmalignant pancreatic tissue. EGF regulates NRP-1 expression through the PI-3K/Akt and MAPK/Erk signaling pathways, and blockade of the EGF receptor is associated with decreased expression of NRP-1 in vivo. NRP-1 may act as a coreceptor for VEGF in pancreatic carcinoma, as it does in other tumor systems, thereby enhancing angiogenesis and the effect of VEGF on the growth of pancreatic adenocarcinoma.     

神经菌毛素在胰腺癌组织及MIA PaCa-Ⅱ细胞系中的表达

目的探讨神经菌毛素（Neuropilin-1，NRP-1）在胰腺导管癌组织及MIA PaCa-Ⅱ胰腺癌细胞系中的表达及意义。方法运用免疫组化和RT-PCR法分别检测在正常胰腺组织、癌旁组织、胰腺癌组织及MIA PaCa-Ⅱ细胞系中Neuropilin-1蛋白及mRNA表达水平。结果蛋白水平：可见正常胰腺组织无表达，癌旁组织轻度表达，而胰腺癌组织及MIA PaCa-Ⅱ胰腺癌细胞中高水平表达。神经组织也可表达Neuropilin-1 mRNA水平见正常胰腺组织呈微量表达，癌旁组织中度表达，而胰腺癌组织及MIA PaCa-Ⅱ胰腺癌细胞中呈高水平表达。结论Neuropilin-1可能与参与了胰腺癌的发生发展，在胰腺癌神经转移中可能起着重要作用。     

神经生长因子促进MIA PaCa-2人胰腺癌细胞的增殖

目的：观察神经生长因子-β(nerve growth factorbeta, NGFβ)对人胰腺癌MIA PaCa-2细胞增殖及细胞周期的影响。方法:体外培养MIA PaCa-2细胞,单独或联合给予不同浓度的NGF-β和K252a (NGF-β受体TrKA的抑制剂),应用克隆平板实验、MTT法和流式细胞术检测NGF-β和K252a对MIA PaCa-2细胞克隆形成率、增殖及细胞周期的影响。结果：NGF-β显著促进MIA PaCa-2细胞的克隆形成（P<0.05）,NGF-β使MIA PaCa2细胞增殖能力明显增强（P<0.01）,K252a抑制MIA PaCa-2细胞增殖（P<0.05）,NGF-β与K252a联合作用对MIA PaCa-2细胞的增殖能力无明显影响。NGF-β作用使MIA PaCa2细胞周期阻滞于S期,K252a作用使其周期阻滞于G0/G1期,两者联合作用使MIA PaCa-2细胞周期阻滞于S期。结论：NGF-β具有促进胰腺癌MIA PaCa-2细胞增殖的作用。     

Neuropilin-1 在胰腺癌组织及细胞系中的表达

 目的 了解Neuropilin-1在胰腺癌组织、癌旁组织及胰腺癌细胞系中的表达情况。方法 运用免疫组化法和RT-PCR法分别检测Neuropilin-1及其mRNA在胰腺癌组织、癌旁组织及细胞系中的表达。结果 免疫组化检测发现18例胰腺癌组织标本中Neuropilin-1均呈高表达，而癌旁组织中无一例表达；RT-PCR检测rnRNA的表达结果显示14例胰腺癌组织中有9例表达，癌旁组织中无一例表达，Panc-1、Asps-1、Bxp-3细胞系中均表达。结论 Neuropilin-1表达于大多数胰腺癌组织及细胞系中，而在癌旁组织中不表达，提示Neuropilin-1的表达可能与胰腺癌的发生发展密切相关。     

Decrease of endogenous vascular endothelial growth factor may not affect glioma cell proliferation and invasion

Vascular endothelial growth factor (VEGF) is abundantly produced by glioma cells especially glioblastoma, the most malignant form of astrocytoma. VEGF, a well known angiogenic factor, acts in a paracrine fashion on endothelial cells to develop tumor vasculature. However, recent studies have found that several tumor cells express VEGF receptors, and an autocrine action of VEGF on tumor cells has been suggested. To test this hypothesis, three human glioma cell lines (U251n, U87 and A172) were checked for VEGF and VEGFR expression. These cells express 0.1-0.6 ng/ml VEGF165 in cell culture medium within 24 hours. Western blot analysis showed that these cells express all of the VEGF receptors, VEGFR-1/Flt-1, VEGFR-2/KDR, Neuropilin-1 (NRP-1) and Neuropilin-2(NRP-2), even though tyrosine kinase receptor VEGFR-2/KDR exhibited baseline levels of expression. VEGF expression was significantly down regulated by phosphorothioate oligodeoxynucleotide (PS-ODN) and VEGF RNAi transfection. However, VEGF RNAi transfection as well as VEGF and VEGFR2 neutralization antibody treatment did not decrease cell proliferation detected by MTT and CyQuant NF proliferation assay except that PS-ODN transfection caused a non-specific decrease on cell proliferation. VEGF RNAi transfection did not alter cell invasion, as demonstrated in a matrigel invasion assay. Matrix metalloproteinase-2 (MMP-2) and MMP-9, facilitating cell invasion and over expressed in glioma cells, were not altered by VEGF RNAi transfection, as shown by zymographic assays. Our data indicate that the decrease of endogenous VEGF expression may not affect glioma cell proliferation and invasion.     

Role of Sp proteins in regulation of vascular endothelial growth factor expression and proliferation of pancreatic cancer cells

Sp proteins play an important role in angiogenesis and growth of cancer cells, and specificity protein 1 (Sp1) has been linked to vascular endothelial growth factor (VEGF) expression in pancreatic cancer cells. RNA interference was used to investigate the role of Sp family proteins on regulation of VEGF expression and proliferation of Panc-1 pancreatic cancer cells. Using a series of constructs containing VEGF promoter inserts, it was initially shown that Sp1 and Sp3 were required for transactivation, and this was primarily dependent on proximal GC-rich motifs. We also showed that Sp4 was expressed in Panc-1 cells, and RNA interference assays suggested that Sp4 cooperatively interacted with Sp1 and Sp3 to activate VEGF promoter constructs in these cells. However, the relative contributions of Sp proteins to VEGF expression were variable among different pancreatic cancer cell lines. Small inhibitory RNAs for Sp3, but not Sp1 or Sp4, inhibited phosphorylation of retinoblastoma protein, blocked G0/G1-->S-phase progression, and up-regulated p27 protein/promoter activity of Panc-1 cells; similar results were observed in other pancreatic cancer cells, suggesting that Sp3-dependent growth of pancreatic cancer cells is caused by inhibition of p27 expression.     

低分子肝素对肿瘤细胞分泌血管内皮生长因子的影响

目的 研究低分子肝素(LIVIWH)对不同肿瘤细胞系分泌血管内皮生长因子(VEGF)的影响,以及其对VEGF所诱导的肿瘤血管内皮细胞增殖的作用.方法 采用酶联免疫吸附(ELISA)法,检测LIVIWH和肝素对肺腺癌A549细胞、肝癌HepG2细胞、结肠腺癌HCT116和HCT8细胞分泌VEGF和肿瘤坏死因子α(TNF-α)蛋白的影响.采用逆转录聚合酶链反应(RT-PCR)和实时定量PCR法,检测LMWH和肝素对HepG2细胞表达VEGF mRNA的影响.采用非放射性细胞增殖检测试剂盒和流式细胞仪,检测肿瘤条件培养基在有无LMWH作用时对脐静脉血管内皮细胞(HUVEC)增殖和细胞周期的影响.结果 对照组、IMWH组和肝素组A549细胞中,VEGF蛋白的表达量分别为(1045.89±165.30)pg/ml、(782.45±67.17)pg/ml和(916.54±71.25)pg/ml;对照组、LMWH组和肝素组HCT116细胞中,VEGF蛋白的表达量分别为(955.76±51.14)pg/ml、(822.89±142.39)pg/ml和(951.77±188.22)pg/ml;对照组、LMWH组和肝素组HCT8细胞中,VEGF蛋白的表达量分别为(1290.62±41.23)pg/ml、(1063.34±63.82)pg/ml和(1257.14±11.40)pg/ml;对照组、LMWH组和肝素组HepG2细胞中,VECF蛋白的表达量分别为(1083.00±134.35)pg/ml、(758.00±84.85)pg/ml和(874.00±22.62)pg/ml.LMWH可抑制不同肿瘤细胞系中VEGF蛋白的表达(均P＜0.05),但对TNF-α蛋白的表达没有影响.LNWH可抑制HepG2细胞中VEGF mRNA的表达.肿瘤条件培养基可以诱导HUVEC增殖,加入LMWH的肿瘤条件培养基中,HUVEC增殖减少,G_0+G_1期细胞比例增加.结论 LMWH可抑制不同的肿瘤细胞系分泌VEGF,并具有抑制VEGF诱导的血管内皮细胞增殖的作用.     

Thymosinalpha1 stimulates cell proliferation by activating ERK1/2, JNK, and increasing cytokine secretion in human pancreatic cancer cells

In this study, we investigated the expression and function of thymosinalpha1 (Thyalpha1) in human pancreatic cancer. We found that human pancreatic cancer cell lines Panc-1, Panc03.27, ASPC-1, and PL45 cells significantly over-expressed the mRNA of Thyalpha1 as compared to the normal human pancreatic ductal epithelium (HPDE) cells.. Thyalpha1 mRNA and protein levels were also over-expressed in clinical pancreatic adenocarcinoma specimens. In addition, synthetic Thyalpha1 significantly promoted Panc-1 cell proliferation and increased phosphorylation of ERK1/2 and JNK. Furthermore, Thyalpha1 increased the secretion of multiple cytokines including IL-10, IL-13, and IL-17 in Panc-1 cells. Thus, Thyalpha1 may have a new role in pancreatic cancer pathogenesis.     

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.     

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.     

Differential dependency of human cancer cells on vascular endothelial growth factor-mediated autocrine growth and survival

Analysis using the public microarray database Gene Expression Omnibus indicates significantly higher mRNA expression of VEGF and VEGFRs in colorectal cancer and high grade astrocytoma but not in hepatocellular carcinoma compared to normal tissue. Human malignant astrocytoma cell lines (U251-MG and U373-MG) and HT-1080 fibrosarcoma cells expressed relatively higher levels of VEGF and VEGFRs compared to hepatocellular and colorectal cancer cell lines. Administration of exogenous VEGF-A induced cell growth in a dose-dependent fashion in astrocytoma and fibrosarcoma cells but not in colorectal and hepatocellular cancer cells. The blockade of VEGF inhibited cell survival only in U251-MG, U373-MG and HT-1080 cells. These results collectively suggest the role of autocrine VEGF signaling in various cancer cells and provide a basis for the variable clinical responses to antiangiogenic therapy observed in different types of malignancies.     

Potential role for vascular endothelial growth factor‐D as an autocrine factor for human gastric carcinoma cells

Vascular endothelial growth factor (VEGF)‐D induces lymphangiogenesis by activating VEGF receptor (VEGFR)‐3, which is expressed mainly by lymphatic endothelial cells. VEGFR‐3 has also been detected in several types of malignant cells, but the significance of VEGFR‐3 expression by malignant cells remains unclear. We examined the expression and function of VEGF‐D/VEGFR‐3 in human gastric carcinoma cells. Expression of VEGF‐D and VEGFR‐3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF‐D on the expression of genes associated with disease progression in VEGFR‐3‐expressing KKLS cells. VEGF‐D‐transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF‐D and VEGFR‐3. In vitro treatment of KKLS cells with exogenous VEGF‐D increased expression of cyclin D1 and Bcl‐2 and stimulated cell proliferation. VEGF‐D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF‐D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms. (Cancer Sci 2010)     

Significance of phospho‐vascular endothelial growth factor receptor‐2 expression in pancreatic cancer

Vascular endothelial growth factor receptors (VEGFRs) are mainly expressed by endothelial cells, but they are also expressed by some cancer cells, including pancreatic cancer. The objective of this study was to evaluate the significance of VEGFRs expression in pancreatic cancer cells. A total of 107 primary pancreatic tumors were stained with antibodies against VEGFR‐1, VEGFR‐2, phospho‐VEGFR‐2 (pVEGFR‐2), VEGFR‐3, VEGF‐A, VEGF‐C, and VEGF‐D. VEGFR‐2 and pVEGFR‐2 expression were positive in 74 (69%) and 54 (50%) of 107 pancreatic cancers. There was a significant correlation (P < 0.001) between VEGFR‐2 expression and pVEGFR‐2 expression. pVEGFR‐2 was significantly associated with invasion to the anterior capsule of pancreas (P = 0.032) and arterial invasion (P = 0.012). In contrast, VEGFR‐1 and VEGFR‐3 expression was only observed in 13 (12%) and 15 (14%) of 107 pancreatic cancers, and was not associated with any clinicopathological features. The prognosis of pVEGFR‐2 positive patients with stage IIA tumors was significantly (P = 0.0441) poorer than that of pVEGFR‐2‐negative patients. VEGF‐A, VEGF‐C, and VEGF‐D expression was positive in 42 (39%), 82 (77%), and 39 (36%) of 107 pancreatic cancers, respectively. The prognosis for VEGF‐A‐positive patients was significantly (P = 0.0425) poor, but not for VEGF‐C‐positive and VEGF‐D‐positive patients. A multivariate analysis indicated pVEGFR‐2 expression to be an independent prognostic factor, but not VEGF‐A. These findings suggested that VEGFR‐2 signaling might therefore be associated with the prognosis of patients with pancreatic cancer. The expression of pVEGFR‐2 might be a novel predictive prognostic marker for patients with pancreatic cancers, especially at clinical stage IIA. (Cancer Sci 2010)     

Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3'-kinase activity

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC(50) of about 5 mumol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3'-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G(1) progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound.     

Pancreatic cancer cell-derived vascular endothelial growth factor is biologically active in vitro and enhances tumorigenicity in vivo

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator that acts by binding to high-affinity transmembrane receptors. Although both VEGF and its receptors are overexpressed in human pancreatic ductal adenocarcinoma (PDAC), this malignancy is not generally considered to be highly vascular. It is not known, therefore, whether the abundance of VEGF in PDAC is biologically relevant. To address this issue, we measured the angiogenic effects of pancreatic cancer cell-derived VEGF in an in vitro endothelial cell proliferation assay and characterized the consequences of suppressing VEGF expression on pancreatic tumor growth in an athymic nude mouse model. We found that human pancreatic cancer cell lines secrete large quantities of biologically active VEGF into conditioned medium (CM). Stable transfection of an anti-sense VEGF(189) (AS-VEGF(189)) expression construct into PANC-1 pancreatic cancer cells resulted in decreased VEGF expression and secretion, a decreased capacity of the resultant CM to enhance endothelial cell proliferation and a significant attenuation of tumor cell proliferation in vitro. Furthermore, when injected into athymic nude mice, AS-VEGF(189)-expressing cells exhibited an 80% decrease in tumor growth compared with control cells. These results support the hypothesis that VEGF promotes pancreatic cancer growth in vivo and suggest that anti-VEGF therapy may be useful in the treatment of this disease.     

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT-PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-kappaB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.     

Expression of vascular endothelial growth factor receptors is closely related to the histological grade of hepatocellular carcinoma

Angiogenesis is important for tumor growth, and is regulated by angiogenetic factors such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether or not expression of VEGF receptors (VEGFRs) is related to the proliferation of tumor cells in hepatocellular carcinoma (HCC). We simultaneously stained proliferation marker Ki-67 antigen and either VEGFR1 (Flt-1) or VEGFR2 (Flk-1) on paraffin-embedded tissue sections from 50 cases of surgically resected human HCC. Based on the staining pattern of VEGFRs, we classified the cases into 4 categories; receptor double-negative, Flt-1 single-positive, Flk-1 single-positive, receptor double-positive. Interestingly, the Ki-67 index was significantly lower in receptor double-negative cases in comparison to that in either Flt-1 single-positive or Flk-1 single-positive cases (P = 0.0491, P = 0.0196, respectively). Moreover, the index was also significantly lower in receptor double-positive cases in comparison to either Flt-1 single-positive or Flk-1 single-positive cases (P = 0.0026, P < 0.0001, respectively). We further investigated 35 cases showing a Ki67 index > 10% to determine the expression of VEGFRs on Ki-67 antigen-positive proliferating cells. Surprisingly, the histological grade of HCC and the expression pattern of VEGFRs showed a characteristic relation; the well-differentiated HCC cases were all distributed in the Flk-1-positive group (7/7), moderately differentiated HCC cases were distributed in either the Flt-1 or Flk-1 single-positive group (20/21), and poorly differentiated HCC cases were predominantly distributed in either the receptor double-negative or double-positive group (6/7). These findings suggest that the expression pattern of VEGFRs influences the histological differentiation of HCC.