The efficacy of the human angiogenetic heparin-binding growth factor I (HBGF-I) to initiate site-directed growth of new blood vessels from the aorta into the myocardium was studied. First, manipulated Escherichia coli bacteria, which had received the human mRNA-transcript for HBGF I into their genetic material, were cultivated. The growth factor derived was purified using heparin-Sepharose affinity chromatography. The separation and characterization of biologically active alpha- and beta-chains was carried out using high pressure liquid chromatography (HPLC) of dialyzed and lyophilized samples from the heparin-Sepharose column. One microgram HBGF I (alpha-ECGF) was bound to polytetrafluoroethylene (PTFE) sponges, precoated with collagen type I, and implanted between the aorta and the myocardium of the left ventricle in experimental rats. Twelve growth factor implants in the experimental group were compared to six controls receiving uncoated PTFE sponges for 9 weeks. Digitized computed angiography showed new blood vessels between the aorta and the myocardium in 11 of the 12 experimental animals, and retrograde coronary perfusion by these "new" vascular structures could be seen. Histology showed no specific structures in the control group (without HBGF I). In the experimental group (with HBGF I) individual vessels with highly differentiated endothelial and smooth muscle cell layers were evident. Our experiments proved the feasibility of induced, site-directed angiogenesis. It is possible to initiate in vivo growth of new "coronary" vascular structures between the aorta and the myocardium.

BACKGROUND

In experimentally induced puromycine aminonucleoside nephrosis (PAN) animal models, nephrotic syndrome with minimal change disease and focal and segmental sclerosis-like nephritis similar to that in human is demonstrated; however, the real mechanism of PAN is not yet elucidated. Platelet derived endothelial cell growth factor (PD-ECGF), an endothelial mitogen protein, is believed to take part in microvessel formation and in stimulation of angiogenesis and its expression has not been totally demonstrated in PAN rats yet. In this study, we aimed to examine PD-ECGF expression in acute and chronic PAN induced in rats and find out the association between its expression and the stages of angiogenesis in kidney.

METHODS

For the experiment, twenty-four Male Wistar Albino rats were used and divided into four groups; control group (n = 6), pre-proteinuria group (n = 6), acute group (n = 6) and chronic group (n = 6). We compared statistically all data by One-way ANOVA Test followed by Dunn Multiple Comparison Test.

RESULTS

Proteinurea levels in control and pre-proteinuria groups were not statistically different; however, it was remarkably higher in the acute nephrosis group and significantly greater in the chronic nephrosis group than control group (p < 0.0025). In pre-proteinuria group, the serum albumin and creatinine clearances also did not significantly differ from the control group. On the other hand, in the acute and chronic nephrosis groups, serum albumin and creatinine clearances progressively decreased (p < 0.05). In our immunohistochemical studies, we showed elevated PD-ECGF expression in glomeruli of acute and chronic PAN rats. Microscopic and ultrastructural appearances of the glomeruli of acute and chronic PAN showed various sequential steps of angiogenesis, macrophages and immature capillaries with primitive lumens and apoptotic endothelial cells in the increased mesangial matrix.

CONCLUSIONS

It is reported that acute and chronic PAN progressively increase PD-ECGF expression and following induction of angiogenesis in the affected glomeruli.

OBJECTIVE

Platelet derived-endothelial cell growth factor (PD-ECGF) is a highly potent angiogenic factor. Although angiogenesis plays an active role in pathophysiology of stroke, the expression pattern of this molecule in ischemic brain has not been investigated. In the present study, therefore, we investigated the change of PD-ECGF expression in the brain after ischemia.

METHODS

Using male Wistar rats, the right middle cerebral artery was occluded by a nylon thread for 90 minutes. The animals were decapitated 3 hours, 1, 4 and 10 days after the reperfusion, and frozen sections were prepared. We then performed immunohistochemistry for PD-ECGF and identified the cell phenotype which strongly expressed it by fluorescent double staining.

RESULTS

In the sham-operated brain, only small numbers of cells slightly expressed PD-ECGF. The number of positively stained cells increased at the peri-ischemic area from hour 3 of reperfusion. Not only small-sized cells but also large-sized cells became stained. The number of stained cells further increased, and peaked at day 4 for large-sized cells and at day 10 as to small-sized cells. Fluorescent double staining revealed that both large-sized and small-sized cells were neurons, indicating that neurons are the main source of PD-ECGF production in the ischemic brain.

CONCLUSIONS

PD-ECGF has a strong angiogenic property without vascular permeability increasing effect. This molecule may have a therapeutic potential for ischemic stroke treatment.

In a tumor microenvironment, endothelial cell migration and angiogenesis allow cancer to spread to other organs causing metastasis. Indeed, a number of molecules that are involved in cytoskeleton re-organization and intracellular signaling have been investigated for their effects on tumor cell growth and metastasis. Alongside that, Amblyomin-X, a recombinant Kunitz-type protein, has been shown to reduce metastasis and tumor growth in in vivo experiments. In the present report, we provide a mechanistic insight to these antitumor effects, this is, Amblyomin-X modulates Rho-GTPases and uPAR signaling, and reduces the release of MMPs, leading to disruption of the actin cytoskeleton and decreased cell migration of tumor cell lines. Altogether, our data support a role for Amblyomin-X as a novel potential antitumor drug.

BACKGROUND

Amb-X: Amblyomin-X; ECGF: endotelial cell growth factor; ECM: extracellular matrix; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cell; LRP1: low-density lipoprotein receptor-related protein; MMP: matrix metalloproteinase; HPI-4: hedgehog pathway inhibitor 4; PAI-1: plasminogen activator inhibitor 1; PMA: phorbol 12-myristate-13-acetate; TFPI: tissue factor pathway inhibitor; uPA: urokinase plasminogen activator; uPAR: uPA receptor.

Human thymidine phosphorylase (dThdPase) is an angiogenic factor identical to platelet-derived endothelial cell growth factor (PD-ECGF). Thymidine phosphorylase is also a converting enzyme of the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) to 5-fluorouracil (5-FU) in tumors. To assess the role of dThdPase in targeting chemotherapy, we examined the relationship between the expression of dThdPase and the sensitivity of 5'-DFUR in cancer cell lines, and also examined whether transfection of dThdPase cDNA enhanced the drug-sensitivity to 5'-DFUR with or without angiogenesis in breast cancer cells. Thirteen human cancer cell lines consisting of 4 breast cancer, 6 gastric cancer, and 3 colon cancer cell lines were used. Expression of dThdPase was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). In vitro drug-sensitivity was assessed by MTT assay, and anti-tumor effect in vivo was assessed using nude mouse xenografts. Intratumoral microvessel density was evaluated by immunohistochemical staining to factor VIII related antigen. Transfection of dThdPase cDNA was performed using pcDNA3 expression vector encoding its cDNA by the lipofection method. An inverse relationship between the expression of dThdPase and the IC50 values of 5'-DFUR was observed (p=0.1278, rho=-0.440) in the 13 cancer cell lines. Transfection of dThdPase cDNA into MCF-7 breast cancer cells resulted in an approximately 2.6- and 10-fold increase of the expression of dThdPase mRNA and its enzyme activity, respectively, compared to the control vector alone. The sensitivity to 5'-DFUR in the transfected cells was increased approximately 20-fold compared to the parent cells and control vector alone, and the sensitivity to 5-FU was also somewhat increased. In contrast, the sensitivity to ADM, CDDP, and VP-16 was not different between the transfected and control cells. In nude mice xenografts of the transfected cells, treatment with 5'-DFUR had a significant anti-tumor effect compared to those of the untreated transfected cells and control vector alone treated with 5'-DFUR (p<0.01). Intratumoral microvessel density in the transfected cells was not significantly increased with or without treatment with 5'-DFUR compared to control vector alone. The high expression of dThdPase was correlated with an increase in the sensitivity to 5'-DFUR in gastrointestinal and breast cancer cell lines. The introduction of dThdPase cDNA in breast cancer cells enhanced the sensitivity to 5'-DFUR without an increase of tumor angiogenesis, and targeting chemotherapy of dThdPase may be a good tumor-specific and personalized therapy for improving the poor prognosis of cancer patients who show high expressions of dThdPase.

Thymidine phosphorylase (TP) first identified as platelet derived endothelial cell growth factor (PD-ECGF) plays a key role in nucleoside metabolism. Human TP (hTP) is implicated in angiogenesis and is overexpressed in several solid tumors. Here, we report the crystal structures of recombinant hTP and its complex with a substrate 5-iodouracil (5IUR) at 3.0 and 2.5A, respectively. In addition, we provide information on the role of specific residues in the enzymatic activity of hTP through mutagenesis and kinetic studies.

BACKGROUND

The management of patients with pT1 G3 bladder cancer remains controversial because of the high incidence of recurrence with muscle invasion. Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. The aim of this study was to determine whether the expression of PD-ECGF/dThdPase in bladder cancer tissue was associated with tumor progression and recurrence in patients with pT1 G3 bladder cancer.

METHODS

Fifteen patients who were pathologically diagnosed as having pT1 G3 transitional cell carcinoma of the bladder were treated with transurethral resection. Sections of paraffin-embedded bladder tissue were immunohistochemically stained with either mAb654-1, a monoclonal antibody against human PD-ECGF or anti-CD34 monoclonal antibody, respectively. When more than 10% of tumor cells were positively stained with mAb654-1, this section was defined as positive in this study.

RESULTS

Eight of 15 sections from patients with pT1 G3 bladder cancer (53%) were positive with PD-ECGF/dThdPase. During follow up, patients in the negative group had no disease progression and only two patients had local recurrence. In contrast, seven of eight positives had recurrence (P < 0.05) and progression was also observed in four recurrent patients. However, there was no statistical relationship between PD-ECGF and CD34 expression in any of the patients.

CONCLUSIONS

The expression of PD-ECGF/dThdPase appears to be an important prognostic factor of pT1 G3 bladder cancer and did not show any significant relationship between PD-ECGF/dThdPase expression and vascular density.

Thymidine phosphorylase (dThdPase)/platelet-derived endothelial cell growth factor (PD-ECGF) is expressed at higher levels in a variety of human carcinomas than in adjacent normal tissue. The higher expression is associated with an increase in intratumoral microvessel density (IMVD) and an unfavorable patient prognosis. This study examined the role of dThdPase in apoptosis, IMVD and p53 expression in human gastric carcinomas, dThdPase expression was noted in 12 (35.3%) of 34 early carcinomas, and in 20 (55.6%) of 36 advanced carcinomas. At least 10 areas consisting of carcinoma cells with diffuse dThdPase expression from the 32 dThdPase-positive tumors (category I), and 10 areas without dThdPase expression from the 38 negative tumors (category II) were selected from each case. For early gastric carcinoma, the mean IMVD was 88.8+/-19.4 in category I and 61.4+/-17.3 in category II carcinomas, while for advanced gastric carcinoma, the mean IMVD was 98.8+/-21.0 in category I and 76.0+/-27.1 in category II carcinomas. The mean IMVD was significantly higher in category I than in category II tumors (P<0.05). The mean apoptotic index (AI: percentage of apoptotic cells) was 1.95+/-1.30 in category I, and 3.76+/-1.49 in category II carcinomas for early gastric carcinoma, and 1.51+/-0.98 in category I and 2.14+/-0.66 in category II carcinomas for advanced gastric carcinoma, the value of the mean AI being significantly (P<0.05) higher in dThdPase-negative tumors (category II) than in the positive tumors (category I), regardless of tumor stage or histological type. There was a significant inverse correlation (P<0.001) between AI and IMVD. These results indicate that dThdPase expression is associated with both an increase in intratumoral microvessels and a decrease in apoptosis in human gastric carcinomas.

We investigated the effects of the angiogenesis inhibitor TNP-470 on human lung squamous cell carcinoma cell lines H226B and H226Br both in vivo and in vitro. H226B was established from human lung squamous cell carcinoma and H226Br was established from a brain metastatic lesion of H226B in nude mice. Nude mice inoculated with these cells were treated with 30 mg / kg of TNP-470 subcutaneously every other day. At this dose, TNP-470 only significantly suppressed the growth of H226Br tumor, but not H226B tumor. Attempts to use a high dose of TNP-470 (100 mg / kg) resulted in a severe loss of body weight. Immunohistochemical studies showed marked tumor vascularization in H226Br tumor, but the formation of new blood vessels was suppressed by 30 mg / kg of TNP-470. Investigation of the mechanism of anti-angiogenic effects of TNP-470 in vivo showed that the expression and the activity of platelet-derived endothelial cell growth factor / thymidine phosphorylase (PD-ECGF / dThdPase) in H226Br tumor was significantly suppressed by 30 mg / kg of TNP-470. Furthermore, TNP-470 inhibited cell growth of cultured H226Br dose-dependently at concentrations of 1 microg / ml. Immunoblot analysis revealed H226Br cells gave a stronger PD-ECGF signal than H226B cells, and the expression of PD-ECGF / dThdPase in H226Br was also suppressed by treatment with TNP-470 at 0.1 microg / ml. No change in basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) was noted in these cell lines. Our results suggested that TNP-470 acts, at least in part, by downregulation of PD-ECGF / dThdPase in this cell line.

The potential of growth, invasion and metastasis of ovarian cancer cells associated with neovascularization, and the expression of platelet-derived endothelial growth factor (PD-ECGF) and its mRNA in ovarian cancers were determined. The relationship between their expression and histopathological types and clinical stages of ovarian cancers was also analyzed. The levels of PD-ECGF and its mRNA were higher in ovarian cancers than in normal ovaries. Furthermore, some endometrioid carcinomas and serous cystadenocarcinomas of the ovary and some ovarian cancers in stages III and IV expressed remarkably high levels of PD-ECGF and its mRNA. Therefore, in some ovarian cancers, PD-ECGF might be related to advanced stages of ovarian cancers associated with neovascularization.

The angiogenic growth factor thymidine phosphorylase/PD-ECGF has been identified as a potential target in the development of anti-cancer drugs. This review firstly discusses the biological rôle of TP/PD-ECGF and its importance in the activation of 5-fluorouracil and its prodrugs. The chemistry and chemotherapeutic potential of TP/PD-ECGF inhibitors are also discussed.

Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding.

Thymidine phosphorylase (TP), also known as platelet derived endothelial cell growth factor (PD-ECGF), is an enzyme involved in thymidine synthesis and degradation and exerts an angiogenic activity, whereas N4 pentyloxy-carbonyl- 5'-deoxy-5-fluorocytidine, commonly called capecitabine (CAP), is a TP-activated oral fluorpyrimidine, which generates 5-fluorouracil (5-FU) within tumours. In addition to its classic antitumour activity, recent studies suggest that CAP may act as an antiangiogenetic molecule. Assessment of tumour microvessel density as expressed by endothelial cell TP positivity may identify the most vascularized and hence CAP-sensitive tumours. This review summarizes: (i) the biochemical and tissue expression of TP; (ii) the pharmacological profile of CAP as an anti-cancer compound and the central role of TP in its activation; (iii) the potential antiangiogenetic role of TP-activated CAP in tumours.

Thymidine phosphorylase (TP) is an enzyme involved in reversible conversion of thymidine to thymine. TP is identical to an angiogenic factor, pletelet-derived endothelial cell growth factor (PD-ECGF) and the expression levels of TP in a variety of malignant tumors were higher than the adjacent non-neoplastic tissues. To investigate the molecular basis for the effect of TP on the metabolic process and the anticancer effect of 5-fluorouracil (5-FU), human gastric carcinoma AZ521 cells and epidermoid carcinoma KB cells were transfected with TP cDNA, and AZ521/TP and KB/TP were cloned. AZ521/TP and KB/TP cells overexpressed TP and were more sensitive to 5-FU than the counterpart parental cells. TPI, a newly synthesized inhibitor for TP (Ki=2.36 x 10(-9) M), decreased the sensitivity to 5-FU of the TP expressing cells but not of the parental cells. 5-Formyl-tetrahydrofolate (leucovorin; LV) stabilized the complex of thymidylate synthase (TS) and 5-fluoro-deoxyuridine-monophosphate (FdUMP), increased the sensitivity to 5-FU of TP expressing AZ521 cells, but not of the parental cells. The levels of FdUMP in TP expressing cells were significantly higher than in parental cells and TPI considerably decreased FdUMP to the level comparable to that in the parental cells. 5-FU increased the expression of early growth response protein-1 (Egr-1) and an angiogenesis inhibitor, thrombospondin-1 (TSP-1), in KB/TP cells but only slightly in KB/CV cells, if any. TPI attenuated the induction of Egr-1 and TSP-1 mRNA by 5-FU, while LV increased the expression of Egr-1 and TSP-1 mRNA in KB/TP cells. These findings demonstrate that the TP has a principal role in the production of FdUMP and the enhanced responses to 5-FU by leucovorin in TP-overexpressing KB and AZ521 cells, and FdUMP but not FUTP is implicated in the induction of Egr-1 and TSP-1 in KB cells.

OBJECTIVE

To investigate the role of biochemical changes in the umbilical cord and placenta in developing preeclampsia (PE).

METHODS

Thirty women with PE and 15 healthy pregnant women as controls were enrolled in this study. Vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 1 (sVEGFR-1), platelet-derived endothelial cell growth factor (PD-ECGF), neutrophil elastase and nitric oxide (NO) were measured.

RESULTS

Both serum (maternal and fetal) and tissue (placenta and umbilical cord) levels of VEGF, sVEGFR-1, PD-ECGF and neutrophil elastase were significantly increased, whereas NO was significantly decreased (except placental tissue showed no changes) in preeclamptic patients. The cord serum level of PD-ECGF was significantly higher in severe PE compared to mild PE and normal pregnant women. The placental and cord tissue levels of PD-ECGF and neutrophil elastase were significantly higher in severe PE, while the cord tissue level of NO was significantly lower in severe PE.

CONCLUSIONS

Our data showed that umbilical cord vessels and stroma can serve as an additional source of vasoactive and angiogenic substances that contribute to the biochemical changes occurring in PE.

Neural regeneration after grafting can be unpredictable. In an effort to enhance the return of function after cable grafting, we studied the effects of an angiogenic factor, endothelial cell growth factor (ECGF), on regenerating nerves. Cable grafts on the sciatic nerve were established in 18 rats and treated with ECGF or a control saline solution. At 5 weeks, nerve conduction studies were performed, and the animals were killed for histologic measurements of graft vascularity and axon counts. A significant increase in vascularity was noted in the treated group versus the control group; neither the axon counts nor the nerve conduction velocities differed significantly between the two groups, although the treated group appeared to show improved neural conduction compared with the control group.

OBJECTIVE

To culture human retinal capillary endothelium cells (HRCECs) in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs.

METHODS

Retinas were digested by 2.5% trypsin and 0.1% collagenase I in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium (HE-SFM BGM) with 10% fetal bovine serum, insulin-transferrin-selenium (ITS) and endothelial cell growth factor (ECGF). The cultured cells were identified by anti-factor VIII related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscopy, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs.

RESULTS

Cultured HRCECs attached in the bottom of dishes in 48 h - 72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor VIII. EGPF positive cells were seen under laser confocal scanning microscopy after 48 h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085 ± 0.021 in normal group and 0.166 ± 0.024 in hypoxia group (t = 3.938, P < 0.05). In normal oxygen condition, the numeric value OA was 0.171 ± 0.011 in normal control group, 0.178 ± 0.016 in rAAV2-EGFP treated group, and 0.169 ± 0.017 in rAAV2-PEDF treated group (F = 0.01, P>> 0.05). In hypoxia condition, the numeric value OA was 0.166 ± 0.013 in CoCl(2) treated group, 0.155 ± 0.012 in CoCl(2) + rAAV2-EGFP treated group, and 0.116 ± 0.015 in CoCl(2) + rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2.3% in normal control group, and 3.3% in rAAV2-EGFP treated group, and 1.7% in rAAV2-PEDF treated group. In hypoxia condition, the ratio of apoptosis was 3.6% in CoCl(2) treated group, 6.7% in CoCl(2) + rAAV2-EGFP treated group, and 36.4% in CoCl(2) + rAAV2-PEDF treated group.

CONCLUSIONS

PEDF gene can stably express in HRCECs after rAAV2-PEDF transfection and can obviously inhibit proliferation of HRCECs in hypoxia.

Endothelial progenitor cells (EPC) can differentiate into both endothelial cells and contractile smooth muscle cells (SMC). Previously we reported that TR-BME2 cells, a model for EPC, developed contractile SMC-like characteristics in culture medium deprived of endothelial cell growth factors (ECGF). The aim of the present study was to clarify the effect of one of these factors, basic fibroblast growth factor (bFGF) on differentiation of EPC. First it was confirmed that bFGF receptor (FGFR-1) mRNA is expressed in TR-BME2 cultured in both ECGF-rich and ECGF-deprived medium. When TR-BME2 cells were cultured in ECGF-deprived medium, they differentiated into contractile SMC. Expression of an undifferentiated state marker, CD133, and proliferation of TR-BME2 were both reduced by ECGF deprivation, but these changes were diminished in the presence of bFGF. mRNA expression of smooth muscle α-actin (SMA) and smooth muscle protein 22 (SM22), which are contractile SMC markers, was induced by deprivation of ECGF and the induction was suppressed by bFGF. In vascular endothelial cell growth factor (VEGF)-induced tube formation assay, TR-BME2 cells formed tube structures in the presence of bFGF, but not in its absence. Our results indicate that bFGF is essential for the maintenance of EPC phenotype, serving to suppress differentiation to contractile SMC.

Primary epithelial cultures (PECs) derived from normal, benign hyperplastic (BPH), and cancerous human prostate tissue were treated with increasing doses of suramin, and assayed for cell proliferation over a period of days. The suramin IC50 (inhibitory concentration 50%) value was 0.5 to 1.0 x 10(-4) M, whereas doses between 2.5 x 10(-4) and 5 x 10(-4) M resulted in total growth inhibition. This inhibition was reversible by exchange with suramin-free medium up to day 6. Concentrations>> or = 5 x 10(-4) M resulted in increased cytotoxicity as exposure time increased. No differential response to suramin could be demonstrated among the prostate PECs derived from different tissues. The established cell lines, PC-3 and DU 145, grown in serum containing medium exhibited IC50s comparable to the PECs grown in serum free medium. EGF, bFGF, alpha, or beta ECGF at the concentrations tested did not reverse suramin inhibition. Increasing concentrations of bovine pituitary extract (BPE) increased cell growth in both the treated and the control cells. However, the percent growth inhibition by suramin at each concentration of BPE remained constant. Flow cytometry examination of cells treated for 7 days with suramin (0-10(-3) M) failed to detect any significant cell cycle alterations compared to control. At high concentrations of suramin >> or = 10(-4) M), large numbers of viable and dead cells were detectable in the medium. The increase in unattached viable cells was most prevalent (80%) in cultures treated with suramin at the time of plating, but also occurred with cells (25-30%) plated hours prior to the addition of suramin. Treatment for several days with low concentrations of suramin (10(-7) to 10(-5) M) transiently enhanced cell growth compared to controls.

A 65-year old woman operated on for verrucous carcinoma of the uterine cervix 13 years previously was found to have multiple small recurrent tumors in the retroperitoneal space. Tumor cell expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) was investigated at the second operation. Expression of VEGF was strongly positive in the tumor cells and expression of PD-ECGF was strongly positive in both the tumor cells and the interstitial cells.

The study was designed to analyse results of alternative methods of myocardial revascularization in "no-option patients" to whom direct revascularization is not indicated. Over 600 cases were treated with the use of transmyocardial laser revascularization (TMLR), angiogenic factors (VEGF, alpha-ECGF), and cellular therapy. It was shown that TMLR is a safe and efficacious procedure increasing perfusion and normalizing myocardial metabolism. It eliminates angina and improves quality of life of the patients. Single bolus administration of angiogenic factors alone does not stabilize perfusion and patients' condition. The use of stem cells provokes the growth of new vessels and promotes restoration of viable myocardium via stimulation of angiogenesis. However, mesenchymal stem cells do not contribute to scar reparation and are inefficient in patients with a critical mass of cicatrically-altered myocardium.

The object of this study was to clarify the association of angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (dThdPase) with clinicopathological factors including tumor angiogenesis and patient outcome in endometrial cancer. There was no correlation between the expression of PD-ECGF in cancer cells and any of the clinicopathological variables. Immunopositivity for PD-ECGF in stroma cells was significantly higher in poorly differentiated adenocarcinomas. The microvessel counts correlated with PD-ECGF positive stroma cells (p<0.0001). Disease-free survival was significantly worse in patients with marked PD-ECGF expression in stromal cells and high microvessel count. A multivariate analysis using Cox's proportional hazard model showed that high microvessel counts independently predicted disease-free survival as well as stage and myometrial invasion. The expression of PD-ECGF in stroma cells may play a crucial role in the promotion of angiogenesis. Tumor angiogenesis can be used to predict prognosis in patients with endometrial cancer.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is associated with angiogenesis and the progression of human ovarian cancer. The enzyme converts thymidine to thymine and 2'-deoxyribose-1-phosphate and can also metabolize the prodrug 5'-deoxy-5-fluorouridine (Furtulon) to 5-fluorouracil and 5'-deoxy-D-ribose-1-phosphate. The aim of this study was to obtain information about the activities of Furtulon in an established three-dimensional model of angiogenesis. The plan was to study partial and complete effects of Furtulon (in the absence and presence of PD-ECGF/TP or ovarian cancer cyst fluids) on the formation and destruction of microvessels from cultured segments of rat aorta in serum-free media. The endpoint was the number and form of microvessels compared with controls after 4, 7, 11, and 14 (and sometimes 17) days in culture. Furtulon (10 micromol/L) gradually reduced the size and number of microvessels over 17 days of culture (100 micromol/L significantly reduced the number by day 4). PD-ECGF/TP (10 ng/ml) and ovarian cancer cyst fluids (2% in medium, v/v) stimulated the production of microvessels. The culture of explants with Furtulon and PD-ECGF/TP or ovarian cancer cyst fluids (from day 1 or day 11 of culture) enhanced the vasoclastic activity of the drug. The effect of Furtulon at the highest dose (1000 micromol/L) or at a lower dose (100 micromol/L) in the presence of ovarian cancer cyst fluid was not reversible after culture day 11.

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial mitogen from platelets and demonstrated to have angiogenic activity and thymidine phosphorylase (TP) activity. It was reported that the overexpression of PD-ECGF occurred with the rapid tumor growth in vivo. In this study, we transfected PD-ECGF into the head and neck squamous cell carcinoma cell line IMC-3 and investigated the property of transfectants in vitro. Highly overexpressed PD-ECGF transfectants rapidly grew compared with parental cells and control vector (CV) transfectants (p<0.05). The expression of cyclin D1 and cyclin E were more enhanced in PD-ECGF transfectants than parental cells and CV transfectants, while the p27kip1 was inhibited in PD-ECGF transfectants. In PD-ECGF transfectants, S and G2/M-phase cells rapidly increased compared with parental cells and CV transfectants. These results showed that the cancer cell line with high expression of PD-ECGF had a rapid cell cycle and consequently facilitated rapid cell growth not only in vivo but also in vitro. Furthermore, the inhibitor of thymidine phosphorylase (TPI) suppressed the cell cycle and rapid cell growth that were acquired by PD-ECGF transfection. Since PD-ECGF was reported to be an independent, poor prognosis factor for head and neck cancer, TPI might be useful for the inhibition of cancer cell growth.