We evaluated the expression of molecular markers in colorectal adenocarcinoma in relation to p53 protein expression. Tissue samples of 54 patients with colorectal adenocarcinoma were obtained at surgery at university hospitals in the years 2000-2003. These were analyzed by immunohistochemical techniques using primary antibodies for p53, Bcl-2, P-gp, topoisomerase II alpha and Thymidylate Synthase (TS), thymidine phosphorylase/PD- ECGF (TP) and LSAB detection kit. The highest prevalence of expression among six analyzed markers were P-gp and p53 with 77% expression and the lowest one was Topo II with 35% expression. No clinicopathological significance was recorded in colorectal cancer patients. Several immunophenotypes were observed between p53 and other molecular markers. Additionally the prevalence of lack of expression of Bcl-2, Topo II and TS was higher in p53+ tumors than in p53-tumors. A significant association (p = 0.021) existed between p53/Bcl-2 coexpression and mean age of patients (63.5 [10.1]y vs. 52.3 [15.2] y) and between p53/TP coexpression and sex (66.7% male; (p = 0.022). Overexpression of mutated p53 seen in tumor samples may alter the expression pattern of other molecular markers that are predictors of tumor response to chemotherapy regimens. Age and sex of patients could also affect the p53 related proteins such as Bcl-2 and TP, which can affect therapeutic outcome and disease prognosis. These findings emphasize the importance of tumor immunophenotypes as valuable prognostic or predictive markers in clinical settings.

BACKGROUND

Vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) and leptin are known as potent angiogenic factors The objective of the study was to evaluate these angiogenic factors VEGF, PD-ECGF/TP and leptin in children with congenital heart disease (CHD) and the factors that lead to angiogenesis in such cases.

METHODS

Sixty CHD children were studied and divided into two groups (n=30); cyanotic-CHD (C-CHD) and acyanotic-CHD (A-CHD). Twenty five healthy children were included as controls.

RESULTS

Significantly higher serum levels of VEGF, PD-ECGF/TP activity and leptin were detected in patients with CHD, particularly in patients with C-CHD. CHD patients with SpO2<90%, pulmonary hypertension (PH), severe pulmonary stenosis (PS), detectable collaterals, cardiomegaly and/or heart failure showed significantly higher levels of these factors than those with higher SpO2 or those without these findings.

CONCLUSIONS

Hypoxia, PH and PS are important factors that lead to harmful angiogenesis. However, angiogenesis could be essential in some cases of CHD as coarctation of aorta to enhance renal perfusion. This may provide new ways for therapeutic strategies aiming at reducing or promoting angiogenesis in CHD to improve patient's outcome.

To clarify biological and clinical significance of tumor angiogenesis in the development of ovarian carcinoma, we investigated the relationship between tumor vascularity, the expression of thymidine phosphorylase (dThdPase), which is an angiogenic factor identical to platelet-derived endothelial cell growth factor (PD-ECGF), and patient outcome in ovarian carcinoma, including serous surface papillary carcinoma (SSPC). Primary tumor specimens (stages I-IV) from 54 patients were examined. Intratumoral microvessel density (IMVD) and dThdPase expression were evaluated immunohistochemically using anti-CD34 and anti-dThdPase antibodies, and results were correlated with clinicopathologic parameters and prognosis. IMVD for the 54 tumors ranged from 22.5 to 120.7 (number/0.73686 mm2/field). Twenty-three tumors were positive, and 31 tumors were negative for dThdPase expression. IMVD positively correlated with the expression of dThdPase (p < 0.01), tumor size, and peritoneal metastases (p < 0.05). However, there was no statistical correlation between IMVD, dThdPase expression, and clinical outcome. Of the 54 patients examined, 30 were diagnosed with International Federation of Gynecology and Obstetrics (FIGO) stage III or IV primary ovarian carcinoma, and 9 were diagnosed with SSPC. There were no significant differences between the two groups with respect to clinicopathologic features, IMVD, dThdPase expression, or patient outcome. In conclusion, angiogenic activity may be necessary for the growth of metastatic implants in ovarian carcinoma and SSPC.

Gliostatin (GLS)/Platelet-derived endothelial cell growth factor (PD-ECGF) is a protein factor that has angiogenic and thymidine phosphorylase activity. It has been recently demonstrated to be related to disease activity in rheumatoid arthritis. However, its physiological role in the gastric mucosa is unknown. In the present study, concentrations of this protein in human gastric mucosa and plasma were evaluated. Further, the effect of purified human GLS/PD-ECGF on experimental ulcer healing was investigated in the rat. The human plasma concentration of GLS/PD-ECGF was significantly higher in patients with intractable gastric ulcer than in patients with significant resolution. The tissue content was significantly higher at the gastric ulcer edge than in either the fundic or pyloric region. GLS/PD-ECGF infusion delayed ulcer healing in a dose-dependent manner. These results suggest that gastric tissue and/or circulating GLS/PD-ECGF may participate in pathology and etiology of gastric ulcers and that this mechanism may relate to the pathogenesis of RA.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45 kDa single chain polypeptide, which stimulates the DNA synthesis and chemotaxis of endothelial cells in vitro and angiogenesis in vivo. Purification from human platelets and cDNA cloning from a human placental cDNA library, revealed that PD-ECGF is a novel type of peptide without sequence similarity to hitherto known proteins. PD-ECGF is present in human platelets and placenta, and is produced by certain normal and transformed cultured cells; it lacks a hydrophobic leader sequence and most of the protein remains inside the producer cells. Analysis of PD-ECGF produced by cultured cells, revealed that it contains nucleotide(s) covalently bound to serine residues. The in vivo function of PD-ECGF is not known; its target cell specificity and tissue distribution suggest roles in angiogenesis of the placenta and in the maintenance of the integrity of the endothelial cell layer of blood vessels. PD-ECGF may have a clinical utility in the stimulation of wound healing and re-endothelialization of vessels.

Small cell carcinoma of the stomach has an aggressive feature, and the survival rate of the patients is poor. The purpose of this study was to determine the clinical course, and effects of histopathologic characteristics of specific tumors including DNA contents and immunohistochemical aspects in patients with small cell carcinoma of the stomach. Medical records of 8 patients who presented with small cell carcinoma of the stomach were retrospectively reviewed. Primary tumors were studied by flow cytometric analysis and immunohistochemical staining for the p53 protein, PCNA (proliferating cell nuclear antigen), factor VIII related antigen (specific for endothelial cells), VEGF (vascular endothelial growth factor) and PD-ECGF (platelet-derived endothelial cell growth factor). DNA aneuploid was observed in 4 cases. Staining for the p53 product was positive in 50% of all the cases. The average PCNA labeling rate (LR) was 71.3+/-9.9%. Positive VEGF expression was found in 7 tumors and positive PD-ECGF expression was found in all tumors. The estimated median survival was 252 days for all the patients. Liver metastases were observed in 4 of the 8 patients, however, surgery and chemotherapy have given us one long-term survivor (43 months). Higher PCNA LR of small cell carcinoma may be an unfavorable characteristic of biological behavior. Moreover, both VEGF and PD-ECGF positivity are well-characterized inducers of hepatic metastasis.

Unstimulated mononuclear cells express IGF-1, PDGF-A and PDGF-B mRNA, but not a number of other genes coding for growth factors or cytokines, as we demonstrated previously. The main focus of the present investigation was to compare gene expression of mononuclear cells unstimulated in suspension with gene expression of monocytes stimulated by adherence. mRNA levels of IGF-1-A and -B, PDGF-A, -B, PD-ECGF, basic FGF, acidic FGF, TGF-alpha, TGF-beta 1, and IGF-2 were sought for and quantified with our sensitive RT-PCR method (3n-PCR). The respective mRNAs of basic FGF, acidic FGF, TGF-alpha and IGF-2 were not detected, independent of the culture conditions. In suspension culture, mRNA levels of IGF-1A and -B, PDGF-A, -B, and CD18 remained unchanged. Monocyte adherence regulated IGF-1A, PDGF-A, and -B mRNA levels. In parallel, mRNA levels of the monocyte adhesion molecule CD18 increased rapidly (4.5-fold). In contrast, independent of the presence of an adherence stimulus, the mRNAs for the cytoskeletal structure protein beta-actin and PD-ECGF remained constant, whereas mRNA for growth factors TGF-beta 1 and IGF-1B, respectively, was increased. Thus, monocyte adherence selectively regulates IGF-1, PDGF-A, PDGF-B and CD18 mRNAs (adherence-responsive genes) in a coordinated manner. This led us to identify two novel consensus elements within their respective functional promoters. Both motifs, an 11 bp purine-rich sequence and a 13 bp pyrimidine-rich segment, respectively, are absent from the genes that were not specifically activated by adherence. The identified elements are potential binding sites for transcription factors that may define a common basis for the regulation of the adherence-responsive genes IGF-1A, PDGF-A, PDGF-B and CD18.

BACKGROUND

Pyrimidine nucleoside phosphorylase (PyNPase) is identical to the protein, platelet-derived endothelial cell growth factor (PD-ECGF), which has angiogenic activity. The physiological roles of PyNPase activity in the uterus and ovary are not known. In this study, we measured PyNPase activity in normal tissues of the uterus, ovary, and lymph nodes, and in benign and malignant lesions of these organs, and we considered the clinical implications of PyNPase activity in the uterus and ovary.

METHODS

Tissue samples were obtained from 163 patients (whose diseases are listed below) during surgery. PyNPase activity was measured spectrophotometrically, by monitoring the formation of 5-fluorouridine.

RESULTS

Mean PyNPase activity in tissues from the lesions of patients with cervical cancer (n = 20), uterine endometrial cancer (n = 26), leiomyoma (n = 23), ovarian cancer (n = 46), ovarian endometriosis (n = 21), and benign epithelial ovarian tumor (n = 27) was significantly greater than that in the corresponding normal tissues. The PyNPase activity in the normal endometrium was significantly higher in the secretory phase than in the proliferative phase. The activity in normal or metastatic lymph nodes was significantly greater than that in normal tissues of the uterus and ovary. Mean PyNPase activity in cancerous cervical tissues was significantly greater than that in cancerous endometrial tissues or cancerous ovarian tissues. There were no significant differences in PyNPase activity in cervical cancer, endometrial cancer, and ovarian cancer tissues according to tumor stage. The enzyme activity appeared to be greater in histopathological G3 grade endometrial cancer than in G1 and G2 endometrial cancer. The enzyme activity in mucinous adenocarcinoma of the ovary was significantly lower than that in serous, endometrioid, and clear cell adenocarcinomas. All patients with cervical squamous cell carcinomas with PyNPase activity greater than 500 nmol/min per mg protein exhibited lymph node metastasis.

CONCLUSIONS

Increased PyNPase activity consistently reflected neoplastic growth, and varying levels of activity were seen in different histologic cell types. This enzyme activity may be involved in cervical squamous cell carcinomas, in normal and metastatic lymph nodes, and in the normal, secretory phase in the endometrium.

Surgical debulking of malignant mesothelioma (MM) ultimately fails due to local recurrence. Suramin, an inhibitor of extracellular growth factors (ECGFs), has demonstrated efficacy in the treatment of malignant mesothelioma in a small case series. Our goal was to study survival benefits and disease progression in several MM animal models treated with suramin as a potential agent for adjuvant therapy. In vitro growth of human (REN, I-45), rat (II-45) and murine (AB12) mesothelioma cell lines were measured with or without suramin exposure. Human and murine MM tumors were implanted subcutaneously into murine flanks or injected intraperitoneally (i.p.) into murine abdominal cavities. Dose and treatment schedules were optimized to reduce the rate of tumor progression and to improve survival curves. Suramin inhibited the in vitro growth of all cell lines, reaching statistical significance (P<0.01) three doubling cycles after suramin administration, with a maximum inhibition of 10-25% of control growth. A significant time- and dose-dependent effect was observed. In vivo, suramin inhibited the growth of MM in the xenogeneic model (55% of control growth, P<0.01), and in the syngeneic model at both the low and high loading doses (46 and 36% of control growth, respectively, P<0.01). Suramin treatment inhibited in vivo growth in the REN intraperitoneal model shown grossly by necropsy of same day deaths comparing treatment and control animals. Tumor inhibition with the higher dose was also reflected by the lower mean tumor burden scores (control: 4.0 and high dose: 3.4). Suramin inhibits the growth of human, murine, and rat MM in vitro, in a time- and dose-dependent manner. Suramin also inhibits the growth of human MM flank and intraperitoneal xenografts in vivo in an immunodeficient host, as well as the growth of syngeneic murine flank tumors in an immunocompetent host. These studies demonstrate that suramin may have the potential to provide effective therapy for MM, and that further studies are necessary to elucidate the survival advantage of suramin mediated MM growth inhibition.

Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor which recently has been shown to be identical to thymidine phosphorylase. We describe here, high levels of expression of PD-ECGF/thymidine phosphorylase in neurons of the peripheral nervous system (PNS) but very little in the central nervous system (CNS). Monoclonal and polyclonal antibodies were used for the staining of sections of dorsal root ganglia, sympathetic cervical ganglia and the enteric plexus as well as the brain and spinal cord. In addition, in situ hybridisation confirmed the results of immunohistochemistry. The possible role of thymidine phosphorylase in the PNS is discussed.

We found that 5'-O-trityl-inosine (KIN59) inhibits recombinant bacterial (E. coli) and human thymidine phosphorylase (TPase) with an IC50 of 44 microM and 67 microM, respectively. In contrast to previously described TPase inhibitors, KIN59 does not compete with thymidine (dThd) at the pyrimidine nucleoside-binding site or with inorganic phosphate (Pi) at the phosphate-binding site of the enzyme. These findings are strongly suggestive for the presence of an allosteric binding site at the enzyme. TPase is identical to the angiogenic protein platelet-derived endothelial cell growth factor (PD-ECGF). As such, PD-ECGF stimulates angiogenesis in the chick chorioallantoic membrane (CAM) assay. This angiogenic response was completely inhibited by KIN59. Inosine did not inhibit the enzyme or the angiogenic effect of TPase, confirming that the 5'-O-trityl group in KIN59 is essential for the observed effect. Our observations indicate that allosteric sites in TPase may regulate its biological activity.

To improve current angiogenic gene therapy with a vascular endothelial growth factor (VEGF)-encoding plasmid (Baumgartner et al. Circulation 1998; 97: 1114-23 [1]; Kusumanto et al. Fifth Annual Meeting of the American Society of Gene Therapy, Boston, 2002, Abstr. 621 [2]), we have generated a combination plasmid, encoding the VEGF gene and the thymidine phosphorylase (TP, also known as platelet-derived endothelial growth factor (PD-ECGF) or gliostatin (GLS)) gene: phVEGF165-TP.MB. Upon transfection in COS-7 cells both gene products were expressed and functional as shown by Western blots, ELISAs and bioassays. Culture supernatants of COS-7 cells transfected with this plasmid were able to induce endothelial proliferation. In an in vitro angiogenesis assay with recombinant proteins, TP was able to increase VEGF-induced tube formation. The phVEGF165-TP.MB plasmid is therefore a promising candidate for in vivo angiogenesis studies.

Aluminum salts have been shown to stimulate 3H-thymidine incorporation in primary cultures of bovine brain microvessel endothelial cells (BMECs). Aluminum chloride or sulfate salts in concentrations between 0.01 to 100 microM were, in general, most effective in stimulation of thymidine uptake by BMECs with maximal effects observed after a 24 hour exposure to the metal. Concentrations of aluminum salts greater than 100 microM inhibited thymidine incorporation. Cell numbers were not affected by exposure to concentrations of the aluminum salts less than approximately 100 microM. Concentrations producing half-maximal stimulation of BMEC thymidine incorporation were approximately, 0.3 microM and 0.5 microM, for aluminum chloride and aluminum sulfate, respectively. These findings indicate that BMECs are sensitive to lower concentrations of aluminum salts than other mammalian cell types. Hydroxyurea completely inhibited thymidine incorporation into BMECs in the presence and absence of aluminum suggesting that thymidine incorporation into BMECs is representative of DNA synthesis. Endothelial cell growth factor (ECGF) stimulated both increased DNA synthesis and BMEC cell numbers in the primary culture system. Aluminum had only slight effects on DNA synthesis in endothelial cell growth factor stimulated BMECs. In contrast to ECGF, aluminum then, appears to provide a stimulus for DNA synthesis but not subsequent mitosis in BMECs. Results from this study are consistent with previous studies in other cell types and with current knowledge of the effects of aluminum on the blood-brain barrier in vivo.

Although a variety of angiogenic growth factors have been isolated, its appropriate in vivo delivery remains problematic due to nonspecific, uncontrolled delivery by conventional methods. We have investigated calcium alginate microbeads as a vehicle for the controlled slow-release of endothelial cell growth factor (ECGF). Three different microbead compositions, dependent on ECGF amount and alginate percentage were studied. Microbeads were incubated in a 1.5% calcium chloride solution and release of ECGF into solution was measured spectrophotometrically at specific timepoints. Our results show release rate and amount released after the first 2 hours are dependent on initial quick delivery of ECGF in the first 2 hours after which a sustained controlled release occurred for 4-5 days. Beyond this point, release at a slower rate was noted for at least approximately 2 weeks. Calcium alginate microbeads demonstrated a controlled and predictable rate of release and that the amount of ECGF delivered can be varied by varying the initial concentration of ECGF in the microbeads. Based on these observations we conclude that calcium alginate microbeads are a convenient and practical vehicle for sustained ECGF delivery.

OBJECTIVE

To investigate the presence of angiogenic factors in benign, premalignant and malignant vulvar lesions.

METHODS

Immunohistochemical demonstration of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in normal vulvar skin, lichen sclerosus, vulvar intraepithelial neoplasia (VIN) and vulvar cancer.

RESULTS

VEGF was found in the majority of vulvar cancers but only a minority of VIN lesions. PD-ECGF was found in the majority of lesions.

CONCLUSIONS

Demonstration of angiogenesis may suggest which preinvasive lesions will progress to invasive cancer.

BACKGROUND

Platelet-derived endothelial cell growth factor (PD-ECGF), whose expression is increased in several cancers, is an endothelial cell mitogen and has chemotactic activity in vitro and angiogenic activity in vivo. Tumors with high PD-ECGF expression tend to have frequent lymph node metastasis and are associated with poor outcome.

METHODS

We screened genes transduced by PD-ECGF transfection to the colon cancer cell line DLD-1 by using a complementary DNA microarray. Cell motility was evaluated by in vitro migration assay. Actin fiber polymerization was visualized by immunofluorescent detection of phalloidin.

RESULTS

Rho-associated coiled-coil domain kinase (ROCK1) was found to be significantly overexpressed in PD-ECGF transfectants compared with mock cells. PD-ECGF transfectants showed higher cell motility than mock cells. The parental cell, DLD-1, with recombinant PD-ECGF showed higher cell motility than that without recombinant PD-ECGF, in which motility was blocked by the neutralizing antibody of PD-ECGF or Y-27632, a specific inhibitor of ROCK1. Moreover, the actin fiber polymerization, which is a marker of activation of ROCK1, was higher in PD-ECGF transfectants than in mock cells.

CONCLUSIONS

PD-ECGF expression may be associated with cancer cell migration via activation of ROCK1. This may explain one mechanism by which tumors with high expression of PD-ECGF show aggressive behavior.

Angiogenesis is thought to play critical roles in local tumor growth and eventual metastasis. No studies have examined the expression of platelet-derived endothelial cell growth factor (PD-ECGF) in prostatic tissues. Prostatic tissues were obtained from 36 prostatic adenocarcinoma patients. We assessed the expression of PD-ECGF using ELISA and immunohistochemistry. The mean level of PD-ECGF in prostatic adenocarcinomas was higher than that in neighboring normal prostatic tissues in ELISA. Immunohistochemistry showed that the expressions of PD-ECGF, which were associated with increase of microvessel count, were found in the endothelial cells, macrophages, lymphocytes, or fibroblasts. These results suggest that PD-ECGF is involved in the development of prostatic adenocarcinoma.

OBJECTIVE

To investigate the expression rates of the three angiogenic factors-basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet-derived endothelial cell growth factor (PD-ECGF) in hepatocellular carcinoma (HCC) and to investigate the relation of their expression rates to the microvessel density (MVD) of the tumor and to the formation of portal vein tumor thrombus (PVTT).

METHODS

Immunohistochemical staining was conducted on 61specimens of hepatic cell carcinoma removed in operation.

RESULTS

The expression rates of dFGF, VEGF, and PD-ECGF in HCC tissue were 45.9%, 75.4% and 70.5% respectively. The incidence rates of PVTT while all of the 3 angiogenic factor were negatively expressed, one of them was positively expressed, two of them were positively expressed, and all of them were positively expressed were 16.7%, 25.0%, 54.2% and 63.2% respectively (P < 0.05, test for trend). The MVD counts in HCC tissue in the above-mentioned four conditions were 99 +/- 56, 113 +/- 27, 140 +/- 347 and 194 +/- 52 respectively (P < 0.001, ANOVA test).

CONCLUSIONS

As the number of positively expressed angiogenic factors in HCC tissue increases, the angiogenic capability of the tumor enhances greatly, and the incidence rate of PVTT increases too.

We investigated the clinical significance of platelet-derived endothelial cell growth factor (PD-ECGF) as measured by enzyme-linked immunosorbent assay in primary epithelial ovarian cancers (EOC), finding amounts to be significantly greater in cancers than in normal ovarian tissue (p<0.01). PD-ECGF was significantly more abundant in stages III and IV than in lower stages (p<0.05), and also was high in tumors with macroscopically evident metastases in the peritoneal cavity (p<0.05), or pelvic (p<0.01) or paraaortic (p<0.01) lymph node metastases. Further, PD-ECGF was significantly lower in mucinous than in serous adenocarcinomas (p<0.05). No significant correlation was seen between PD-ECGF and histologic grade, maximum intraperitoneal metastatic tumor diameter (<2 vs.>2 cm), or presence of demonstrable malignant cells in peritoneal fluid. In stage III disease, PD-ECGF exhibited significant correlation with recurrence (p<0.05). Our data suggested that results of PD-ECGF assays in primary tumors can predict progression and recurrence of EOC.

Tumour associated neovascularisation is a complex interplay between inhibitory and stimulatory angiogenic factors. Despite intense research in this field, little is known about the interaction between endothelial and chemoresistant cancer cells. For this purpose, we assessed the impact of cellular supernatants of the primary adenocarcinoma cell line CCL228, its lymph node metastasis CCL227, and four subclones resistant to different levels of 5-fluorouracil on the growth of microvascular and macrovascular endothelial cells. The growth of endothelial cells in vitro was affected to a moderate degree by supernatants from colon cancer cell lines. This effect was independent of the degree of chemoresistance. The stimulation of endothelial cells by the growth factors VEGF, bFGF, and PD-ECGF in the presence of supernatants from cancer cell lines was generally higher in macrovascular endothelial cells when compared with microvascular cells. The secretion of VEGF from colon cancer cells in vitro was inversely related to the degree of chemoresistance with the low chemoresistance phenotype producing VEGF 8.7-fold higher than the high resistance subclone. With a maximal secretion of 1500 pg VEGF/ml cell supernatant, the concentration necessary to directly stimulate the growth of endothelial cells was not achieved. In conclusion, chemoresistance affects the interaction between colon cancer cells and endothelial cells dependant on the endothelial cell type. Although the level of chemoresistance has a profound impact on the production of VEGF by cancer cells with low, intermediate or high resistance, it does not differentially affect growth stimulation or inhibition of endothelial cells in vitro.

Prostacyclin (PGI2) synthesis in human vascular tissue is mainly regulated by the activity of enzymes that metabolize arachidonic acid, and especially by the activity of cyclooxygenase. The activity of this enzyme depends upon the balance between its synthesis and its inactivation by peroxidated metabolites or drugs. Cyclooxygenase in vascular endothelial cells is strongly inhibited by aspirin, but is rapidly resynthesized. The resynthesis proceeds more rapidly in endothelium than in smooth muscle cells. Growth factors for vascular cells strongly influence cyclooxygenase production and activity. PDGF both releases arachidonate and stimulates cyclooxygenase synthesis in fibroblasts and smooth muscle cells, and ECGF enhances new enzyme synthesis. By contrast, we find that the endothelial growth factor depresses PGI2 synthesis in human endothelial cells in a time and concentration-dependent manner. This inhibitory effect is potentiated by heparin. The inhibition correlates with a decrease in the content of immunoreactive cyclooxygenase within the endothelial cells when they are grown in the presence of ECGF and heparin. These findings suggest that proliferation selectively suppresses the function of prostaglandin synthesis in endothelial cells.

Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 micrograms/ml) and endothelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No change in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of heparin on EC FN synthesis was independent of whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cells. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-translationally regulated.

The fibroblast growth factors (FGF), including endothelial cell growth factor (ECGF)/acidic FGF and basic FGF, are important modulators of endothelial cell replication in vitro and in vivo. Premature infants and adults with lung injuries are often treated with high levels of inspired O2, which can be necessary for survival but potentially injurious to developing lungs and in tissue repair following injury. Human umbilical artery and vein endothelial cells were grown in ECGF- or FGF-supplemented Medium 199 and exposed to ambient levels of O2 from 10 to 95%. Endothelial cell growth, measured by [3H]thymidine incorporation, was inhibited by increasing levels of O2 and ceased above 50% O2. Vein endothelial cells could recover from up to 24 h of hyperoxic exposure when given fresh medium, but not after 48 h. Artery-derived cells were more sensitive to O2 than were vein-derived cells. Complete medium without endothelial cells, preincubated 24 h in 95% O2, lost its ability to support cell growth under normoxic conditions. Exposing individual medium components to high O2 demonstrated that purified natural ECGF and recombinant acidic or basic FGF were all inactivated by O2. Human recombinant superoxide dismutase prevented FGF inactivation. O2 inactivation of essential growth factors could thus have major consequences for lung development or repair of injured capillaries in infants or adults inspiring high levels of O2.

A full-length cDNA encoding the porcine monocyte chemoattractant protein-1 (pMCPC-1) was isolated from growth-stimulated porcine cerebral capillary endothelial cells (cEC); the pMCP-1 cDNA showed 89% identity to human MCP-1 and was isolated by use of subtractive hybridization and differential screening of two phenotypically different sub-populations of cloned cEC. pMCP-1 was abundantly expressed in cEC grown in the presence of FCS, ECGF and heparin whereas lower expression was observed in cEC kept in FCS-supplemented medium only. As shown by Northern blot analysis, no pMCP-1 transcripts were present in total RNA derived from freshly isolated brain capillaries, large brain vessels or whole brain homogenate. MCP/JE expression was also demonstrated in ECGF/heparin-treated murine cEC. Astrocytes and smooth muscle cells grown in FCS-supplemented medium did not show MCP-1 expression. Treatment of porcine cEC with TNF-alpha increased pMCP-1 mRNA levels in a dose-dependent manner. These data further support the notion that cerebral capillary endothelial cells actively participate in processes of CNS host defense.