The mechanism of neovascularization secondary to renal interstitial fibrosis is not well understood. Platelet-derived endothelial cell growth factor (PD-ECGF) is known to promote angiogenesis. We examined the expression of PD-ECGF immunohistochemically in 9 normal kidneys and 26 scarred kidneys secondary to urinary tract diseases. To estimate up-regulation of angiogenesis, microvessels were counted by immunostaining endothelial cells for CD34. Immunostaining of PD-ECGF was observed in most Bowman's capsules, occasional tubules, and some interstitial mononuclear cells in normal kidneys. A remarkable increase of immunostained PD-ECGF was found in the tubules and interstitial mononuclear infiltrates in the scarred kidneys. The predominant cell type in the infiltrate was T cells (CD3(+)). The microvessel count and mean numbers of PD-ECGF(+) tubular and interstitial mononuclear cells increased with increasing interstitial fibrosis. A significant correlation was noted between microvessel count and the number of PD-ECGF(+) tubular cells (P = 0.0002) or PD-ECGF(+) interstitial mononuclear cells (P < 0.0001). Immunostaining of endogrin, a marker of endothelial proliferation, increased in the microvessels located in the fibrotic interstitial spaces. These results suggest that angiogenesis may play a critical role in the progression of tubulointerstitial injuries and that up-regulation of PD-ECGF may contribute to neovascularization.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45 kDa single chain polypeptide which stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo. Analysis of a full length PD-ECGF cDNA revealed an open reading frame coding for 482 amino acids without homology to other known proteins. No signal sequence was observed, and analysis of the biosynthesis and processing of PD-ECGF in a thyroid carcinoma cell line revealed that PD-ECGF is released only very slowly. PD-ECGF becomes covalently associated with nucleotide triphosphates (e.g., ATP) in vivo, as well as in vitro. The physiological significance of this posttranslational modification remains to be elucidated. The tissue distribution and target cell specificity of PD-ECGF suggest roles in angiogenesis (e.g., during wound healing and in the developing placenta), as well as in the maintenance of the integrity of the endothelial cell lining of large vessels.

OBJECTIVE

The purpose of this study was to examine how gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) is involved in the molecular mechanism of cartilage degradation in rheumatoid arthritis (RA) with special reference to the GLS-induced gene expression and protein synthesis of matrix metalloproteinase (MMP)-1 (collagenase-1) and MMP-3 (stromelysin-1).

METHODS

Fibroblast-like synoviocytes (FLSs) obtained from RA patients were cultured and stimulated by GLS. Changes in the expression levels of GLS, MMP-1 and MMP-3 were assessed by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) for GLS, and by RT-PCR and enzyme-linked immunosorbent assay for MMPs and tissue inhibitor of metalloproteinase 1.

RESULTS

GLS demonstrated a self-induction of mRNA in cultured RA FLSs. GLS evoked a dose-dependent induction of MMP-1 and MMP-3 mRNAs, and subsequently their extracellular secretion.

CONCLUSIONS

These findings suggest that GLS is a plausible pathogenic factor causing the extensive joint destruction in RA mediated via MMPs.

BACKGROUND

To evaluate the roles of angiogenic factors in the development and progression of odontogenic tumors, expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) and of angiopoietins in ameloblastic tumors as well as in tooth germs.

METHODS

Tissue specimens of 11 tooth germs, 44 ameloblastomas, and five malignant ameloblastic tumors were examined immunohistochemically with the use of antibodies against PD-ECGF/TP and angiopoietin-1 and -2.

RESULTS

Immunohistochemical reactivity for PD-ECGF/TP was detected in mesenchymal cells in tooth germs and stromal cells in ameloblastic tumors, and the level of immunoreactivity for PD-ECGF/TP was significantly higher in ameloblastomas than in tooth germs. Granular cell ameloblastomas showed PD-ECGF/TP reactivity in granular neoplastic cells as well as in stromal cells. Immunoreactivity for angiopoietin-1 and -2 was detected predominantly in odontogenic epithelial cells near the basement membrane in tooth germs and in benign and malignant ameloblastic tumors. Malignant ameloblastic tumors had decreased angiopoietin-1 reactivity and ameloblastic carcinomas had increased angiopoietin-2 reactivity as compared with the respective levels in tooth germs and ameloblastomas. Immunohistochemical reactivity for angiopoietin-2 was slightly higher in follicular ameloblastomas than in plexiform ameloblastomas.

CONCLUSIONS

Expression of PD-ECGF/TP and angiopoietin-1 and -2 in tooth germs and ameloblastic tumors suggests that these angiogenic factors participate in tooth development and odontogenic tumor progression by regulating angiogenesis. Altered expression of PD-ECGF/TP and angiopoietins in ameloblastic tumors may be involved in oncogenesis, malignant potential, and tumor cell differentiation.

Platelet-derived endothelial cell growth factor (PD-ECGF) is expressed in the lining epithelial cells of ovarian endometriomas, and in interstitial cells of the subepithelial area with angiogenesis. The expression of PD-ECGF persists in endometriotic endometrium during the menstrual cycle. This might suggest that PD-ECGF contributes to the growth of ovarian endometriomas via subepithelial angiogenesis independently of the sex steroidal milieu.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa peptide mitogen which is present in platelets and placenta and produced by certain cultured cell lines. Immunoprecipitation of A431 cells metabolically labeled with [32P]orthophosphate revealed the incorporation of 32P radioactivity into PD-ECGF. Phosphoamino acid analysis showed that serine residues of PD-ECGF were phosphorylated in vivo. Forskolin, 12-O-tetradecanoylphorbol-13-acetate, and epidermal growth factor had no effect on the in vivo phosphorylation of PD-ECGF. Moreover, incubation of pure PD-ECGF with [gamma-32P]ATP led to labeling of PD-ECGF. Optimal labeling was achieved by incubation at 95 degrees C for 5 min in the presence of sodium dodecyl sulfate, dithiothreitol, and Mg2+ or Mn2+. PD-ECGF was also labeled with [2,8-3H]ATP, [2,5',8-3H]ATP, or [alpha-32P]ATP. ATP and GTP were the preferred nucleotide substrates by comparison with other nucleotides and related components. Partial amino acid hydrolysis liberated a significant amount of O-[32P]phosphoserine from PD-ECGF labeled in vitro with [gamma-32P] ATP. Furthermore, 32P-radiolabeled nucleotides were released after snake venom phosphodiesterase or piperidine treatment from PD-ECGF labeled in vitro with [alpha-32P]ATP or [gamma-32P]ATP, as well as from PD-ECGF labeled in vivo with [32P]orthophosphate. These data indicate that serine residues of PD-ECGF can be covalently linked to phosphate groups of nucleotides, resulting in a nucleotidylated protein. The functional significance of this post-translational modification remains to be determined.

Platelet-derived endothelial cell growth factor (PD-ECGF) was purified to homogeneity from human term placenta, an organ characterized by extensive angiogenesis. N-terminal amino acid sequencing revealed that placental PD-ECGF was proteolytically processed at Thr-6, in contrast to PD-ECGF purified from human platelets, which is processed at Ala-11. The purified factor stimulated porcine aortic endothelial cells as well as two choriocarcinoma cell lines. Immunohistochemical staining revealed that PD-ECGF was present in the connective tissue cells of the placenta. The possibility that PD-ECGF is involved in the development of the placenta is discussed.

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.

BACKGROUND

Thymidine phosphorylase (TP) is the key enzyme for capecitabine activation in tumour cells.

OBJECTIVE

To examine whether TP expression in tumour cells and stroma is predictive of the tumour response to capecitabine plus docetaxel chemotherapy in patients with advanced non-small cell lung cancer (NSCLC).

METHODS

Tumour samples were available from 30 of 39 patients enrolled in a previous phase II study of capecitabine/docetaxel chemotherapy in patients with advanced NSCLC. Stromal and tumour cell TP expression was evaluated by immunohistochemistry using monoclonal antibody PD-ECGF.

RESULTS

High tumour cell TP expression was found in 13 of 30 cases and was negatively associated with stromal TP expression (p = 0.000). High stromal TP expression was found in 16 of 28 cases and was strongly associated with intense macrophage infiltration (p = 0.002), suggesting that macrophages are the major component of TP expression in the stroma. Tumour response to capecitabine/docetaxel was significantly associated with high tumour cell TP expression (p = 0.004) and low stromal TP expression (p = 0.009). Moreover, high tumour cell TP expression was significantly associated with severe hand-foot syndrome, a toxic side effect of capecitabine (p = 0.01). Improved survival was seen for high tumour cell and low stromal TP expression, although results were not significant (p = 0.6 and 0.3, respectively).

CONCLUSIONS

In advanced NSCLC, TP expression in tumour cells and stroma is associated with tumour response to capecitabine/docetaxel chemotherapy, and might be a useful predictor of tumour response to capecitabine based chemotherapy. A large scale prospective study is needed to confirm the prognostic significance of TP expression in NSCLC.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), is an enzyme that catalyzes the reversible dephosphorylation of thymidine, deoxyuridine and their analogs. TP has also angiogenic properties, although the precise mechanism by which it promotes angiogenesis is not known. We examined TP expression using immunohistochemistry (654-1 Mab) in 182 invasive breast carcinomas (67 N0 and 115 N1/2; median follow-up 78 months [range, 3-177]; 51 patients treated with adjuvant systemic cyclophosphamide, methotrexate and 5-fluorouracil [CMF] chemotherapy and 82 with tamoxifen). High TP expression was found in 142 cases (78%) and correlated with lower histologic grade and low p53 expression. No correlation was found between TP expression and vascular density. TP-positive tumors had a significant increase in both disease-free survival (DFS; p = 0.0025) and overall survival (OS; p = 0.0070) in the total cohort of patients and in the subgroups of node-positive patients and patients treated with CMF adjuvant therapy; no significant difference in either DFS or OS was observed in patients without CMF treatment. Our findings suggest that TP has little effect on tumor angiogenesis of breast carcinoma, whereas it could represent an interesting marker that could predict response to CMF chemotherapy.

Human tissue contents of gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF) and its drug-induced expression in tumor cells were currently examined by a sandwich enzyme immunoassay (EIA) system and a reverse transcription-polymerase chain reaction (RT-PCR) method. Gliostatin/PD-ECGF was found to distribute in rather ubiquitous than specific human tissues and organs, with a relatively high levels in the tissues of digestive system (esophagus and rectum), brain, spleen, bladder and lung, but not in gall bladder, aorta, muscle, fat and kidney. Most of examined human tumor cell lines showed 4- or 5-fold higher contents (21.5 +/- 3.9 ng/mg protein) than normal tissue contents (4.4 +/- 1.1 ng/mg protein) on the average. While gliostatin/PD-ECGF is known to lack a signal sequence, some tumor cells (A431 and MKN74) appeared to release it into the conditioned medium. Expression of gliostatin/PD-ECGF in epidermoid carcinoma cell (A431) and stomach cancer cell (MKN45) was induced by dibutyryl cyclic AMP and phorbol ester, and uniquely in MKN45 by hydrocortisone. In particular, this hydrocortisone specifically caused an increase of the apparent secretion of MKN74 without its cytotoxic effects, suggesting a possible secretion of gliostatin/PD-ECGF in the restricted but not universal cell line. Biological significance on the chemical induction of gliostatin/PD-ECGF in tumor cells and on its extracellular secretion are discussed.

OBJECTIVE

Platelet-derived endothelial cell growth factor (PD-ECGF), which has angiogenic activity, is identical to thymidine phosphorylase. Tumor vascularization is considered to be an important prognostic factor. Nitric oxide synthases (NOSs) are a kind of enzyme that generates nitric oxide. Nitric oxide has not only a self defense against neoplastic cells but also tumor growth stimulation by promoting new blood vessel formation. Our purpose was to investigate the correlation between the expression of PD-ECGF or inducible NOS (iNOS) in cancer cells and prognosis.

METHODS

Formaldehyde-fixed and paraffin-embedded biopsy specimens excised from 71 cervical squamous cell carcinoma patients who were treated with radiotherapy alone were investigated using an immunohistochemical method.

RESULTS

Cancer cells that were positive for PD-ECGF showed intranuclear and cytoplasmic staining patterns. Of the 71 patients, 40 (56%) were positive for PD-ECGF and 31 (44%) were negative. The 5-year survival of the PD-ECGF-positive patients was significantly better than that of the PD-ECGF-negative patients (p = 0.026). Cancer cells that were positive for iNOS showed a cytoplasmic staining pattern. Twenty-seven patients (38%) were positive for iNOS and 44 (62%) were negative. No significant prognostic correlation was observed between iNOS-positive and iNOS-negative patients.

CONCLUSIONS

PD-ECGF positivity in cancer cells is a predictive factor for a good prognosis in cervical squamous cell carcinoma treated with radiotherapy alone.

In this chapter we have described one of the more complex hemopoietic factors, M-CSF. The single-copy M-CSF gene is almost 21 kb in length and is arranged into 10 exons and 9 introns. Expression of the gene at the RNA level is heterogeneous, and several species of M-CSF mRNA have been found in human and murine cells and tissues. In human cells the different mRNAs arise from alternative splicing of the nuclear RNA precursor in both coding and noncoding regions. This results in mRNAs encoding two distinct M-CSF proteins, 256 and 554 amino acids in length. In murine cells only a 552-amino-acid form has been found thus far. All forms of M-CSF have a 32-amino-acid signal peptide and a 23-amino-acid hydrophobic region near the carboxy-terminus, which resembles a transmembrane domain. A large portion of the carboxy-terminal end, including the hydrophobic region, is not found in the mature protein. Thus, the primary translation product of M-CSF is a prepropolypeptide, with processing occurring at both amino- and carboxy-terminal ends. The exact size of the mature protein is still somewhat in doubt, but deletion mutagenesis from the carboxy-terminal end indicates that the protein may be as small as 150 amino acids and still be functional. Site-directed mutagenesis has also shown that the first seven cysteines in the mature molecule are probably necessary for biological activity, whereas the next two cysteine residues are not. In spite of the heavy glycosylation found in the native protein, removal of the N-linked glycosylation signals does not seem to affect activity to any great degree. The M-CSF gene and its receptor, C-FMS, are tightly linked on the long arm of chromosome 5, a unique finding in the ligand/receptor field. This region also contains the genes for GM-CSF, IL-3, ECGF, and the receptor for PDGF. A similar situation may exist on chromosome 11 of the mouse. The close linkage of these factors and receptors is the probable cause for the disorders of hemopoiesis that arise when deletions occur in this area. The preceding discussion has shown how quickly the area of M-CSF molecular biology has advanced in the past 2-3 years. A great deal of effort is now being directed toward expressing M-CSF at high levels in a variety of prokaryotic and eukaryotic systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-derived endothelial cell growth factor (PD-ECGF), identical to thymidine phosphorylase, has been reported as an angiogenic factor in human malignancies. However, the role of PD-ECGF in human hepatocellular carcinoma (HCC) is still unconfirmed. Herein, we studied the expression of PD-ECGF in 27 human HCC cases by immunohistochemistry, to clarify the relationship to tumor angiogenesis. The immunoreaction of PD-ECGF in HCC cells was scored in both the staining percentage and intensity. CD34, an endothelial cell marker, was used to evaluate the intratumoral microvessel density (IMVD). PD-ECGF expression was noted in carcinoma cells in 14 (51.9%) of 27 HCCs. In these cases, the carcinoma cells showed heterogeneous staining in both the nucleus and cytoplasm. Tumor-associated stroma cells and infiltrating lymphocytes were also stained. Kupffer cells in non-tumor areas were strongly positive. Statistically, the expression of PD-ECGF increased in HCC specimens with high Edmondson grades (III-IV) or portal vein tumor thrombosis (PVTT) (P<0.05). Additionally, the IMVD of PD-ECGF-positive HCC specimens (136.071+/-31.008, mean +/- SD) was higher than that of the PD-ECGF-negative HCC specimens (61.077+/-15.795) (P<0.05). These findings may suggest that PD-ECGF is one of the angiogenic factors in human HCCs. Furthermore, with the increasing expression of PD-ECGF, HCC cells show poor differentiation and invasive behavior.

The role of CD77, inflammatory cytokines and endothelial cell growth factor (ECGF) in verotoxin (VT)-induced apoptosis in human umbilical vein endothelial cells (HUVECs) was studied. Apoptosis was detected using annexin V and propidium iodide staining. The expression of CD77 antigen was measured on a FACStar flow cytometer using specific monoclonal antibodies. Our experiments showed that HUVECs had very low initial levels of CD77 and were resistant to VT. Treatment with tumour necrosis factor alpha (TNF-alpha) resulted in a significant upregulation of CD77 expression and sensitized endothelial cells to VT-mediated apoptosis. HUVECs incubated with a combination of cytokines [TNF-alpha and interferon gamma (IFN-gamma), both 500 U/ml] showed more pronounced upregulation of CD77 expression >> sixfold at 48 h) and underwent apoptosis, suggesting that TNF-alpha and IFN-gamma have a synergistic effect on CD77 expression in HUVECs and can induce apoptosis without VT. Cells pretreated with TNF-alpha and IFN-gamma and incubated with VT showed the most pronounced (14-fold) increase in CD77 expression. ECGF had a partial protective effect against cytokine- and VT-induced apoptosis. Taken together, our data suggest that CD77 antigen is involved in the regulation of endothelial cell apoptosis.

Serum platelet-derived endothelial cell growth factor (PD-ECGF) in patients with uterine cervical cancers revealed a significantly positive correlation with clinical stage and tumor size and with the advancement indicators lymph node metastasis, parametrial involvement, and vessel permeation in both squamous cell carcinomas and adenocarcinomas. The prognosis of the patients with high serum PD-ECGF was extremely poor, whereas the 36-month survival rate of the other patients with low serum PD-ECGF was 81.3% in squamous cell carcinomas and 80.0% in adenocarcinomas. Our data indicate that serum PD-ECGF levels reflect the status of advancement of uterine cervical cancers and thus may be recognized as a novel tumor marker for both squamous cell carcinomas and adenocarcinomas of the uterine cervix.

OBJECTIVE

Comedo carcinoma is generally regarded as the subtype of ductal carcinoma in situ (DCIS) most likely to progress to invasive carcinoma. Increased angiogenesis could be associated with an enhanced risk of progression and might therefore be a marker of poor prognosis, as can be demonstrated for invasive breast tumours. Therefore, the present study investigates the correlations between the expression of oncoproteins (HER2, HER1/EGFR), angiogenic growth factors (VEGF and PD-ECGF/TP) and microvessel density (MVD) in DCIS.

RESULTS

Forty-six breast cancer specimens of DCIS were tested immunohistochemically for the expression of angiogenic factors and oncoproteins. Different vascular distribution patterns of DCIS were examined semiquantitatively. Our results showed a significantly inverse correlation between HER1/EGFR and comedo-type DCIS (P = 0.048), but HER1/EGFR expression seemed to be independent of HER2 overexpression. VEGF expression was significantly associated with endoglin expression (P = 0.031) and the cuffing phenomenon (P = 0.017).

CONCLUSIONS

The significantly inverse correlation between HER1/EGFR and comedo-type DCIS and the observation that VEGF and the other angiogenic factors tested are independent of HER2 overexpression, suggest that progression of comedo-type DCIS and angiogenesis in breast carcinoma are not regulated via the HER1/EGFR or HER2 pathway.

Human umbilical vein endothelial cells (HUVEC) are inducible for tissue factor (TF) activity in culture. Based on experiments using ECGF (4-20 micrograms/ml) with heparin (90 micrograms/ml), we obtained the following results: 1) In confluent HUVEC cultures, ECGF had essentially no influence on the levels of inducible TF. 2) In growing HUVEC cultures, ECGF reduced the TF response shortly after seeding but full response was regained when cells were kept confluent for 2-3 days. 3) Although secondary cultures responded best to TF induction in the absence of ECGF, the response was essentially equal over at least 8 passages in the presence of ECGF. 4) Of total cellular TF induced in HUVEC, about 25% was available on the surface, and less than 4% was released with the shed plasma membrane vesicles. The proportion of total TF activity available on the surface of intact cells was not influenced by the presence of ECGF. 5) T1/2 for the decay of TF activity induced was 8.3-9.5 h, whereas in HUVEC when protein synthesis was blocked after TF induction a T1/2 of about 30 h was found.

Since the galactose-fed dog is an animal model that develops the advanced stage of proliferative retinopathy, the effects of vascular endothelial growth factor (VEGF) on cell growth, receptor expression and the activation of mitogen-activated protein (MAP) kinase pathway of dog retinal capillary endothelial cells were investigated. Dog retinal endothelial cells were cultured at 37 degrees C under 5% carbon dioxide atmosphere in CS-C medium supplemented with endothelial cell growth factor (ECGF). VEGF receptor expression was examined by RT-PCR, and activation of MAP kinase was examined with antibody against phospho-Elk-1 (Ser383). When growth factors were removed from the culture medium, cell survival of dog endothelial cells was significantly reduced. Addition of VEGF protected these cells from cell death induced by growth factor starvation. VEGF also enhanced tube formation in dog endothelial cells and increased the expression of two VEGF receptors, Flt-1 and KDR/Flk-1. Cells treated with VEGF also displayed the phosphorylation of the transcription factor, Elk-1. Addition of the tyrosine kinase inhibitor, genistein, eliminated VEGF-induced cell growth and Elk-1 phosphorylation. These data confirm that cell growth and tube formation of dog retinal capillary endothelial cells are stimulated by VEGF. VEGF also increases the expression of the receptors, KDR and Flt-1, and activates the p44/42 MAP kinase pathway.

The rapid establishment of an endothelial cell (EC) monolayer on the luminal surface of small-diameter vascular grafts may be necessary to prevent early thrombosis and failure. We have studied procedures used to promote EC coverage of vascular grafts and have compared preclotting prosthetic surfaces with ECs in platelet-rich plasma (seeding) with plating ECs onto a preestablished clot (sodding). We evaluated the rate of monolayer formation, the subsequent resistance to shear stress, and the effects of EC growth factors (ECGF and heparin) on these functions. Woven Dacron was seeded or sodded at a density of 2 x 10(5) cells/cm2 with human adult microvessel ECs derived from adipose tissue. In the presence of ECGF-heparin, the immediate establishment of an EC layer after sodding was observed, whereas seeded grafts required almost 48 hours for cells to reach the surface. In the absence of ECGF-heparin, sodded grafts still exhibited a complete monolayer of EC, whereas ECs were not observed at the surface of seeded grafts after 48 hours. After exposure to shear stress (up to 20 dynes/cm2) for 2 hours, most freshly sodded EC remained attached; however, the loss of loosely adherent cells did occur. EC seeded grafts remained covered with fibrin matrix after exposure to shear stress. We conclude that the use of a microvessel sodding technique as an alternative to previously reported seeding techniques is necessary for the immediate formation of an EC monolayer before implantation.

Angiogenesis contributes to the growth and secondary spreading of solid tumours. Platelet-derived endothelial cell growth factor (PD-ECGF) is identified as such an angiogenic factor. In the present study, the prognosis of the patients with high PD-ECGF uterine cervical cancers was worse than those with low PD-ECGF cancers, and PD-ECGF expression correlated with cellular proliferation and with vascular density and venous invasion in uterine cervical cancers. Therefore, PD-ECGF might contribute to the growth of uterine cervical cancers via angiogenesis related to vascular spreading. Furthermore, PD-ECGF and its mRNA had a wide range and were highly expressed in uterine cervical cancers, especially squamous cell carcinoma, regardless of clinical stage. Therefore, PD-ECGF in uterine cervical cancers might play a role of basic angiogenesis in all processes of advancing of uterine cervical cancers. This indicates that 5'-deoxy-5-fluorouridine might be highly effective in squamous cell carcinoma of the cervix, which possesses a high activity of thymidine phosphorylase to convert 5'-deoxy-5-fluorouridine to 5-fluorouracil, and that some angiogenic inhibitors of new capillary formation might be effective in the inhibition of tumour growth and spreading associated with angiogenesis.

Minoxidil directly promotes hair growth via the stimulation of dermal papilla (DP) and epithelial cells. Alternatively, there is little evidence for indirect promotion of hair growth via stimulation of adipose-derived stem cells (ASCs). We investigated whether minoxidil stimulates ASCs and if increased growth factor secretion by ASCs facilitates minoxidil-induced hair growth. Telogen-to-anagen induction was examined in mice. Cultured DP cells and vibrissae hair follicle organ cultures were used to further examine the underlying mechanisms. Subcutaneous injection of minoxidil-treated ASCs accelerated telogen-to-anagen transition in mice, and increased hair weight at day 14 post-injection. Minoxidil did not alter ASC proliferation, but increased migration and tube formation. Minoxidil also increased the secretion of growth factors from ASCs, including chemokine (C-X-C motif) ligand 1 (CXCL1), platelet-derived endothelial cell growth factor (PD-ECGF), and platelet-derived growth factor-C (PDGF-C). Minoxidil increased extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation, and concomitant upregulation of PD-ECGF and PDGF-C mRNA levels were attenuated by an ERK inhibitor. Subcutaneous injection of CXCL1, PD-ECGF, or PDGF-C enhanced anagen induction in mice, and both CXCL1 and PDGF-C increased hair length in ex vivo organ culture. Treatment with CXCL1, PD-ECGF, or PDGF-C also increased the proliferation index in DP cells. Finally, topical application of CXCL1, PD-ECGF, or PDGF-C with 2% minoxidil enhanced anagen induction when compared to minoxidil alone. Minoxidil stimulates ASC motility and increases paracrine growth factor signaling. Minoxidil-stimulated secretion of growth factors by ASCs may enhance hair growth by promoting DP proliferation. Therefore, minoxidil can be used as an ASC preconditioning agent for hair regeneration.

BACKGROUND

PD-ECGF/TP is an essential enzyme in converting 5'DFUR and 5FU to their active metabolites and can be up-regulated by some cytokines.

METHODS

PD-ECGF/TP mRNA and protein expressions were determined by RT-PCR and Western blot, respectively. The cytotoxicity of 5FU, 5'DFUR or MMC against RT-4 and T24 cells was evaluated by MTS assay. The PD-ECGF/TP expressions in primary bladder cancers were also analyzed.

RESULTS

Levels of PD-ECGF/TP mRNA and protein were concomitantly elevated in RT-4 and T24 cells after IFN gamma treatment. IFN gamma decreased the IC50 of 5FU and 5'DFUR in both cell lines, while it did not alter the IC50 of MMC, which is not a substrate of PD-ECGF/TP. PD-ECGF/TP expression correlated with tumor stage and grade in primary bladder cancers.

CONCLUSIONS

IFN gamma enhances the cytotoxicity of 5FU and 5'DFUR against human bladder cancer cells through induction of PD-ECGF/TP. The results imply that an IFN gamma/5FU or IFN gamma/5'DFUR combination therapy may be applicable to clinical bladder cancers.