Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.

Platelet-derived endothelial cell growth factor (PD-ECGF) has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The fusion protein was purified by one-step affinity chromatography on glutathione-agarose beads, and recombinant PD-ECGF was proteolytically cleaved with thrombin from its GST leader peptide to yield pure protein. Recombinant PD-ECGF stimulated [3H]methylthymidine uptake by endothelial cells in vitro; however, we were unable to detect stimulation of cell proliferation under a wide variety of conditions. We confirm that in accord with the recent report that PD-ECGF and human thymidine phosphorylase are products of the same gene [Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., & Akiyama, S. I. (1992) Nature 356, 668] recombinant PD-ECGF has thymidine phosphorylase activity comparable to that of E. coli thymidine phosphorylase. Further, E. coli thymidine phosphorylase was able to mimic the activity of recombinant PD-ECGF in the [3H]methylthymidine uptake assay, and it appears that recombinant PD-ECGF's effect on the uptake of thymidine by endothelial cells may be due to modulation of cellular thymidine pools. The mechanism by which PD-ECGF stimulates angiogenesis remains to be elucidated.

Angiogenesis is the formation of new blood vessels from the existing vascular bed. It is a complex multi-step process controlled by a number of angiogenic factors. One such factor is platelet-derived endothelial cell growth factor (PD-ECGF), recently shown to be thymidine phosphorylase (TP), which is angiogenic in several in vivo assays and tumour systems. PD-ECGF/TP catalyses the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine. Since PD-ECGF/TP has an important role in cellular metabolism and in angiogenesis and its expression has been only partially characterized, we raised a monoclonal antibody against recombinant PD-ECGF/TP and used an immunohistochemical approach to examine the expression of PD-ECGF/TP in a comprehensive range of normal human tissues. The clone P-GF44.C, which recognizes recombinant PD-ECGF/TP and cell lysates transfected with a plasmid expressing PD-ECGF/TP cDNA on Western blotting, was selected for its ability to stain routinely processed tissue. Staining was observed in both the cytoplasm and/or the nucleus. Immunoreactivity was strongly expressed by macrophages, stromal cells, glial cells, and some epithelia. Gastrointestinal epithelium, smooth muscle, adrenal, lung, and testis were negative. Although endothelial cell expression was observed, there was no correlation with sites of new vessel growth. This pattern of expression suggests tight PD-ECGF/TP regulation and that cellular thymidine pools may serve to control its different functions. Thus, in the nucleus it might modulate the pool for DNA synthesis, whilst in the cytoplasm it could control other effects through different enzyme systems. The high expression present in macrophages and skin might be important for total body thymidine homeostasis.

Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.

BACKGROUND

It has been reported that angiogenic factors play an important role in proliferation and metastasis in various cancers.AIMTo investigate the expression of angiogenic factors and microvessel density (MVD) in pancreatic ductal carcinoma and to examine the correlations among expression of angiogenic factors, clinicopathologic parameters, and clinical prognosis.

METHODS

Paraffin-embedded specimens from 55 patients with pancreatic ductal carcinoma were immunostained for vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), platelet-derived endothelial cell growth factor (PD-ECGF), and CD34. The correlations among the expression of individual angiogenic factors and MVD, the clinicopathologic features, and the clinical prognoses were analyzed statistically.

RESULTS

Immunostaining demonstrated that 70.8% of pancreatic ductal carcinomas were positive for VEGF, 60.9% for FGF-2, and 57.2% for PD-ECGF. A significant correlation was observed between VEGF expression and MVD (p < 0.05) but not between FGF-2 or PD-ECGF and MVD. Although the expression of each angiogenic factor had no correlation with clinicopathologic features, the patients with tumors that showed high expression of VEGF and FGF-2 had significantly shorter survival times than those with low or no such expression (p < 0.05).

CONCLUSIONS

These observations suggest that the expression of VEGF closely correlates with MVD and with a poor prognosis in pancreatic ductal carcinoma.

Thymidine phosphorylase (TP), also known as "platelet-derived endothelial cell growth factor" (PD-ECGF), is an enzyme, which is upregulated in a wide variety of solid tumors including breast and colorectal cancers. TP promotes tumor growth and metastasis by preventing apoptosis and inducing angiogenesis. Elevated levels of TP are associated with tumor aggressiveness and poor prognosis. Therefore, TP inhibitors are synthesized in an attempt to prevent tumor angiogenesis and metastasis. TP is also indispensable for the activation of the extensively used 5-fluorouracil prodrug capecitabine, which is clinically used for the treatment of colon and breast cancer. Clinical trials that combine capecitabine with TP-inducing therapies (such as taxanes or radiotherapy) suggest that increasing TP expression is an adequate strategy to enhance the antitumoral efficacy of capecitabine. Thus, TP plays a dual role in cancer development and therapy: on the one hand, TP inhibitors can abrogate the tumorigenic and metastatic properties of TP; on the other, TP activity is necessary for the activation of several chemotherapeutic drugs. This duality illustrates the complexity of the role of TP in tumor progression and in the clinical response to fluoropyrimidine-based chemotherapy.

The human genes for the hematopoietic growth factors interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been mapped to 5q23-31. We present in situ hybridization evidence that the human IL-4 gene is located at 5q23.3-31.2, suggesting that the four cytokine genes may be closely linked. We used pulsed-field gel electrophoresis to prepare subchromosomal restriction maps surrounding these genes to define this possible linkage more precisely. The IL-4 and IL-5 genes are tightly linked, being 90 to 240 kilobases (kb) apart, as has been shown for the IL-3 and GM-CSF genes, which are only 9 kb apart. Possible overlap of the map containing the IL-4 and IL-5 genes with restriction sites 5' to the IL-3 gene suggests that the four cytokine genes may be localized within 500 kb of each other. The endothelial cell growth factor gene (ECGF), which has also been localized to the 5q31 region, did not appear to be close to the cytokine genes. Linkage of the IL-3, IL-4, IL-5, and GM-CSF genes has important implications in the evolutionary origin and regulation of expression of these genes. The four cytokine genes are located in the region of the long arm of chromosome 5, which is deleted in the 5q- anomaly. The present study provides a basis for further investigations of this disorder.

Two polypeptides from secretory products of human hepatoma cells were isolated and characterized on the basis of their stimulation of maintenance and growth of human endothelial cells in serum-free cell culture. Both factors were purified to homogeneity by a combination of reverse-phase, ion exchange, and molecular filtration high performance liquid chromatography. One factor (endothelial cell growth factor (ECGF-2a) had Mr approximately 6,500 and pI near 6. The second (ECGF-2b) had Mr = 27,000 and a pI below 4.0. Both ECGF-2a and ECGF-2b exhibited single NH2-terminal sequences. The first 25 NH2-terminal residues of ECGF-2a and the first 49 residues of ECGF-2b were determined by gas-phase microsequencing. All clearly determined residues of ECGF-2a were identical with human pancreatic secretory trypsin inhibitor. All assignable residues of ECGF-2b were identical with urinary glycoprotein proteinase inhibitor (HI-30/EDC1). Both proteins are absent or at low levels in normal plasma and urine, but appear during acute inflammatory disease and cancer. Amino acid composition of ECGF-2a and ECGF-2b was also similar to human pancreatic secretory inhibitor and HI-30/EDC1, respectively. Both ECGF-2a and ECGF-2b inhibited bovine pancreatic trypsin (2 micrograms/ml) by 50% at 750 ng/ml. ECGF-2a and ECGF-2b stimulated endothelial cell number at a half-maximal dose of 50 ng/ml (8 nM) and 80 to 130 ng/ml (5 to 9 nM) protein, respectively. When assayed under identical conditions, no effect of either factor on human smooth muscle cells, human hepatoma cells, or human, rat, and mouse fibroblasts could be detected.

We report that hypoxia regulates and influences the level of the angiogenic enzyme platelet-derived endothelial cell growth factor (PD-ECGF), also called thymidine phosphorylase, in vitro and in vivo. Levels of PD-ECGF protein increased 6-fold in the breast cancer cell line MDA 231 after 16 h of growth in 0.3% oxygen. A simultaneous increase in enzyme activity was observed. Immunohistochemical staining of MDA 231 tumors grown in nu/nu mice showed increased expression of PD-ECGF in those parts of the tumor that are proximal to the areas of necrosis. In addition, increased and widespread staining for PD-ECGF protein was obtained when the tumor vascular supply was occluded for 2 h by clamping. Lowering the media pH to 6.3-6.7 in vitro also resulted in an increase in PD-ECGF protein levels. This study demonstrates that tumor microenvironmental factors can result in the specific up-regulation of an angiogenic enzyme that can also activate 5-fluorouracil prodrugs and hence is exploitable therapeutically.

Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line (1 microM dThd increased the IC50 value 1000-fold), but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 (1 microM dThd raised the IC50 value 3-fold). We observed a substantial 'bystander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.

Platelet-derived endothelial cell growth factor (PD-ECGF), a protein which stimulates angiogenesis in vivo, is shown to have a 39.2% amino acid sequence similarity over a 439 amino acid region with the thymidine phosphorylase of Escherichia coli (E. coli). Using recombinant human PD-ECGF, we show that PD-ECGF has thymidine phosphorylase activity. Analysis by gel chromatography revealed that recombinant human PD-ECGF occurs as a 90 kDa homodimer, similar to other thymidine phosphorylases. In addition to a possible effect on DNA synthesis, PD-ECGF was shown to affect [3H]thymidine assays in a manner which is not related to cell proliferation. The in vitro and in vivo effects of PD-ECGF may thus occur by an indirect mechanism through its enzymatic activity.

To investigate the relationship between endothelial cells and organ-associated vascular physiology, microvascular endothelial cells were isolated from murine brain, lung, and liver tissues. During culture, these endothelial cells maintained certain differentiated characteristics common to all endothelial cells, but also showed organ-specific characteristics, with distinct patterns of responsiveness to various growth factors. Microvascular endothelial cells from all organs responded to endothelial cell growth factor (ECGF), but lung (LE-1) and brain (MBE-12) endothelial cells showed different responsiveness to thrombin (10-60 nM), combinations of thrombin and ECGF, or thrombin and extracellular matrix. Liver sinusoidal endothelial cells (HSE) were relatively unresponsive to thrombin, but were the most responsive of the endothelial cells to EGF. Endothelial cells isolated from lung and brain, where fluxes in vascular permeability are observed following injury, showed dramatic morphological alterations in response to nanomolar concentrations of thrombin. These cells also exhibited the highest amount of 125I-thrombin binding at these concentrations. Scatchard analysis of 125I-thrombin binding indicated that LE cells have the highest affinity for thrombin, followed by MBE, with HSE exhibiting significantly lower affinity. The binding of 125I-thrombin to these cells was inhibited by the TR-9 monoclonal antibody directed against fibroblast high-affinity thrombin receptors involved in thrombin-stimulated mitogenesis. The results suggest that the differences in growth stimulation observed between organ-derived endothelial cells in response to thrombin, ECGF, and EGF may relate to differential expression of receptors for these factors. These observations demonstrate yet another aspect of the functional heterogeneity of the microvascular endothelium.

Endothelial cell growth factor (ECGF) can be rapidly purified from bovine brain to high specific activity using heparin-Sepharose affinity chromatography. Purification of the mitogen by this method results in relatively high yields of the polypeptide (10 to 100 micrograms/kg of tissue) with biological activity on murine and human endothelial cells in the picogram range. The product obtained is a mixture of two single-chain polypeptides with apparent molecular weights of 17,000 (alpha-ECGF) and 20,000 (beta-ECGF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two forms of ECGF can be separated by either NaCl gradient elution from heparin-Sepharose or reversed-phase high pressure liquid chromatography. The two polypeptides are related on the basis of similar: amino acid compositions, affinity for heparin-Sepharose, cyanogen bromide and trypsin-derived cleavage products, and biological activity. Furthermore, the cyanogen bromide fragments derived from the two forms of ECGF also possess similar amino acid compositions and mobilities on sodium dodecyl sulfate gels. These data suggest that there are at least two discrete molecular forms of ECGF in bovine brain and that these two molecules are structurally related.

OBJECTIVE

Both platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) are known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We aimed at evaluating the gene expression of PD-ECGF and VEGF in hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT).

METHODS

Surgical specimens (28 HCC, 28 nontumorous liver tissues and 18 PVTT) were studied by Northern blot analysis. The levels of PD-ECGF mRNA and VEGF mRNA expression were measured by densitometric scanning of the autoradiographs, and they were normalized to the level of expression of an internal control (glyceraldehydephosphate dehydrogenase) mRNA.

RESULTS

The expression rates of PD-ECGF mRNA in PVTT, HCC and nontumorous liver tissues were 77.8% (14/18), 67.9% (19/28) and 35.7% (10/28), being 88.9% (16/18), 75.0% (21/28) and 17.9% (5/28) respectively for VEGF mRNA. The expressions of PD-ECGF mRNA and VEGF mRNA were higher in HCC with PVTT than when PVTT was absent (P < 0.05). The PVTT was more often seen in patients with positive expression of both PD-ECGF mRNA and VEGF mRNA in HCC than in patients who were positive for only one of these factors or negative for both (P < 0.05).

CONCLUSIONS

Both PD-ECGF and VEGF correlated well with the formation of PVTT of HCC.

This study was designed to determine the relative activity of basic fibroblast growth factor (bFGF), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (PD-ECGF), hepatocyte growth factor (HGF), and interleukin-8 (IL-8) in regulating endothelial cell division, migration, degradation of the extracellular matrix (ECM), morphogenesis, and survival. Human umbilical vein endothelial cells (HUVEC) were treated with different concentrations of the six cytokines. bFGF was the most potent mitogen followed by VEGF/VPF and PD-ECGF. VEGF/VPF and bFGF also enhanced the survival of the endothelial cells in serum-free medium. Interstitial collagenase (MMP-1) and urokinase plasminogen activator (uPA) were significantly upregulated only by bFGF. HGF, bFGF, and VEGF/VPF induced chemotactic migration of the endothelial cells, but only HGF (scatter factor) enhanced nondirectional motility. The organization of endothelial cells to form tubes on Matrigel was induced by bFGF and, to a lesser extent, by VEGF/VPF and IL-8. Permeability across endothelial cell monolayers was induced only by VEGF/VPF. These data demonstrate that different angiogenic molecules differentially regulate distinct steps in the process of angiogenesis, suggesting that any given molecule may be necessary but in itself insufficient for establishment of a viable vasculature.

Vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) are known to be angiogenic growth factors in vitro and in vivo. In this study, we have investigated the relationship between VEGF expression and PD-ECGF expression in human breast cancer tissues using immunocytochemical methods. Of 152 primary breast cancers, 84 (55.3%) and 71 (46.7%) were positive for VEGF and PD-ECGF, respectively. Fifty-three (63. 1%) of 84 VEGF-positive tumors had a PD-ECGF-positive phenotype, whereas only 18 (26.5%) of 68 VEGF-negative tumors had a PD-ECGF-positive phenotype. There was a significant correlation between the VEGF expression and PD-ECGF expression (P < 0.01). As a single factor, VEGF expression and PD-ECGF expression were significantly associated with an increase in the microvessel density assessed by the immunocytochemical analysis using antifactor VIII-related antigen mAb. Interestingly, in addition, of 53 tumors with more than 100 microvessel counts/1 mm2, 40 (75.5%) had both VEGF- and PD-ECGF-positive phenotypes. It was found that VEGF and PD-ECGF were frequently coexpressed in highly vascularized tumors with high microvessel counts. It is suggested that VEGF and PD-ECGF might cooperatively function in the neovascularization of human breast cancer.

Angiogenesis is a recently described prognostic factor in non-small-cell lung cancer. Platelet-derived endothelial cell growth factor (PD-ECGF), shown to be the enzyme thymidine phosphorylase (TP), induces angiogenesis in vitro and in vivo. High intracellular levels of the enzyme are associated with increased chemosensitivity to pyrimidine antimetabolites. PD-ECGF/TP expression was evaluated immunohistochemically in surgically resected specimens from 107 patients with operable non-small-cell lung cancer using the P-GF,44C monoclonal antibody. High expression of PD-ECGF/TP was found in 25% of cases and was associated with high vascular grade (P = 0.01). Fourteen of 32 (44%) high vascular grade tumours showed a positive reactivity for PD-ECGF/TP vs 13/75 (17%) of low/medium vascular grade. Positive expression was observed more frequently in T2-staged cases than in T1 (P = 0.04). While overall survival was not affected (P = 0.09), subset analysis revealed that node-negative patients with positive PD-ECGF/TP expression had a worse prognosis (P = 0.04). The results suggest that PD-ECGF/TP may be an important molecule involved in angiogenesis in non-small-cell lung cancer. Up-regulation of the enzyme defines a more aggressive tumour phenotype in patients with node-negative disease. Assessment of vascular grade and PD-ECGF/TP expression should be taken into account in the design of randomized trials assessing the role of adjuvant chemotherapy in non-small-cell lung cancer.

OBJECTIVE

Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role.

METHODS

Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera.

RESULTS

Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis.

CONCLUSIONS

T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.

Two endothelial cell growth factors (ECGF) have been purified from bovine brain and termed alpha- and beta-ECGF [Burgess, W. H., Mehlman, T., Friesel, R., Johnson, W. V. & Maciag, T. (1985) J. Biol. Chem. 260, 11389-11392]. Amino acid sequence analysis indicates that beta-ECGF represents a 20 amino acid amino-terminal extension of alpha-ECGF and a 14 amino acid amino-terminal extension of acidic fibroblast growth factor. These data indicate that both alpha-ECGF and acidic fibroblast growth factor may be derived from beta-ECGF by posttranslational processing. Analysis of the amino-terminal 14 residues of beta-ECGF by fast-atom-bombardment mass spectrometry established the amino acid sequence of this region and the identity of the blocking group at the amino terminus (acetyl).

We have previously shown that platelet-derived endothelial cell growth factor (PD-ECGF) is associated with angiogenesis of human colon cancer; this factor is expressed at high levels in vascular tumors that express low levels of vascular endothelial growth factor (VEGF). In these colon cancers, the major source of PD-ECGF is the infiltrating cells. In this study, we examined the role of PD-ECGF in the angiogenesis of human gastric cancer. Immunostaining for PD-ECGF was done on 93 gastric cancers previously stained for VEGF, basic fibroblast growth factor, and factor VIII-related antigen (specific for endothelial cells). To determine the cell type expressing PD-ECGF, double staining was done using antibodies to both PD-ECGF and CD68 (specific for macrophages). PD-ECGF was expressed more frequently in infiltrating cells (positive CD68 staining; 53.8%) than in tumor epithelium (9.7%; P < 0.0001). Infiltrating cells simultaneously stained positive for both PD-ECGF and CD68. An association between PD-ECGF expression in infiltrating cells, VEGF expression in tumor epithelium, and vessel count was observed in intestinal-type gastric cancer but not in diffuse-type gastric cancer. Vessel count was greater in tumors with high expression of both PD-ECGF and VEGF than in those with high expression of either factor alone (P = 0.002). Multiple angiogenic factors expressed by both tumor cells and infiltrating cells may play a role in the regulation of angiogenesis in intestinal-type gastric cancer.

Human platelet-derived growth factor (PDGF) is a potent mitogenic polypeptide which is believed to be a heterodimer of A- and B-chains stabilized by interchain disulphide bonds. The B-chain of PDGF is encoded by the c-sis gene, the normal cellular homologue of the transforming gene of the simian sarcoma virus (SSV). cDNA clones of the B-chain from both normal and transformed cells have mutually consistent DNA sequences. Recently, an A-chain cDNA clone (D-1) was isolated from a transformed human glial cell cDNA library. We report the complete sequence of an A-chain cDNA clone (BT-1) isolated from a normal human umbilical vein endothelial (HUVE) cell cDNA library. BT-1 differs from the sequence of the D-1 clone by a 69 base pair deletion containing the predicted carboxy terminus of the protein. The mRNA levels of the A- and B-chains of PDGF in HUVE cells were analysed and shown to respond differently to the endothelial cell growth factor (ECGF).

Compelling experimental and clinical data support the concept that breast carcinoma, as most of the other solid tumors, needs to develop the angiogenic phenotype for invasiveness, progression and metastasis. Several studies have determined intratumoral microvessel density by panendothelial markers and immunohistochemical techniques, with most of them showing that the degree of vascularity is associated with prognosis of the patients operated of early-stage invasive breast cancer. More recently, certain angiogenic peptides have been assessed in human breast cancer: vascular endothelial growth factor (VEGF), platelet derived-endothelial cell growth factor (PD-ECGF, also known as thymidine phosphorylase, TP) and fibroblast growth factor family (FGFs). Among these, the most studied is VEGF, which appears to be a powerful prognostic indicator. Little data are available on the clinical significance of naturally occurring antiangiogenic factors, with few studies reporting on interleukin-12 and thrombospondins. In vivo techniques for dynamic assessment of tumor blood network are presently under extensive research, in particular for monitoring activity of inhibitors of angiogenesis. The methods of assessment of angiogenic activity and the results of published clinical studies in peer reviewed Journals with a computerized overview of literature will be presented. Overall, the results of the reported studies suggest that human breast cancer is an angiogenic-dependent tumor for which antiangiogenic therapy represents a promising novel antitumoral therapeutic strategy.

BACKGROUND

Recurrence after resection of hepatocellular carcinoma (HCC) is a major obstacle to improve prognosis. Therefore, further improvement of long-term survival may depend on prevention and treatment of the recurrent tumor.

OBJECTIVE

To evaluate the progress of surgery for HCC, the risk factors for recurrence, and clinical and basic studies on the prevention and management of recurrence and metastasis after resection of HCC.

METHODS

A review of currently available data in the mentioned areas.

RESULTS

Encouraging changes in the prognostic pattern were observed when the primary liver cancer (PLC) data of 1958-1967 (n=118), 1968-1977 (n=356), 1978-1987 (n=715) and 1988-1997 (n=2038) were compared. The 5-year survival was 2.8%, 7.3%, 27.1% and 52.5%, respectively, and the 10-year survival 2.8%, 4.3%, 19.8% and 39.9%, respectively. Risk factors for recurrence included symptomatic patient, high gamma-glutamyl-peptidase (gamma-PGT), large tumor size, portal vein embolus, advanced tumor stage, etc. Active hepatitis activity in the nontumorous liver and perioperative transfusion enhanced the recurrence. Molecular research into the invasiveness of HCC identified some factors positively related to invasiveness, P16 and P53 mutation, H-ras, c-cerbB2, mdm2, transforming growth factor (TGF), epidermal growth factor receptor (EGF-R), matrix metalloproteinase-2 (MMP-2), urokinasetype plasminogen activator (uPA), its receptor (uPA-R) and inhibitor (PAI-1), intercellular adhesion molecule-1 (ICAM-1), vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor (PD-ECGF), and basic fibroblast growth factor (bFGF). In contrast, some factors were negatively related to HCC invasiveness: nm23-H1, Kai-1, tissue inhibitor of metalloproteinase-2 (TIMP-2), integrin 5, and E-cadherin. Re-resection of subclinical recurrence yielded a 5-year survival of 56.0% calculated from the first resection (n=202). Postoperative transarterial chemoembolization (TACE, n=103), hepatic artery cannulation during operation (n=105), postoperative biotherapy (n=49), and cryohepatectomy (cryosurgery followed by immediate resection of the frozen tumor, n=84) might decrease the recurrence rate, and the 3-year recurrence rate was 7.6%, 18.0%, 11.1%, and 30.1%, respectively. Minimal intraoperative blood loss and transfusion could reduce postoperative recurrence, although the exact mechanism remains to be elucidated.

CONCLUSIONS

HCC invasiveness is the major topic to be studied, particularly in the molecular level. Anti-angiogenesis, biotherapy, novel approach based on molecular findings, and multidisciplinary interventions might also be important for HCC.

Tumor angiogenesis is a complicated process for which the mechanisms remain unclear. The aim of this study was to elucidate the clinical significance of several angiogenic factor expression as a predictor of the invasive/metastatic potential and of the prognosis of advanced colorectal carcinoma (CRC) in relation to their intratumoral histologic heterogeneity. One hundred fifty two patients who had undergone surgical resection for advanced CRC entered this study. PD-ECGF, VEGF, and VEGF-C were examined immunohistochemically with antibodies 1C6-203, A-20, and C-20, respectively. Tumor microvessel density (MVD) was determined immunohistochemically with anti-CD34 antibody. Expression of PD-ECGF, of VEGF, and of VEGF-C at the deepest invasive site were detected in 77 (50.7%), 62 (30.8%), and 71 (46.7%) of the 152 lesions, respectively. PD-ECGF, VEGF, and VEGF-C expression at the deepest invasive site in lesions with liver metastasis (77, 67, and 70%) was significantly higher than that in those without liver metastasis (44, 34, and 41%). In cases with curative surgery, patients with PD-ECGF, VEGF, and VEGF-C expression at the deepest invasive site had a significantly poorer prognosis than those without PD-ECGF, VEGF, and VEGF-C expression at the deepest invasive site. PD-ECGF, VEGF, and VEGF-C expression at the deepest invasive site correlated significantly with MVD. Multivariate analysis with logistic regression for 5-year survival in patients with curative surgery showed that lymph node metastasis and VEGF expression were significant risk factors. Expression of PD-ECGF, VEGF, and VEGF-C was correlated significantly with metastatic potential and prognosis in relation to MVD. Of the several angiogenic factors, VEGF expression at the deepest invasive site of tumor was the most statistically significant indicator of prognosis in advanced CRC.