The platelet-derived endothelial cell growth factor (PD-ECGF) level was significantly (P < 0.05) increased in 8 of 40 metastatic lymph node lesions of uterine cervical cancers. The prognosis of the eight patients with high PD-ECGF (>10,000 pg/mg protein) in metastatic lymph node lesions was extremely poor. On the other hand, the 24-month survival rate of the 32 patients with low PD-ECGF (<10,000 pg/mg protein) in metastatic lymph node lesions was 75%. This indicates that PD-ECGF may contribute to the advancement of metastatic lesions, and that the PD-ECGF level in metastatic lesions may be a prognostic indicator.

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

We investigated the correlation between thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor (PD-ECGF) expression, angiogenesis, and prognosis in renal cell carcinoma (RCC) patients. We prepared paraffin block specimens from 56 postradical nephrectomy RCC patients. The preparations were immunohistochemically stained using anti-CD34 antibody and anti-TP antibody. Angiogenic findings were evaluated based on both microvessel density (MVD) and renal arteriography findings as classified by Roosen et al. TP expression showed heterogeneity in 56 patients: 11 (19.6%) were negative, 28 (50.0%) weak, and 17 (30.4%) positive. There was no correlation between TP expression, MVD, and renal arteriography. There was no TP expression in chromophobe types. Univariate analysis showed a significant correlation between survival and TP expression, patient age, tumor infiltration type, pathologic T- and N-stages, venous involvement, distant metastasis, and tumor grade. There was no correlation between survival and MVD or renal arteriography. Multivariate analysis showed a significant correlation between survival and pathologic T-stage, distant metastasis, tumor infiltration type, and TP expression. TP expression in RCC may be an independent prognostic factor rather than just an index for angiogenesis.

Platelet-derived endothelial cell growth-factor (PD-ECGF) is similar to the pyrimidine enzyme thymidine phosphorylase (TP). A high TP expression at tumor sites is correlated with tumor growth, induction of angiogenesis, and metastasis. Therefore, high TP is most likely associated with a poor prognosis. TP is not only expressed in tumor cells but also in tumor surrounding tissues, such as tumor infiltrating macrophages. TP catalyzes the conversion of thymidine to thymine and doxyribose-1-phosphate (dR-1-P). The latter in its parent form or in its sugar form, deoxyribose (dR) may play a role in the induction of angiogenesis. It may modulate cellular energy metabolism or be a substrate in a chemical reaction generating reactive oxygen species. L-deoxyribose (L-dR) and thymidine phosphorylase inhibitor (TPI) can reverse these effects. The mechanism of TP induction is not yet completely clear, but TNF, IL10 and other cytokines have been clearly shown to induce its expression. The various complex interactions of TP give it an essential role in cellular functioning and, hence, it is an ideal target in cancer therapy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant. Health effects have been studied intensively, but low-dose effects are quite complex and not yet fully understood. In many studies, the immune system was identified as the most sensitive target. Here, we demonstrate changes of protein expression in liver and thymus of male marmosets (Callithrix jacchus) which were subjected to a single dose of a subcutaneous injection of 100 ng/kg body weight TCDD. Histopathological examination revealed myocardial fibrosis, but there were no significant findings in pathology and histopathology of liver and thymus. In order to detect more subtle treatment-related changes, we performed a comparative proteomic investigation of liver and thymus using a 2-D gel electrophoresis based proteomics approach. Fluorescence labeling and automated image analysis was used to enhance sensitivity and reproducibility. In both organs, distinct changes of protein expression were detected which were more pronounced in thymus, where the pattern of deregulated proteins could be clearly related to immune responses. In the thymus of treated animals, several toxicologically relevant factors were increased, including chaperones, glycerol-3-phosphate dehydrogenase, and adseverin. Among others, vimentin, Ca-dependent protease and protein disulfide isomerase were downregulated. In the liver, transferrins, lamin A and HSP70 were upregulated, whereas thymidine phosphorylase (synonyms: endothelial cell growth factor, PD-ECGF, gliostatin) was significantly reduced. Comparative analysis of deregulated proteins in both organs revealed a pattern of related functions, which fits well into the existing knowledge of the toxic processes and mechanisms underlying TCDD-mediated toxicity.

Cytokine expression and production by human megakaryocytic cells were studied using the CMK cell line as a model for cytokine gene expression by cell line as a model for cytokine gene expression by polymerase chain reaction (PCR) and for cytokine protein synthesis by specific radioimmunoassays. CMK cells at all stages of maturation were found to constitutively express moderate mRNA levels for tumor necrosis factor (TNF-alpha), transforming growth factor beta (TGF-beta), interleukin (IL) 1 beta, and endothelial cell growth factor (ECGF) transcripts. After 6-h treatment with the phorbol ester PMA, gene expression for IL-1 alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and the IL-6 receptor were increased. After 24 h of exposure to PMA, levels for most cytokines declined to baseline, except for IL-6 which appeared as a new transcript. PMA-stimulated CMK lines synthesized low levels of TNF-alpha and IL-6, and higher levels of GM-CSF, IL-1 beta, and IL-1 alpha protein. These observations suggest that cells of megakaryocytic lineage are capable of producing a repertoire of cytokines which could mediate an autocrine role as well as modulate the replication and function of other hematopoietic cells.

We have previously shown that administration of endothelial cell growth factor (ECGF) significantly accelerates revascularization in the ischemic rabbit hindlimb model. Nevertheless, much remains to be learned as to the effectiveness and limitations of this approach. The present study was designed, therefore, to determine whether a dose-response relationship could be demonstrated for this agent, whether it was effective if systemically administered, and finally, whether it affected vascularization in a nonischemic limb. Our established unilateral ischemic rabbit hindlimb model and ECGF administration protocol were used to examine these questions. Three groups of animals were studied to determine a dose-response relationship of ECGF in the ischemic limb. Revascularization was assessed by measurement of calf blood left to right (L/R) pressure ratio before (Day 0) and after ECGF injections (Days 1, 10, and 20) and quantitative vascularization was assessed by angiography at Day 20 when the study was terminated. The results of the dose-response study were [formula: see text] Two other groups of animals were also studied. In Group 4, intramuscular ECGF was injected in the left front limb remote from the ischemic hindlimb and in Group 5 it was injected into the left hindlimb of a normal animal. In neither group was any significant effect on vascularization evident. Thus, our data suggest that the relationship of ECGF and revascularization of the ischemic limb is dose dependent and is demonstrable only when it is administered directly into the limb in the presence of ischemia.

Previous studies suggested that arterial smooth muscle cells (SMC) may be involved in regulating the growth of capillaries into atherosclerotic plaques. In the present study, we determined the effect of SMC products on porcine aortic endothelial cell (EC) replication in vitro. Quiescent or slowly growing EC in medium without endothelial cell growth factor (ECGF) were stimulated to proliferate in the presence of porcine aortic SMC conditioned medium, while the same conditioned medium inhibited the growth of rapidly dividing EC in high serum concentrations or with ECGF. The magnitude of both activities depended on SMC conditioned medium concentration. The dose-dependent increase in EC number stimulated by ECGF was completely inhibited by SMC conditioned medium. This effect was not due to a direct interaction of conditioned medium with ECGF because SMC conditioned medium inhibited the growth of EC that were rapidly proliferating in 10% serum without ECGF. The inhibitory activity was retained by an ultrafiltration membrane with an exclusion limit of 1000 daltons; the stimulatory activity was recovered in the ultrafiltrate and remained stable after boiling, treatment with acid or base and trypsin, and repeated freezing and thawing, but was removed by activated charcoal. The growth-promoting activity could not be accounted for by release of cell contents from lysed cells or of thymidine into the medium. Conditioned medium from SMC incubated in the presence of serum contained less EC growth-stimulatory activity but more growth-inhibitory activity than that from SMC in serum-free medium.

The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD). The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form. The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism. A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level. The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base. To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates. Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate. Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine. For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine. Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form. These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors.

The mechanisms of excessive body weight gain after diet-restriction are still unclear. In this study, we investigated expression of angiogenic factors in adipose tissue and skeletal muscle of rabbits which had rebound weight gains; trying to make inquiries into the mechanisms of this rebound weight gain. Ten rabbits were divided into two groups. One group had free food intake (group C), and the other group had restricted food intake until day 40 of the experiments and then had free food intake (group DR). Specimens of adipose tissue and skeletal muscle were collected from each rabbit on days 20, 40, and 60 after the initial examination, and expressions of CD34, vascular endothelial growth factor (VEGF), and platelet-derived endothelial cell growth factor (PD-ECGF) were investigated. Expression of VEGF was significantly strong in the adipose tissue of group DR at the recovery period of body weight. In conclusion, rebound weight gain after a restricted-diet may be associated with angiogenesis in adipose tissue, and the angiogenesis may be induced by VEGF.

BACKGROUND

Angiogenesis is essential for development, growth and advancement of solid tumors. During angiogenesis, ETS-1 is strongly expressed in vascular endothelial cells and the adjacent interstitial cells, while the inhibition of ETS-1 expression leads to suppression of angiogenesis. This prompted us to study the clinical implications of ETS-1 in relation to angiogenesis in uterine endometrial cancers.

METHODS

Sixty patients underwent resection for uterine endometrial cancers. From the tissues of 60 uterine endometrial cancers, the levels of ets-1 mRNA, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF) and interleukin (IL)-8 were determined by competitive RT-PCR using recombinant RNA and enzyme immunoassay, and the localization and counts of microvessel were determined by immunohistochemistry.

RESULTS

There was a significant correlation between microvessel count and ets-1 gene expression levels in uterine endometrial cancers. Immunohistochemical staining revealed that the localization of ETS-1 was similar to that of vascular endothelial cells. The level of ets-1 mRNA tended to increase with increasing disease stage. Furthermore, the level of ets-1 mRNA correlated with levels of VEGF in well-differentiated adenocarcinomas (G1) and of bFGF in moderately differentiated adenocarcinomas (G2) and poorly differentiated adenocarcinomas (G3).

CONCLUSIONS

ETS-1 is a possible angiogenic mediator in uterine endometrial cancers.

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial cell mitogen from platelets. In this study, we investigated the expression of PD-ECGF and counted microvessels in 58 oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique to examine their prognostic significance and performed tumor in vitro sensitivity to 5-fluorouracil (5-FU) and cisplatin as determined by a bioluminescence assay of the ATP values of tumor cells after continuous exposure. The percentage of PD-ECGF-positive tumor cells (PD-ECGF score) was correlated with the frequency of the recurrence of disease (P=0.0043) but not with sex, tumor size, metastasis, or clinical stage. Overall survival of the high PD-ECGF expression group (>40% PD-ECGF score) was shorter than the low expression (<40%) group (P=0.0365). Vessel count was correlated with lymph node metastasis and clinical stage. The survival of patients with hypervascularity (more than the median of intratumor vessel counts, >82) was shorter than that of those with hypovascularity (vessel count <81, P=0.0446). However, there was no association between PD-ECGF expression and vessel count. Cox proportional multivariate analysis showed that PD-ECGF expression was the most significant independent prognostic indicator for overall survival. The susceptibility to 5-FU cytotoxicity in the extremely high PD-ECGF expression groups (>70% of PD-ECGF score) was significantly higher than that in the low group, whereas there was no difference in their sensitivity to cisplatin. These results showed that carcinoma cells with high PD-ECGF expression were sensitive to 5-FU in spite of poor prognosis. These data provide further information when deciding on adjuvant therapy for oral and oropharyngeal SCCs.

BACKGROUND

In order to elucidate the roles of tumor angiogenesis in lung carcinogenesis, the expressions of several angiogenic factors in lung carcinoma tissues were examined.

METHODS

Tissue specimens from 112 cases of resected non-small cell lung cancer (NSCLC) were studied. The expressions of platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) were examined immunohistochemically. Microvessel density (MVD) was also evaluated.

RESULTS

VEGF-positive cases were observed more frequently in advanced stage lung cancers than in early cancers, and VEGF-positive tumors had higher MVD than VEGF-negative tumors, while such differences were not observed for PD-ECGF. In squamous cell carcinoma, the patients with high-MVD tumor had significantly worse survival than those with low-MVD tumor.

CONCLUSIONS

Our results suggest that VEGF plays an important role in angiogenesis of lung cancers, while the contribution of PD-ECGF may be limited.

Biodegradable pellets releasing 20 ng/day of endothelial cell growth factor alpha (alpha ECGF) or a- or b-fibroblast growth factor (FGF) and 90 micrograms/day of heparin were implanted beneath the renal capsule in rats and dogs and the muscularis/serosal border of the pyloric stomach in dogs to test for angiogenesis in a potential pancreatic islet transplant site. These factors were also tested in vitro to determine whether the capillary bed of the isolated islet could be preserved. alpha ECGF was superior to a- or bFGF in promoting endothelial cell growth and capillary formation in isolated islets. Both a- or bFGF and alpha ECGF induced the development of a dense capillary bed in the dog stomach, whereas in the kidney site alpha ECGF was more effective in the rat than was a- or bFGF. Priming the isolated islet as well as the transplant site prior to islet transplantation resulted in islet blood flow being established within 3 days in contrast to 7-14 days in controls.

The expression of smooth muscle (sm) alpha-actin was studied in cloned capillary cerebral endothelial cells of two phenotypes. Type I cells were cultured in medium containing 10% FCS, heparin and ECGS (or alpha-ECGF) and stained positive for a specific endothelial cell marker (Bandeiraea simplicifolia). Depletion of heparin and ECGS resulted in a smooth muscle-like appearance after 2-3 days. Cells of this phenotype, (type II) stained positive for the endothelial cell marker and for sm alpha-actin. In contrast to type I cells, type II cells expressed sm alpha-actin protein and mRNA as evidenced by Immunoblots and Northern blots. This phenotypic switch was shown to be reversible and so was the expression of sm alpha-actin.

OBJECTIVE

To study the apoptosis induced by preoperative oral 5'-DFUR administration in gastric adenocarcinoma and its mechanism of action.

METHODS

Sixty gastric cancer patients were divided randomly into three groups (20 each group) before operation: group one:5'-DFUR oral administration at the dose of 800-1200 mg/d for 3 - 5 d, group two: 500 mg 5-FU + 200 mg/d CF by venous drip for 3 - 5 d,group three (control group). One or two days after chemotherapy, the patients were operated. Fas/FasL,PD-ECGF and PCNA were examined by immunohistochemistry and apoptotic tumor cells were detected by in situ TUNEL method. Fifty-four patients received gastrectomy, including 12 palliative resections and 42 radical resections. Six patients were excluded. Finally 18 cases in 5'-DFUR group, 16 cases in CF+5-FU group, and 20 cases in control group were analyzed.

RESULTS

There was no significant difference in patient mean age, gender, white blood cell count, haematoglobin (HB),thromboplastin, perioperative complication incidence, radical or palliation resection, invasion depth (T), lymphonode involvement (N),metastasis (M) and TNM staging among the three groups. However,the PCNA index (PI) in 5'-DFUR group (40.51+/-12.62) and 5-FU+CF group (41.12+/-15.26) was significantly lower than that in control group (58.33+/-15.69) (F=9.083, P=0.000). The apoptotic index (AI) in 5'-DFUR group (14.39+/-9.49) and 5-FU+CF group (14.11+/-9.68)was significantly higher than that in control group (6.88+/-7.37) (F=4.409, P=0.017).The expression rates of Fas and FasL in group one and group three were 66.7% (12/18) and 50% (9/18), 43.8% (7/16) and 81.3% (13/16), 45.0% (9/20) and 85% (17/20), respectively. The expression rate of FasL in 5'-DFUR group was significantly lower than that in the other two groups (chi2=6.708, P=0.035). Meanwhile, the expression rate of PD-ECGF was significantly lower in 5'-DFUR group (4/18,28.6%) than in CF+5-FU group(9/16,56.3%)and control group (13/20,65.0%) (chi2=7.542, P=0.023). The frequency of Fas expression was significantly correlated with palliative or radical resection (chi2=7.651, P=0.006), invasion depth (chi2=8.927, P=0.003), lymphatic spread (chi2=4.488, P=0.034) and UICC stages (chi2=8.063, P=0.045) respectively. By the end of March 2005,45 patients were followed up. The 0.5-, 1-, 2-, 3-year survival rates were 96%,73%,60%,48%, respectively, which were related with T, N, M and Fas expression, but not with PD-ECGF and FasL expression.

CONCLUSIONS

Preoperative oral 5'-DFUR administration may induce apoptosis of gastric carcinoma cells and decrease tumor cell proliferation index,but cannot improve the prognosis of patients with gastric cancer.Down-regulation of FasL and PD-ECGF expression mediated by 5'-DFUR may be one of its anti-cancer mechanisms.Fas expression correlates with the progression of gastric carcinoma and may be an effective prognostic factor.

Hepatocytes can be successfully transplanted into highly vascular sites such as the spleen, liver, and lungs. Subcutaneous sites lack adequate vascularization to nutritionally support transplanted hepatocytes. We recently reported that matrix-immobilized angiogenic growth factors, e.g., endothelial cell growth factor (ECGF), can induce a high degree of neovascularization. Using this technique, we explored the possibility of transplanting isolated fetal porcine hepatocytes to create liver tissue organoids at a specific subcutaneous site. We evaluated chitosan as a scaffold biomaterial because of its structural similarity to glycosaminoglycans; glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. Freshly isolated fetal porcine hepatocytes (FPH) (viability greater than 97%) were cultured on modified chitosan scaffolds and transplanted into rat groin fat pads with or without ECGF-induced neovascularization. Cell density and attachment kinetics on chitosan were examined by scanning electron microscopy (SEM) and quantified using a flavianic acid binding assay. Hepatocyte viability and liver organoid formation were examined immunohistochemically. FPH transplanted without prior neovascularization died within 1 day post-transplantation. When transplanted after ECGF-induced neovascularization, FPH thrived for at least 2 weeks and formed liver tissue like structures. Immunohistochemical analysis revealed the presence of hepatocyte-specific cytokeratin staining as well as the presence of alpha-fetoprotein. Light microscopy and SEM revealed that FPH did not change their morphology after attachment to the chitosan surfaces. Thus, chitosan-based biomaterial surfaces have good hepatocyte attachment properties. However, extensive neovascularization is essential for hepatocyte survival and organoid formation. In the future, chitosan-based biomaterials may be useful as scaffolds for creating liver tissue organoids.

Endothelial cells (EC) from human aorta, umbilical vein and pulmonary artery were grown in Medium 199 supplemented with 20% human serum (HS), endothelial cell growth factor (ECGF) from bovine and human brain (200 micrograms/ml) and heparin (100 micrograms/ml) in gelatin-coated flasks. Under these conditions cells rapidly proliferated and survived 15-25 passages (40-60 cumulative population doublings). When cells were cultured on plastic substrate and without growth factors a capillary-like network appeared after 3-4 weeks of growth. According to TEM, this network consisted of tubes with the lumen encircled by one or several cells. The reduction of serum concentration in the medium or the replacement of plasma-derived serum (PDS) for HS reduced the time of network formation to 3-5 days. S-180 conditioned medium mitogenic for EC induced a rapid spreading of the cells and a partial reversion to a two-dimensional monolayer structure. Trypsin inhibitor did not abolish the effect of tumour conditioned medium. Other EC mitogens, e.g. ECGF and fibroblast growth factor (FGF), also disorganized the capillary-like network. In a day or two the network was completely restored. In contrast, culturing EC on gelatin-coated substrate is a sufficient condition for monolayer formation from tubes and long-term maintenance. We suggest that mitogens can influence the EC morphology but that it is the nature of the substrate that determines the stage of large vessel EC differentiation.

Schizandrol A (SA) is an bioactive component isolated from the Schisandra chinensis (Turcz.) Baill., which has been used as a remedy to prevent oxidative injury. However, whether the cardioprotective effect of SA is associated with regulating endogenous metabolites remains unclear, thus we performed comprehensive metabolomics profiling in acute myocardial ischemia (AMI) mice following SA treatment. AMI was induced in ICR mice by coronary artery ligation, then SA (6 mg·kg^-1·d^-1, ip) was administered. SA treatment significantly decreased the infarct size, preserved the cardiac function, and improved the biochemical indicators and cardiac pathological alterations. Moreover, SA (10, 100 M) significantly decreased the apoptotic index in OGD-treated H8c2 cardiomycytes in vitro. By using HPLC-Q-TOF/MS, we conducted metabonomics analysis to screen the significantly changed endogenous metabolites and construct the network in both serum and urine. The results revealed that SA regulated the pathways of glycine, serine and threonine metabolism, lysine biosynthesis, pyrimidine metabolism, arginine and proline metabolism, cysteine and methionine metabolism, valine, leucine and isoleucine biosynthesis under the pathological conditions of AMI. Furthermore, we selected the regulatory enzymes related to heart disease, including ecto-5'-nucleotidase (NT5E), guanidinoacetate N-methyltransferase (GAMT), platelet-derived endothelial cell growth factor (PD-ECGF) and methionine synthase (MTR), for validation. In addition, SA was found to facilitate PI3K/Akt activation and inhibit the expression of NOX2 in AMI mice and OGD-treated H9c2 cells. In conclusion, we have elucidated SA-regulated endogenous metabolic pathways and constructed a regulatory metabolic network map. Furthermore, we have validated the new potential therapeutic targets and underlying molecular mechanisms of SA against AMI, which might provide a reference for its future application in cardiovascular diseases.

ECGF, a polypeptide of bovine hypothalamic derivation, is the most potent endothelial cell mitogen known, with mitogenic and chemotactic effects well demonstrated in vitro on human endothelial cells. These effects are synergized by heparin. In vivo re-endothelialization of blood-contacting biomaterials may be enhanced by bonding ECGF and heparin to prosthetic surfaces. Long woven Dacron (24 mm) and woven PDS vascular prostheses were treated first with human plasma fibronectin (10 micrograms/cm2). Porcine sodium heparin (20 micrograms/cm2) was added by means of fibronectin's heparin affinity. Pure 125I-ECGF (95% alpha, 5% beta; 1 ng/cm2) was next fixed by the heparin affinity of ECGF and followed by a second heparin layer (20 micrograms/cm2) to synergize with and stabilize ECGF. 125I-ECGF adherences were determined by scintillation counts. Attachment efficiency averaged 25%. Prostheses were interposed into rabbit aortas and harvested in triplicate from 0 to 30 days to establish in vivo washout curves. After explantation, residual 125I-ECGF was eluted from prostheses, and intact ECGF was identified by SDS gel electrophoresis. Similarly prepared but nonradioiodinated Dacron and PDS prostheses were explanted after 7 days and their ECGF eluted off for in vitro activity documentation. This ECGF retained its mitogenic properties, causing a 1000% to 1200% increase in 3H-thymidine incorporation into newly synthesized DNA in test murine LE-II cells. Fibronectin-heparin-ECGF fixation to blood-contacting biomaterials may enhance spontaneous re-endothelialization and/or hasten the confluence of transplanted endothelial cells.

The proliferation of mouse submandibular gland carcinoma YT-12 cells was stimulated by endothelial cell growth factor (ECGF)/bovine brain-derived acidic fibroblast growth factor (aFGF) and recombinant human aFGF. To determine whether aFGF was capable of modifying salivary gland carcinogenesis, the effect of brain-derived aFGF was examined in vivo. Mice in Groups 1 and 2 were injected with 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received bovine brain-derived aFGF and Group 2 mice received vehicle subcutaneously for 10 weeks. Group 3 and 4 mice received either bovine brain-derived aFGF or vehicle only. Sixteen weeks after the start of the experiment, the incidence of submandibular gland carcinomas in Group 1 was significantly greater than that in Group 2. Immunohistochemical study indicated that ducts in the normal submandibular glands and carcinomas showed positive staining with anti-aFGF antibody. Immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression of aFGF in these tissues. FGF receptor (FGFR)-1 and FGFR-4 were detectable in the mouse submandibular glands and carcinomas. These findings suggest that bovine brain-derived aFGF stimulates the proliferation of submandibular gland carcinoma cells and promotes mouse submandibular gland carcinogenesis.

Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.

Epithelial hyperplasia and dysplasia have been diagnosed as precancerous lesions and have been discussed in relationship to carcinogenesis. We analyzed the immunohistochemical expression of granulocyte colony-stimulating factor receptor (G-CSFR) and platelet-derived endothelial cell growth factor (PD-ECGF) in oral and oropharynx; 33 samples of normal epithelium, 28 samples of hyperplasia, 16 samples of dysplasia and 58 samples of squamous cell carcinoma. Also, we examined mean vessel density (MVD) by using CD34 staining and proliferating cell nuclear antigen (PCNA) staining. Dysplasia and head and neck Squamous Cell Carcinoma (SCC) exhibited higher G-CSFR expression and MVD than normal or hyperplastic epithelium (p <0.01). In the PD-ECGF staining, significant differences were found between SCC and normal epithelium, hyperplasia and dysplasia (p<0.001). In dysplasia and hyperplasia, PD-ECGF expression was significantly correlated with PCNA expression (r=0.345, p=0.025), however it was not correlated with the MVD. G-CSFR expression was not correlated with either PCNA or MVD. These results suggest that G-CSFR and PD-ECGF might be concerned with different carcinogenesis pathways of the squamous cells in the oral region and that PD-ECGF may be concerned with epithelial proliferation rather than angiogenesis.

Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor and in some studies PD-ECGF/dTdRPase expression levels in several kind of cancers were higher than in their surrounding normal tissues. In this study, we evaluated PD-ECGF/dTdRPase expression in bladder cancer by an immunohistological method and determined whether it correlated with tumor stage, grade and recurrence. PD-ECGF/dTdRPase expression was correlated with tumor grade and stage. Furthermore, among the superficial tumors, PD-ECGF/dTdRPase expression was correlated with a recurrence-free rate and thus it might be a prognostic factor for the recurrence of superficial bladder cancer.