OBJECTIVE

The aim of this study was to evaluate the platelet-derived endothelial cell growth factor (PD-ECGF) and VEGF expressions of tumor cells as prognostic factors for radiotherapy outcome in patients with adenocarcinoma of the uterine cervix.

METHODS

In 47 formalin fixed, paraffin-embedded tissues from adenocarcinoma of the uterine cervix which had been treated with radiation (1970-1995), PD-ECGF and VEGF expressions were determined using immunohistochemistry, and the relationships between PD-ECGF or VEGF expressions and local control or survival were assessed.

RESULTS

PD-ECGF and VEGF expressions were successfully detected in the cytoplasm and/or nucleus of adenocarcinoma cells of the uterine cervix. Of the 47 patients, 44.6 (21/47 cases) and 57.4% (27/47 cases) were positive for PD-ECGF and VEGF, respectively. There was no correlation between PD-ECGF or VEGF expressions and age, grade, or histologic subtypes. Stage and high expression of PD-ECGF showed a significant correlation to local control (P = 0.0025, P = 0.0057, respectively) and were significant independent prognostic factors for 5-year survival in multivariate analysis (P = 0.0039, P = 0.0032, respectively).

CONCLUSIONS

This study demonstrated that PD-ECGF expression was a significant prognostic factor for radiotherapy outcome in patients with adenocarcinoma of the uterine cervix. Preradiation assessment of PD-ECGF expression may be helpful in selecting high-risk patients, providing them with opportunities to receive more sophisticated and individualized treatments.

OBJECTIVE

To investigate the expression of platelet-derived endothelial cell growth factor (PD-ECGF) in liver cancer and explore its related intervention with Capecitabine for the growth and metastasis of liver cancer.

METHODS

The protein level of PD-ECGF was determined using immunohistochemical method in 61 HCC samples and the mRNA level was detected using Northern blot analysis. Capecitabine was administered orally in 24 nude mice bearing LCI-D20 tumor. All treatments lasted 3 weeks. The tumor size was calculated by the following formula: V = a x b2 x 0.5. Lung metastasis was evaluated by HE staining in lung samples.

RESULTS

The PD-ECGF expression rate in 61 HCC and paratumoral liver tissues was 70.5% and 47.5%, respectively. The rate was higher in HCC tissues with advanced TNM stage than in those with early TNM stage (81.1% vs 54.2%, P < 0.05). The mRNA level of PD-ECGF was related well to its protein expression. The tumor size on day 3 after last treatment measured in the control, and low dose (1.05 mmol.kg-1.day-1), moderate dose (2.10 mmol.kg-1.day-1) and high dose (3.15 mmol.kg-1.day-1) of Capecitabine treatment groups was 447 mm3 +/- 159 mm3, 414 mm3 +/- 97 mm3, 240 mm3 +/- 119 mm3 and 209 mm3 +/- 150 mm3, respectively. High doses of Capecitabine increased the inhibitory effect on the growth and lung metastasis of HCC implant.

CONCLUSIONS

PD-ECGF is highly expressed in HCC and correlates with the TNM staging. Capecitabine may inhibit the growth and metastasis of HCC.

Platelet-derived endothelial cell growth factor (PD-ECGF) has been identified to be an angiogenic factor, and a close relationship between the expression of PD-ECGF and tumor development has been postulated. This study was designed to assess both the role of PD-ECGF in human colorectal polyps as well as its relationship to the expression of other oncogenes during colorectal carcinogenesis. One hundred twenty patients with colon polyps who had undergone a polypectomy were studied. The polyps were classified based on the pathological findings as nonneoplastic or sporadic adenoma. The polyps were immunostained for PD-ECGF and vascular endothelial cell growth factor (VEGF), as well as for Ki-67 antigen and p53. The correlations between expression of PD-ECGF and clinicopathologic factors were examined. PD-ECGF was expressed at significant levels only in adenomas: in 4 of the 20 polyps with severe dysplasia (20%), and in 5 of the 20 cases of carcinoma in adenoma (25%). PD-ECGF was not detected in the nonneoplastic polyps and in adenomas with low-grade dysplasia. The intensity of immunostaining for PD-ECGF in adenomas correlated with the expression of Ki-67 antigen (P < 0.001) but not with that of p53. VEGF was not detected in any types of polyps. Angiogenic factors in colorectal adenomas might play an important role in carcinogenesis. The correlated expression of PD-ECGF and Ki-67 antigen suggests that PD-ECGF might not only act as an angiogenic factor, but also as a tumor growth factor.

Expression of various endothelial growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), Platelet-derived endothelial cell growth factor (PD-ECGF) and hepatocyte growth factor (HGF) was investigated in human breast carcinoma tissues, and the results were compared to the intratumoral microvessel density evaluated by the immunostaining to anti-factor VIII related antigen. VEGF and PD-ECGF were examined by immunostainings, and bFGF and HGF were assessed by enzymatic immunoassays. As a result, VEGF and PD-ECGF were significantly associated with the increment of microvessel density, although no significant correlation was found with bFGF and HGF. In addition, interestingly, a tendency of co-expression between VEGF and PD-ECGF was demonstrated. It was suggested that VEGF and PD-ECGF play important roles in the promotion of angiogenesis in human breast cancer.

Endothelial cell growth factor (ECGF) stimulates the synthesis of t-PA and u-PA by confluent, diploid human lung fibroblasts, and this activity is potentiated considerably by heparin. In contrast, the malignant cell lines, Bowes melonoma and CALU-3, producers of t-PA and u-PA, respectively, are insensitive to ECGF. Studies with metabolic inhibitors and direct measurements of PA-specific mRNAs show that ECGF-mediated production of PA by human lung fibroblasts is dependent on de novo protein and RNA synthesis. The mechanism by which heparin potentiates this effect is thought to reside in its ability to prolong or strengthen the interaction of ECGF with cell surface receptors. The results raise the possibility that endogenous ECGF or related polypeptides (and heparin) may act to regulate PA synthesis by lung fibroblasts and possibly other responsive target cells in vivo.

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).

Myofibroblasts (Mfs) from rat fat tissues produced a potent endothelial cell growth factor (Mf-ECGF). The growth factor activity found in the conditioned media from primary cultures of Mfs, was labile to heat (80 degrees C for 10 min) and proteinase (trypsin), and did not bind to heparin in the presence of 0.2 M NaCl. Mf-ECGF was partially purified 4760-fold with a recovery of 25% from serum-free conditioned media by sequential carboxymethyl (CM) ion-exchange column chromatography and gel filtration. This Mf-ECGF activity was recovered from the 40 kD region of a non-reducing SDS-PAGE, and from the pH region between 6.5 and 7 of isoelectric focusing, with recoveries of 20% and 65%, respectively. These results indicated that a major portion of ECGF activity in the conditioned media was clearly distinct from other well-known endothelial cell growth factors including fibroblast growth factors (FGFs).

The formation of new blood capillaries (angiogenesis) occurs in response to angiogenic factors released by either normal or tumoral cells. In the present study, we cultured human umbilical vein endothelial cells (HUVEC) on collagen gels and aimed to clarify the effects of cyclic nucleotides on angiogenesis induced by endothelial cell growth factor (ECGF). HUVEC invaded the underlying collagen matrix and formed tube-like structures when ECGF was added. ECGF (9.4 to 75 micrograms/ml) induced angiogenesis in a concentration-dependent manner; the effect reached a plateau at 75 micrograms/ml. Cyclic AMP (10(-3) M), dibutyryl cyclic AMP (10(-3) M), 8-bromo cyclic AMP (10(-5) M) and Sp-cAMPS (10(-3) M), a stimulator of cyclic AMP-dependent protein kinase, each significantly inhibited ECGF-induced angiogenesis by 64.2, 86.1, 46.5, 74.7%, respectively. Forskolin and cholera toxin, which are activators of adenylate cyclase, did not inhibit ECGF-induced angiogenesis. Dibutyryl cyclic GMP (10(-4), 10(-3) M) also did not affect the formation of capillary-like tubes induced by ECGF. In conclusion, cyclic AMP, but not cyclic GMP, inhibits angiogenesis in vitro. This antiangiogenic activity may be applicable to the treatment of such conditions as solid tumors, diabetic retinopathy and rheumatoid arthritis in which the suppression of angiogenesis is important.

Protein expression patterns of morphologically different cloned capillary endothelial cells from porcine and murine brain cortices were examined. Type I cells, grown in medium containing heparin and endothelial cell growth factor (ECGF), exhibited a polygonal, cobblestone appearance and appeared to replicate the cells of the blood-brain barrier endothelium. Type II cells, grown in medium without heparin and ECGF, were elongated and appeared to replicate capillaries in central nervous system tissue. Cells of both phenotypes stained positive by the specific endothelial cell marker Bandeiraea simplicifolia lectin. The expression of alpha smooth-muscle actin (mRNA and protein) was taken as a marker for type II cells. By use of 2-D gel images and the GELLAB II system, a data base was created revealing that two proteins (90 kDa, pI 5.1, and 35 kDa, pI 5.7) were exclusively expressed in type I cells. Furthermore, the synergistic action of ECGF and heparin in respect to the phenotypic determination of cerebral endothelial cells was demonstrated.

OBJECTIVE

Platelet-derived endothelial cell growth factor (PD-ECGF) is one of the angiogenic factors. The aim of this study was to examine the PD-ECGF concentrations in hepatocellular carcinoma, background liver, and normal liver tissues, and to elucidate their significance on clinicopathological outcomes.

METHODS

The concentration of PD-ECGF in the tissue extract was determined by enzyme-linked immunosorbent assay.

RESULTS

PD-ECGF concentrations were significantly higher in hepatocellular carcinoma and background liver tissues compared with normal control liver (p = 0.003, p = 0.001, respectively). PD-ECGF concentrations in hepatocellular carcinoma tissues were positively correlated with intratumoral arteriole densities (r = 0.667, p = 0.009), and were higher in less differentiated carcinomas (p = 0.039). However, tumor PD-ECGF concentration did not affect the patients' disease-free survival rates. Those in the background liver tissues were positively correlated with histological activity index scores (r = 0.650, p = 0.001) and serum alanine aminotransferase levels (r = 0.0452, p = 0.035).

CONCLUSIONS

PD-ECGF is up-regulated in hepatocellular carcinoma and the corresponding hepatitis liver. The PD-ECGF concentrations in hepatocellular carcinoma correlated positively with microvessel density, lower differentiation, yet not with patients' prognosis. The concentrations of PD-ECGF in the corresponding hepatitis liver correlated positively with the degree of active hepatitis.

BACKGROUND

Angiogenesis is essential for the continuous growth of tumour cells under unfavourable conditions in patients. Experimentally, platelet-derived endothelial cell growth factor (PD-ECGF) promotes tumour proliferation by stimulating angiogenesis. However, the clinical significance and regulating mechanism of its production in colorectal cancer are not well understood.

METHODS

The tissue concentration of PD-ECGF in colorectal neoplasm and normal mucosa was determined. The systemic oxygenation and nutritional status of the patients were also evaluated.

RESULTS

The mean concentration of PD-ECGF in the cancer was significantly higher than that in the normal mucosa or adenoma. The tissue concentration of PD-ECGF in the cancer was associated with the clinicopathologic findings, including the tumour size, serosal invasion, lymphatic vessel involvement, and lymph node metastasis. It was also correlated with the patient's age, levels of PO2 and O2 saturation in arterial blood, and the variables reflecting nutritional status. The multivariate regression model showed that the serum concentration of cholinesterase, the arterial level of PO2, lymph node metastasis, and the tumour size were the independent factors that influenced the tissue concentration of PD-ECGF in colorectal cancer. In contrast, these factors were not associated with the PD-ECGF concentration in normal mucosa.

CONCLUSIONS

PD-ECGF may play an important role in the progression of colorectal cancer. Systemic deterioration of oxygenation and nutritional condition in wasted patients may also lead to local activation of PD-ECGF specifically in the cancer tissue. PD-ECGF may be indispensable for maintaining relentless growth of colorectal cancer, and the control of its expression may be of therapeutic importance.

A series of epichlorohydrin-couple-linked cellulose-gelatin composite films (ECGF) was fabricated in NaOH/urea aqueous solution using a process involving homogenous blending, coupling, dialysis, self-dispersion, microgel solution casting, and evaporation. Their structure and properties were characterized with elemental analysis, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), scanning electron microscope (SEM), atomic force microscopy (AFM), ultraviolet-visible (UV-vis) spectroscopy, water vapor permeability, and mechanical testing. The results showed that the self-dispersed cellulose-gelatin microgels were successfully prepared and the coupling interactions existed in the inter- and intra-molecules of the corresponding composite films during the fabrication process. The water vapor permeability of the ECGF films was improved when the protein content was higher than 30 wt% in composite films at 75% relative humidity. Interestingly, compared with the cellulose/protein composite films prepared via phase separation method, ECGF films exhibited more homogeneous surface and compact cross-section structures, as well as presented higher light transmittance at 400 nm of about 88% and relative lower swelling ratio. Moreover, ECGF films displayed higher tensile strength compared with that of water-soluble cellulose derivatives/protein composite films in dry and wet states.

The anti-cancer drug, 5'-deoxy-5-fluorouridine (5'DFUR) is known to have antitumor activity through the conversion to 5FU by thymidine phosphorylase (TP). Recently, TP was demonstrated to be identical to angiogenic molecule platelet-derived endothelial cell growth factor (PD-ECGF) by cDNA cloning and subsequent biochemical analyses. We have examined the relationship between the clinical response of 5'DFUR and TP/PD-ECGF expression determined by immunocytochemical analysis in 24 recurrent breast cancer patients. Of 24, 13 were TP/PD-ECGF positive and 11 were TP/PD-ECGF negative. In 13 TP/PD-ECGF positive patients, 4 showed objective response (OR) and 3 showed stable disease (SD) with 5'DFUR treatment, however only one case showed OR and no case showed SD in 11 TP/PD-ECGF negative patients, suggesting that 5'DFUR was likely to be effective for TP/PD-ECGF positive patients. In another group of recurrent breast cancer patients treated by adriamycin containing regimen, no significant correlation was observed between the response of 5'DFUR and the status of TP/PD-ECGF expression. It was indicated that an angiogenic enzyme TP/PD-ECGF expression might be a predictor of the effect of 5'DFUR treatment in human breast cancer.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial-cell growth factor (PD-ECGF), is an enzyme that catalyzes the reversible dephosphorylation of thymidine, deoxyguridine and their analogs. TP also has angiogenic properties, although the precise mechanism by which it promotes angiogenesis is not known. TP expression is elevated in many solid tumors including ductal carcinoma in situ and invasive carcinoma of the breast. This has led to intensive study to ascertain whether TP is a biological marker in breast carcinoma; however, the clinical work has produced conflicting results. Some studies have suggested that TP is angiogenic in breast carcinoma, however, we and others have found that TP has little effect on tumor angiogenesis of invasive breast carcinoma. However, increasingly clinical results suggest that TP could represent an interesting marker that could respond to pyrimidine analogues. Widespread application of TP in prognostic testing would require greater uniformity in scoring techniques and determination of the cut-off levels which could distinguish individuals at high and low risk of cancer recurrence and death.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) is an enzyme involved in thymidine metabolism and homeostasis, and its catalytic activity appears to play an important role in angiogenesis. Here we describe the cloning and expression of a His-tagged human TP/PD-ECGF and its assay with uracil and thymine analogues. We present the design, synthesis, and biological evaluation of novel 6-(phenylalkylamino)uracil derivatives which, at micromolar concentrations, inhibit both catabolic and anabolic reactions of human TP in vitro. These base analogues are not converted by the enzyme into the nucleoside form, thus representing pure nonsubstrate inhibitors of the enzyme.

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF), an angiogenesis factor. We investigated the correlation between dThdPase activity in gastric cancer tissue and clinicopathological factors. Thirty-three cancer tissue specimens and 23 adjacent normal gastric mucosal specimens were obtained from surgery. Measurement of dThdPase activity was based on the amount of 5-fluorouracil formed from 5'-deoxy-5-fluorouridine by dThdPase. Mean dThdPase activity in cancer tissue was approximately 3.2 times higher than that in normal tissue. Cancerous tissues with venous invasion had about twice the dThdPase activity as cancerous tissues without venous invasion. Other clinicopathological features were not related to dThdPase activity. A correlation between dThdPase activity and immunosuppressive acidic protein level was observed (r = 0.532, P = 0.005). dThdPase activity in gastric cancer cells was found to be correlated with venous invasion, supporting previous findings that it plays a role in tumor angiogenesis.

Thymidine phosphorylase (dThdPase) is reportedly identical to platelet-derived endothelial cell growth factor (PD-ECGF). We conducted immunohistochemical staining of dThdPase to assess correlation between its expression in cancer tissue and efficacy of a combination therapy with 5'-DFUR, radiotherapy and sizofilan (SPG) in uterine cervical cancer patients. No difference in response rates was observed between dThdPase positive and negative tumor and stromal cells. Survival curves significantly differed between stromal dThdPase positive and negative groups (p=0.032). Results showed that dThdPase immunostaining is possibly prognostic and predictive in determining success of the combination therapy.

We investigated the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) in primary bladder cancer, its association with clinicopathologic findings, and their prognostic value. mRNA was extracted from 20 bladder cancer specimens and 6 normal bladder mucosal tissues. Relative amounts of PD-ECGF/TP mRNA were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared with the level of glyceraldehyde-3-phosphate dehydrogenase mRNA (used as an internal standard). PD-ECGF/TP expression was examined by immunohistochemistry in 85 patients who underwent cystectomy for bladder cancer. Serum PD-ECGF/TP levels were measured in 23 patients using a sandwich-type enzyme-linked immunosorbent assay. By RT-PCR analysis, expression of PD-ECGF/TP was found to be 7-fold higher in invasive tumors than in superficial tumors (P<0.01) and 9-fold higher than in normal bladder (P<0.01). Out of 85 transitional cell carcinoma tissue samples, 69 (81%) were evaluated as PD-ECGF/TP-positive by immunohistochemical staining. PD-ECGF/TP expression correlated significantly with tumor grade (P = 0.001), depth of invasion (P = 0.012), and lymphatic invasion (P = 0.01). No correlation was found between expression of PD-ECGF/TP and the number of tumors, tumor configuration, lymph node involvement, venous invasion, c-erbB-2 expression, or overall survival. We could not detect a significant serum level of PD-ECGF/TP in any patient. The results suggest that PD-ECGF/TP might give valuable information for bladder cancer management, though it may not be a good new tumor marker for bladder cancer.

Skin-cartilage composite grafts are invaluable tissues used in facial reconstruction, yet their survival is unpredictable beyond a 1-cm diameter. In this study, the angiogenic growth factors basic fibroblast growth factor (bFGF) and endothelial cell growth factor (ECGF) and a penetrance enhancer (dimethyl sulfoxide [DMSO]) were applied to composite grafts to determine their effects on survival and vascularization. We applied ECGF, bFGF, and DMSO either topically or by intradermal injection to 120 auricular composite grafts (3.0 cm diameter) in New Zealand White rabbits. Dermabrasion was performed in 2 groups to attempt to increase transdermal delivery. Graft viability and vascularity were evaluated 3 weeks later by template analysis and angiography. In the results, ECGF and bFGF, when grouped together, had a 40% increase in vascular ingrowth as compared to controls (p < .001). However, neither ECGF nor bFGF increased graft survival. A coincidental finding was that DMSO with dermabrasion significantly improved graft viability (>100%) with or without an angiogenic agent (p < .02). The potential of DMSO with dermabrasion to increase composite graft viability warrants further investigation.

Thymidine phosphorylase is an enzyme involved in pyrimidine salvage pathway that is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is enormously up regulated in a variety of solid tumors. Furthermore, surpassing of TP level protects tumor cells from apoptosis and helps cell survival. Thus TP is identified as a prime target for developing novel anticancer therapies. A new class of exceptionally potent isatin based oxadiazole (1-30) has been synthesized and evaluated for thymidine phosphorylase inhibitory potential. All analogs showed potent thymidine phosphorylase inhibition when compared with standard 7-Deazaxanthine, 7DX (IC50 = 38.68 ± 1.12 µM). Molecular docking study was performed in order to determine the binding interaction of these newly synthesized compounds, which revealed that these synthesized compounds established stronger hydrogen bonding network with active site of residues as compare to the standard compound 7DX.

Angiogenesis refers to the formation of new blood vessels from an existing vasculature and is recognised as a necessary requirement for most tumours to grow beyond 1-2 mm in diameter. Factors established as playing a role in angiogenesis may be divided into two principal groups: (a) those that stimulate endothelial cell proliferation and/or elongation, migration and vascular morphogenesis including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived endothelial cell growth factor (PD-ECGF) and the tie and tek receptors, and (b) proteases and their receptors involved in the breakdown of basement membranes and the extracellular matrix (ECM) including the matrix metalloproteinases (MMPs), cathepsins and those involved in the plasmin cascade. Angiogenesis has been identified as a potential target for development of anticancer agents. The discovery of a range of naturally-occurring factors which negatively regulate angiogenesis, including the thrombospondins, angiostatin and endostatin, and the tissue inhibitors of MMPs (TIMPs), has given added impetus to this approach. Synthetic anti-angiogenic compounds have been developed, including TNP-470, carboxyamidotriazole, VEGF-tyrosine kinase inhibitors and MMP inhibitors (MMPI) which, like the naturally-occurring anti-angiogenic factors, inhibit angiogenesis in vitro and in vivo, and tumour development, growth and metastasis in vivo. Anti-angiogenic agents also enhance the antitumour activity of many conventional cytotoxic chemotherapeutic agents. Such combinations may have a particular role as adjuvant therapies following surgical resection of primary tumours. Unlike tumour cells, tumour associated endothelial cells do not develop resistance to anti-angiogenic agents. Furthermore, anti-angiogenic agents are generally cytostatic rather than cytotoxic. As such, these agents are, in general, likely to be administered over long periods of time. Therefore, as well as having proven antitumour efficacy, an anti-angiogenic compound will need to be well-tolerated if it is to become established in the clinical management of patients with malignant disease.

Several N-phenylhomophthalimide derivatives were prepared and their inhibitory activity on thymidine phosphorylase/ platelet-derived endothelial cell growth factor (TP/PD-ECGF) was assessed. Among them, 2-(2,6-diethylphenyl)-7-nitro-1,2,3,4-tetrahydroisoquinoline-1,3-dione (9) was found to be a more potent inhibitor than the classical inhibitor, 5-nitrouracil (1). Lineweaver-Burk plot analysis indicated that 9 shows mixed-type competitive inhibition of TP/PD-ECGF, while 1 is a competitive inhibitor.

In examined the influence of endothelial cell growth factor (ECGF) withdrawal upon the growth and PGI2 production of human endothelial cells (HECs) maintained in either fetal bovine serum (FBS) or human serum (HS). For the study of growth, enzymatically harvested cells were passed twice, divided into seven groups and maintained in medium containing 20% FBS with ECGF (0, 0.1, 1, 10, 100 micrograms/ml) or 20% HS with ECGF (0, 100 micrograms/ml) (n = 8). Growth curve was generated from daily cell counts for ten days. Cells were dead in medium containing FBS with ECGF in concentration of 1 micrograms/ml or less, however, cells were sustained and grew in HS without ECGF. For the study of PGI2 production, harvested HECs were divided into 2 groups and cultured in medium containing 20% FBS or HS (ECGF: 100 micrograms/ml) (n = 12). Second passage cells were divided into 3 groups respectively and cultured in different ECGF concentration (0, 1, 100 micrograms/ml) for 48 hours. Cells were stimulated with thrombin(2U/ml) and PGI2 production was measured by radioimmunoassay. Cells cultured in medium containing HS produced more amount of PGI2 than in FBS in any concentration of BCGF (19 +/- 11 pg/10(5) cells in FBS v.s. 89 +/- 80 pg/10(5) cells in HS: p < 0.05, ECGF: 100 micrograms/ml) Second passage HECs cultured in medium containing HS were viable and maintained the ability of PGI2 production even without ECGF.

Since the half-life of most angiogenic growth factors is several hours or less, sustained-release delivery would be optimal for their future clinical use. Two fibroblast growth factors, basic fibroblast growth factor (bFGF) and endothelial cell growth factor (ECGF), were delivered in two sustained-released modalities (poloxamer 407 and a gelatin sponge [Gelfoam]) to attempt to increase soft tissue vascularity. In vitro bioactivity of ECGF-poloxamer formulations was also tested on endothelial cell cultures. Among vascular-compromised skin flaps in rabbits, ECGF-poloxamer (N = 26), bFGF-poloxamer (N = 5), ECGF-poloxamer (N = 9, irradiated), and bFGF-Gelfoam flaps (N = 22) did not demonstrate significant differences in viability and vascularity compared to controls (p>> .05). Irradiation had a detrimental effect on both flap vascularity and viability (p = .02). Future efforts for sustained delivery of angiogenic proteins are critical in order to make them clinically useful in wound healing.