OBJECTIVE

To correlate levels of angiogenic growth factors with Doppler ultrasound parameters in pregnancies complicated by pre-eclampsia and intrauterine growth restriction (IUGR).

METHODS

In 16 women with pre-eclampsia and 15 women with isolated IUGR, pulsatility indices (PI) in the umbilical and uterine arteries were measured by Doppler ultrasonography. At delivery, maternal and fetal blood (umbilical vein and artery separately) was sampled and angiogenic growth factors measured by means of enzyme linked immunosorbent assay (ELISA).

RESULTS

Umbilical artery PI was significantly higher in women with IUGR than in those with pre-eclampsia, whereas uterine artery PI was not statistically significantly different. Maternal soluble fms-like tyrosine kinase-1 (sFlt-1) levels were higher in women with pre-eclampsia than in those with IUGR (P < 0.0001). Umbilical vein basic fibroblast growth factor (bFGF) levels were lower in women with pre-eclampsia than in those with IUGR (P < 0.05). Placental growth factor (PlGF) levels in the umbilical vein were below the detection limit in nearly all samples of IUGR fetuses and lower than in those with pre-eclampsia (P < 0.001). Maternal PlGF levels were inversely correlated with PI values of both vessels. In the umbilical vein sFlt-1 was positively and soluble kinase insert domain receptor (sKDR) negatively correlated with umbilical artery PI. No correlation could be found in the serum of the umbilical artery for all growth factors and for vascular endothelial growth factor (VEGF) in all compartments.

CONCLUSIONS

The correlations between maternal and fetal angiogenic growth factor serum levels and Doppler ultrasound indices of uterine and umbilical arteries in pre-eclampsia and IUGR reflect the severity of the disorders especially for the fetus. A combination of both measurements may be useful in future screening for early prediction of pregnancy complications. Published by John Wiley & Sons, Ltd.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) is markedly stabilized by heparin. Partially due to the heterogeneity of heparin preparations, the nature of the aFGF polyanion binding site is still ill-defined. We have, therefore, investigated a wide variety of well-defined polyanions in terms of their ability to stabilize human recombinant aFGF (<em>15</em>-<em>15</em>4) against thermal denaturation. The specificity of the interaction between aFGF and polyanions is shown to be remarkably weak with a surprising number of polyanions (including small phosphorylated and sulfated compounds as well as highly charged biopolymers) able to induce physical stability. Temperature-dependent fluorescence and circular dichroism measurements show that many of these polyanionic compounds stabilize aFGF to the same extent as heparin. The ability of these agents to protect the three free thiol groups of aFGF from copper-catalyzed oxidation was also explored and significant protection was observed. The extent and electrostatic requirements of the protein's polyanion binding site were probed by the use of a series of well-defined heparin fragments and differentially phosphorylated inositol compounds. A tetrasaccharide fragment of heparin is the smallest unit of heparin capable of stabilizing aFGF against thermal denaturation. Increasing phosphorylation of inositol compounds (up to six phosphate groups per molecule) enhances the thermal stability of aFGF. These results are discussed in the context of a model of human aFGF based on the X-ray crystal structure of the bovine protein and previous studies by others of the heparin binding site of both acidic and basic FGF.

There are 600 million betel quid (BQ) chewers in the world. BQ chewing is a major etiologic <em>factor</em> of oral cancer. Areca nut (AN) and arecoline may inhibit the <em>growth</em> of oral mucosal <em>fibroblasts</em> (OMF) and keratinocytes. In this study, AN extract (100-800 microg/ml) and arecoline (20-120 microM) inhibited the <em>growth</em> of oral KB cells by 36-90 and <em>15</em>-75%, respectively. Exposure to arecoline >> 0.2 mM) for 24 h induced G(2)/M cell cycle arrest of OMF and KB cells. Areca nut extract >> 400 microg/ml) also induced G(2)/M arrest of KB cells, being preceded by S-phase arrest at 7-h of exposure. No evident sub-G(0)/G(1) peak was noted. Marked retraction and intracellular vacuoles formation of OMF and KB cells were observed. Glutathione (GSH) level, mitochondrial membrane potential (Deltabetam) and H(2)O(2) production of KB cells were measured by flow cytometry. GSH level [indicated by 5-chloromethyl-fluorescein (CMF) fluorescence] was depleted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 microg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1-1.2 mM) and AN extract (800-1200 microg/ml) induced decreasing and increasing H(2)O(2) production (by 2',7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of Deltabetam (increasing of rhodamine uptake) was noted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 microg/ml). AN extract (100- 1200 microg/ml) and arecoline (0.1-1.2 mM) induced little DNA fragmentation on KB cells within 24 h. These results indicate that AN ingredients are crucial in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer by differentially inducing the dysregulation of cell cycle control, Deltabetam, GSH level and intracellular H(2)O(2) production, these events being not coupled with cellular apoptosis.

Cutaneous infantile hemangioma progresses through proliferation and involution phases. Since treatment with interferon, a negative regulator of angiogenesis, accelerates the involution phase, we hypothesized that cutaneous infantile hemangioma is associated with an imbalance between endogenous positive and negative regulators of angiogenesis. We examined 30 specimens of cutaneous hemangioma [proliferative phase (n=<em>15</em>), involuting phase (n=8), and involuted phase (n=7)] and control human skin (n=17), fixed in formalin and embedded in paraffin. Routine histology, immunohistochemistry, and an mRNA in situ hybridization technique were used to measure expression of the positive angiogenic molecules basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em>/vascular permeability <em>factor</em> (VEGF/VPF), and an endogenous inhibitor of angiogenesis, interferon-beta (IFN-beta). Proliferative phase hemangiomas expressed high levels of bFGF and VEGF/VPF but not IFN-beta (mRNA and protein). The epidermis directly overlying proliferating hemangiomas was hyperplastic, contained numerous dividing cells, and expressed bFGF and VEGF/VPF but not IFN-beta. Epidermis from normal individuals and epidermis directly overlying involuted tumors or at sites distant to the proliferating hemangioma was not hyperplastic and expressed normal levels of bFGF, VEGF/VPF, and IFN-beta. These data suggest that the proliferation of cutaneous hemangiomas and adjacent epidermis is associated with an imbalance between positive and negative angiogenic <em>factors</em> expressed by the neoplasm and adjacent normal tissue.

OBJECTIVE

The objective was to study expression of alphav- and beta1-integrin subunits in effusions, primary tumors, and solid metastases of ovarian carcinoma patients, as well as to evaluate its potential association with previously studied metastasis-associated molecules and clinicopathologic parameters.

METHODS

Sections from 121 malignant effusions and 30 corresponding primary and metastatic lesions were evaluated for protein expression of the alphav- and beta1-integrin subunits using immunohistochemistry (IHC). A subset of effusions was additionally studied using immunoblotting (IB) and flow cytometry (FCM). mRNA in situ hybridization (ISH) was performed in 58 effusions and 30 biopsies.

RESULTS

Protein expression of alphav- and beta1-integrin subunits was detected in carcinoma cells in 116/121 (96%) and 113/121 (93%) effusions, respectively. alphav protein expression was limited to carcinoma cells. IB and FCM confirmed IHC results. mRNA for alphav- and beta1-integrin subunits was detected in carcinoma cells in 37/58 (64%) and 33/58 (57%) effusions, respectively. Both protein and mRNA expression were higher in peritoneal effusions, significantly for alphav mRNA (P = 0.042) and beta1 protein (P = 0.023). beta1 protein expression in effusions was more frequently detected in better-differentiated tumors (P = 0.006). alphav-integrin subunit expression correlated with that of the previously studied matrix metalloproteinase-9 (MMP-9) (P = 0.006) and the MMP inducer EMMPRIN (P = 0.001). Expression of beta1-integrin subunit showed an association with that of EMMPRIN (P = 0.029), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (P < 0.001), and the MMP inhibitor TIMP-2 (P = 0.025). In carcinoma cells of solid lesions, alphav protein was uniformly present, while beta1 expression was limited to <em>15</em>/30 (50%) specimens. As in effusions, protein expression of alphav subunit was cancer-specific, while beta1 protein was detected also in stromal <em>fibroblasts</em> and endothelial cells.

CONCLUSIONS

The alphav- and beta1-integrin subunits are frequently expressed in ovarian carcinoma cells in effusions, and the alphav-integrin subunit is a powerful diagnostic marker for carcinoma cells. The reduced expression of the beta1-integrin subunit in solid lesions may be attributed to the role of other subunits at these stages, such as the beta3 subunit as part of the alphavbeta3-vitronectin receptor. The high expression of integrin subunits with a role of binding mesothelium, invasion, and angiogenesis in carcinoma cells in both peritoneal and pleural effusions suggests that cells at both sites have metastatic potential.

SNT was originally described as a approximately 90 kilodalton protein in neuronal precursor cells which bears affinity for the yeast cell cycle protein p13sucl and which undergoes rapid tyrosine phosphorylation following stimulation with <em>growth</em> <em>factors</em> which trigger terminal differentiation, but not by other <em>growth</em> <em>factors</em> which promote proliferation (Rabin et al., 1993). We show here that similarly sized SNT-like proteins (SLPs) are expressed in <em>fibroblast</em>, myoblast, and lymphoid cell lines, and undergo robust tyrosine phosphorylation in response to several mitogenic ligands, including <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). SLPs are tyrosine phosphorylated within <em>15</em> s of FGF stimulation, are predominantly membrane-associated, and are weakly associated with activated FGF receptor-1, suggesting that these proteins may be direct targets of the receptor kinase. Kinetic analysis of SLP phosphorylation and studies with serine/threonine kinase and phosphatase inhibitors suggest that SLPs are no larger than 70 000 kilodaltons, and that serine/threonine phosphorylation follows tyrosine phosphorylation to substantially retard gel electrophoretic mobility. SLPs are associated with the Grb-2 adaptor and are the major tyrosine phosphorylated proteins associated with the Ras guanine nucleotide exchange <em>factor</em> Sos in FGF-stimulated <em>fibroblasts</em>, suggesting that SLP-Grb2-Sos complexes modulate the activity of Ras proteins.

MSCs are promising candidates for stem cell therapy and regenerative medicine. Umbilical cord is the easiest obtainable biological source of MSCs and the Wharton's jelly of the umbilical cord is a rich source of fetus-derived stem cells. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs. Here we have optimized conditions for scaling up of WJ-MSCs. Low seeding density along with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) supplementation in the <em>growth</em> medium, which is DMEM-KO, resulted in propagation of more than 1 x 10(8) cells within a time period of <em>15</em> days from a single umbilical cord. The upscaled WJ-MSCs retained their differentiation potential and immunosuppressive capacity. They expressed the typical hMSC surface antigens and the addition of bFGF in the culture medium did not affect the expression levels of HLA-DR and CD 44. A normal karyotype was confirmed in the large-scale expanded WJ-MSCs. Hence, in this study we attempted rapid clinical-scale expansion of WJ-MSCs which would allow these fetus-derived stem cells to be used for various allogeneic cell-based transplantations and tissue engineering.

Healing of chronic wounds such as diabetic foot ulcers is a significant clinical problem. Methods of accelerating healing in these difficult lower extremity sites include use of <em>growth</em> <em>factor</em>-loaded gels, hyperbaric oxygen, grafts, and artificial skin replacements. Nitric oxide (NO) has been proposed as a possible active agent for enhancing wound healing. This study examines the in vitro and in vivo responses to a novel hydrogel that produces therapeutic levels of NO. A hydrogel wound dressing was fabricated using ultraviolet light-initiated polymerization from poly(vinyl alcohol) with a NO donor covalently coupled to the polymer backbone. NO release from the NO-modified hydrogel was shown to occur over a time period of up to 48 hours, and there was no associated decrease in <em>fibroblast</em> <em>growth</em> or viability in vitro associated with NO hydrogels. <em>Fibroblasts</em> in culture with NO hydrogels had an increased production of extracellular matrix compared with cells cultured without the NO hydrogels. Preliminary animal studies in a diabetic mouse, impaired wound healing model were conducted comparing low (0.5 mM) and high (5 mM) doses of NO. Time to complete closure was similar in control wounds and NO-treated wounds; however, at 8 days control wounds were significantly smaller than NO-treated wounds. By days 10 to 13 this delay was no longer apparent. Granulation tissue thickness within the wounds at days 8 and <em>15</em> and scar tissue thickness after wound closure were increased in animals exposed to higher dose NO hydrogels. The results of this study suggest that exogenous NO released from a hydrogel wound dressing has potential to modulate wound healing.

The insulin-like <em>growth</em> <em>factor</em> signalling pathway is an important regulator of skeletal muscle <em>growth</em>. We examined the mRNA expression of components of the insulin-like <em>growth</em> <em>factor</em> (IGF) signalling pathway as well as <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> 2 (FGF2) during maturation of myotubes in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The transcriptional regulation of IGFs and IGFBP expression by amino acids and insulin-like <em>growth</em> <em>factors</em> was also investigated. Proliferation of cells was <em>15</em>% d(-1) at days 2 and 3 of the culture, increasing to 66% d(-1) at day 6. Three clusters of elevated gene expression were observed during the maturation of the culture associated with mono-nucleic cells (IGFBP5.1 and 5.2, IGFBP-6, IGFBP-rP1, IGFBP-2.2 and IGF-II), the initial proliferation phase (IGF-I, IGFBP-4, FGF2 and IGF-IRb) and terminal differentiation and myotube production (IGF2R, IGF-IRa). In cells starved of amino acids and serum for 72 h, IGF-I mRNA decreased 10-fold which was reversed by amino acid replacement. Addition of IGF-I and amino acids to starved cells resulted in an 18-fold increase in IGF-I mRNA indicating synergistic effects and the activation of additional pathway(s) leading to IGF-I production via a positive feedback mechanism. IGF-II, IGFBP-5.1 and IGFBP-5.2 expression was unchanged in starved cells, but increased with amino acid replacement. Synergistic increases in expression of IGFBP5.2 and IGFBP-4, but not IGFBP5.1 were observed with addition of IGF-I, IGF-II or insulin and amino acids to the medium. IGF-I and IGF-II directly stimulated IGFBP-6 expression, but not when amino acids were present. These findings indicate that amino acids alone are sufficient to stimulate myogenesis in myoblasts and that IGF-I production is controlled by both endocrine and paracrine pathways. A model depicting the transcriptional regulation of the IGF pathway in Atlantic salmon muscle following feeding is proposed.

The circulating levels of hyaluronate were determined in 36 patients with scleroderma and in 36 control subjects matched for age and sex. The mean serum hyaluronate concentration in patients with progressive systemic sclerosis (n = 25) was 131 +/- 67 (SD) microgram/l and significantly greater (p less than 0.001) than that of the controls (mean level 49 +/- 21 (SD) microgram/l). Hyaluronate levels in patients with localised scleroderma (n = 4) were 141 +/- 47 (SEM) microgram/l and in patients with scleroderma-associated overlap syndromes (n = 7) 202 +/- 54 (SEM) microgram/l. The increase in serum hyaluronate probably reflected an enhanced synthesis or outflow of hyaluronate from the connective tissue, or both; it could not be explained by affection of the liver, which is the catabolic site of hyaluronate. The hyaluronate values were not related to certain serological indicators of inflammatory activity or to the extent of the skin lesions or the severity of internal organ manifestations. A positive correlation was noted between circulating platelet counts and hyaluronate levels (p less than 0.001). Plasma beta-thromboglobulin was measured in <em>15</em> of the patients with systemic sclerosis and found to correlate positively with platelet counts. Raised levels of beta-thromboglobulin were associated with the highest hyaluronate values. Platelet-derived <em>growth</em> <em>factor</em>, which stimulates connective tissue cells and is stored in the alpha-granules of platelets together with beta-thromboglobulin, was shown to enhance hyaluronate synthesis in <em>fibroblast</em> cultures. The results suggest an involvement in scleroderma of connective tissue activating substances released from platelets.

We examined the role of the hepatocyte <em>growth</em> <em>factor</em> (HGF)/c-met system on invasion and metastasis of oral squamous cell carcinoma (SCC) cells. In monolayer culture, exogenous HGF marginally affected the <em>growth</em> of oral SCC cells (BHY, HN, IH) and human gingival epithelial cells (GE). In type I collagen matrix, however, HGF significantly enhanced the invasive <em>growth</em> of the cancer cells (p < 0.05). We detected the expression of c-met (HGF receptor) mRNA in all of the cancer cells, but not in human gingival <em>fibroblasts</em> (GF). Oral SCC cells did not secret HGF protein into the medium, but GF secreted a large amount of HGF protein (<em>15</em> ng/ml). Furthermore, HGF markedly enhanced the migration of cancer cells in a Transwell invasion chamber. Then, we examined the serum levels of HGF in oral SCC patients, or HGF concentrations in oral cancer tissues. Serum levels of HGF in the patients were significantly higher than those in healthy volunteers (p < 0.05). After initial treatment, all of the tumor-free survivors showed a marked decline in the serum HGF levels. Furthermore, HGF concentrations in metastatic cancer tissues were significantly higher than those of non-metastatic cancer tissues and normal gingiva (p < 0.01). These results suggest that HGF plays an important role in invasion and metastasis of oral SCC cells as a paracrine <em>factor</em>, and an elevated HGF level in the cancer tissue can be a predictive marker for metastasis formation in patients with oral SCC.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>15</em> (human homolog, FGF19) is an endocrine FGF highly expressed in the small intestine of mice. Emerging evidence suggests that FGF<em>15</em> is critical for regulating hepatic functions; however, the role of FGF<em>15</em> in liver regeneration is unclear. This study assessed whether liver regeneration is altered in FGF<em>15</em> knockout (KO) mice following 2/3 partial hepatectomy (PHx). The results showed that FGF<em>15</em> KO mice had marked mortality, with the survival rate influenced by genetic background. Compared with wild-type mice, the KO mice displayed extensive liver necrosis and marked elevation of serum bile acids and bilirubin. Furthermore, hepatocyte proliferation was reduced in the KO mice because of impaired cell cycle progression. After PHx, the KO mice had weaker activation of signaling pathways that are important for liver regeneration, including signal transducer and activator of transcription 3, nuclear <em>factor</em>-κB, and mitogen-activated protein kinase. Examination of the KO mice at early time points after PHx revealed a reduced and/or delayed induction of immediate-early response genes, including <em>growth</em>-control transcription <em>factors</em> that are critical for liver regeneration. In conclusion, the results suggest that FGF<em>15</em> deficiency severely impairs liver regeneration in mice after PHx. The underlying mechanism is likely the result of disrupted bile acid homeostasis and impaired priming of hepatocyte proliferation.

We identified a mutation in the Diet1 gene in a mouse strain that is resistant to hyperlipidemia and atherosclerosis. Diet1 encodes a 236 kD protein consisting of tandem low-density lipoprotein receptor and MAM (meprin-A5-protein tyrosine phosphatase mu) domains and is expressed in the enterocytes of the small intestine. Diet1-deficient mice exhibited an elevated bile acid pool size and impaired feedback regulation of hepatic Cyp7a1, which encodes the rate-limiting enzyme in bile acid synthesis. In mouse intestine and in cultured human intestinal cells, Diet1 expression levels influenced the production of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>/19 (FGF<em>15</em>/19), a hormone that signals from the intestine to liver to regulate Cyp7a1. Transgenic expression of Diet1, or adenoviral-mediated Fgf<em>15</em> expression, restored normal Cyp7a1 regulation in Diet-1-deficient mice. Diet1 and FGF19 proteins exhibited overlapping subcellular localization in cultured intestinal cells. These results establish Diet1 as a control point in enterohepatic bile acid signaling and lipid homeostasis.

Recently, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been found to increase trabecular bone mass and connectivity in the proximal tibial metaphyses (PTM) in osteopenic rats. The purpose of this study was to determine the bone anabolic effects of bFGF in the lumbar vertebral body (LVB), a less loaded skeletal site with a lower rate of bone turnover than the PTM. Six-month old female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated and untreated for 8 weeks to induce osteopenia. Then group 1 (sham) and group 2 (OVX) were treated subcutaneously (s.c.) with vehicle, and OVXed groups 3 and 4 were treated s.c. with PTH [hPTH (1-34) at 40 microg/kg, 5x/week] and bFGF (1 mg/kg, 5x/week), respectively, for 8 weeks. At sacrifice, the fifth LVB was removed, subjected to micro-CT for determination of trabecular bone structure and then processed for histomorphometry to assess bone turnover. The sixth LVB was used for mechanical compression testing (MTS, Bionix 858). The data were analyzed with the Kruskal-Wallis test followed by post-hoc testing as needed. After 16 weeks of estrogen deficiency, there were significant reductions in vertebral trabecular bone volume and trabecular thickness. Treatment with either bFGF or hPTH (1-34) increased BV/TV in OVX animals. Human PTH (1-34)-treated animals had significant increases in trabecular (48%) and cortical thickness (30%) and bone strength [maximum load (53%) and work to failure (175%)] compared to OVX + Vehicle animals. Treatment of osteopenic rats with bFGF increased bone volume (<em>15</em>%), trabecular thickness (13%), maximum load (45%) and work to failure (140%) compared to OVX + Vehicle animals (all P <0.05). Basic FGF increased trabecular bone volume in the lumbar vertebral body of osteopenic rats by restoring trabecular number, thickness and connectivity density. Also, bFGF improved bone mechanical properties (maximum force and work to failure) compared to the OVX + Vehicle group. Therefore, increasing the number, thickness and connections of the trabeculae contributes to increased bone strength in this small animal model of osteoporosis.

OBJECTIVE

To test whether Müller glial cells sense, and respond to, mechanical tension in the retina.

METHODS

A device was designed to stretch the retina at right angles to its surface, across retinal layers. Pieces of retina were mounted between two hollow tubes, and uniaxial force was applied to the tissue using a micrometer-stepping motor. Müller cells were selectively stained with the fluorescent, calcium-sensitive dye X-Rhod-1 and were monitored in real time during retinal stretch in vitro. Immunohistochemistry was used to study protein levels and activation of intracellular pathways in stretched retinas.

RESULTS

Müller cells responded acutely with transient increases in fluorescence during stretch, indicative of increased intracellular calcium levels. All the Müller cells elongated uniformly, and there was no apparent difference between retinal layers in resistance against mechanical deformation. After stretch, Müller cells showed fast activation of extracellular signal-regulated kinase (after <em>15</em> minutes), upregulation of transcription <em>factor</em> c-Fos (after 1 hour), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (after 3 hours). No changes in intermediate filament protein expression were observed in Müller cells up to 3 hours after stretch.

CONCLUSIONS

A novel technique was developed for real-time monitoring of Müller cells during retinal stretch, which allowed the identification of Müller cells as a mechanoresponsive cell type. Mechanical stress triggers molecular responses in Müller cells that could prevent retinal damage.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) stimulated sn-1,2-diacylglycerol (DAG) mass formation in Swiss 3T3 <em>fibroblasts</em> with a lag time of some 30 s. The response was biphasic, with the second phase being sustained over time. PDGF also stimulated the formation of Ins(1,4,5)P3 with a similar lag time to the DAG response, suggesting that DAG is derived from PtdIns(4,5)P2 hydrolysis at this time point. PDGF-stimulated phosphatidylcholine (PtdCho) hydrolysis in Swiss 3T3 <em>fibroblasts</em>, as measured by the formation of water-soluble choline metabolites and phosphatidylbutanol (PtdBut) accumulation, was by a phospholipase D (PLD)-catalysed pathway which was kinetically downstream of initial PtdIns(4,5)P2 hydrolysis. Accumulation of PtdBut increased up to <em>15</em> min, suggesting that PLD activity is not rapidly densitized in response to PDGF. The kinetics of PtdCho hydrolysis closely paralleled the second phase of DAG formation, strongly suggesting that during prolonged stimulation periods PtdCho is a major source of DAG in these cells. However, since PtdIns(4,5)P2 breakdown was also prolonged, PDGF-stimulated DAG may be derived from both phospholipids. Down-regulation of protein kinase C (PKC), by pre-treatment with phorbol 12-myristate 13-acetate, abolished both [3H]choline and [3H]PtdBut formation, suggesting that PLD-catalysed PtdCho hydrolysis may be dependent on PKC activation, supporting its dependence on prior PtdIns(4,5)P2 hydrolysis.

We recently reported that acidic (aFGF) and basic (bFGF) <em>fibroblast</em> <em>growth</em> <em>factors</em> confer a broad spectrum chemoresistance in solid tumors, and that suramin, an inhibitor of multiple <em>growth</em> <em>factors</em> including aFGF and bFGF, enhanced the in vitro antitumor activity of several anticancer drugs including paclitaxel (Song, S., et al., Proc. Natl. Acad. Sci. USA, 97: 8658-8663, 2000). The present study investigated in vitro and in vivo interaction between paclitaxel and suramin, using human PC3-LN cells which, upon i.v. injection into immunodeficient mice, yielded lung metastases in 100% of animals. In in vitro studies, conditioned medium (CM) obtained from histocultures of rat lung metastases induced a 3-fold resistance. The addition of suramin had no effect in the absence of CM but reversed the CM-induced resistance; calculations based on the IC(50) values indicate a complete reversal in the presence of <20 microM suramin. Analysis by the combination index method indicates a synergistic interaction between paclitaxel and suramin. In in vivo studies, animals with well-established lung metastases (at least five nodules of 1 mm in diameter) were treated i.v. with paclitaxel (<em>15</em> mg/kg) and suramin (10 mg/kg) administered twice weekly for 3 weeks. Single-drug therapy with paclitaxel or suramin did not reduce body weight. Suramin alone had no antitumor activity. Paclitaxel alone reduced the tumor size by approximately 75%, reduced the density of nonapoptotic cells by approximately 70% in residual tumors, and enhanced the fraction of apoptotic cells by approximately 3-fold. The addition of suramin to paclitaxel therapy enhanced the antitumor effect, resulting in an additional 5-fold reduction of tumor size, an additional 9-fold reduction of the density of nonapoptotic cells, and an additional 30% increase in the apoptotic cell fraction. These data indicate significant enhancement of the efficacy of paclitaxel by suramin and support the use of nontoxic doses of suramin with paclitaxel in the treatment of lung cancer.

The binding of iodinated basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to low-density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane-like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled <em>factor</em> and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10-<em>15</em> higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123-130, 1987) in the case of the low-affinity binding sites present on the surface of baby hamster kidney (BHK) cells.

Extracellular matrix scaffolds derived from porcine small intestinal submucosa (SIS-ECM) have been shown to promote the formation of site-specific tissue in a number of preclinical animal studies. However, this constructive remodeling process requires that the scaffold be subjected to a site-specific mechanical environment. The specific quantitative effects of mechanical loading on the gene expression patterns of <em>fibroblasts</em> seeded on SIS-ECM are unknown and yet very important in the tissue remodeling process. The objective of the present study was to evaluate the expression of collagen type I (Col I), collagen type III (Col III), smooth muscle actin (SMA), tenascin-C (TN-C), matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1), and transforming <em>growth</em> <em>factor</em>-beta3 (TGF-beta3) by <em>fibroblasts</em> subjected to various magnitudes (0%, 5%, 10%, and <em>15</em>%) and frequencies (0.1 Hz, 0.3 Hz, and 0.5 Hz) of stretch. A new cyclic-stretching tissue culture (CSTC) system was developed. This system consists of eight independently controlled culture chambers that can be operated in a sterile incubator. Each chamber includes a load cell so that the load in each scaffold can be monitored. It was found that different stretching regimens led to complex and distinctive patterns of gene expression by <em>fibroblasts</em> seeded onto SIS-ECM. In general, the <em>fibroblasts</em> increased expression of Col I up to 5-fold and decreased that of Col III with increased frequency of stretch. In addition, the <em>fibroblasts</em> exhibited a contractile phenotype with increased expression of SMA, TN-C, and TGF-beta1. These findings support the concept that the mechanical environment of a remodeling ECM scaffold may have substantial effects on the behavior of cells within the scaffold and contribute to the site-specific tissue remodeling that has been observed in in vivo studies.

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day <em>15</em>-20), during which the blood vessels undergo remodeling. To examine directly the influence of AT2 receptor expression in the developmental biology of VSMC, we studied cultures of VSMC from fetal and postnatal wild-type (Agtr2(+)) and AT2 receptor null (Agtr2(-)) mice. Consistent with in vivo data, AT2 receptor binding in cultured Agtr2(+) VSMC increased by age, peaking at embryonic day 20, and decreased dramatically after birth. Angiotensin II-induced <em>growth</em> in Agtr2(+) VSMC (embryonic day 20) was increased by the AT2 receptor blocker PD123319, indicating that the AT2 receptors are functional and exert an anti<em>growth</em> effect in Agtr2(+) VSMC. <em>Growth</em> of VSMC in response to serum decreased age dependently and was higher in Agtr2(-) than in Agtr2(+), inversely correlating with AT2 receptor expression. However, serum-induced <em>growth</em> in Agtr2(+) and Agtr2(-) VSMC and the exaggerated Agtr2(-) VSMC <em>growth</em> was maintained even in the presence of PD123319 or losartan, an AT1 receptor blocker. Moreover, Agtr2(-) VSMC showed greater <em>growth</em> responses to platelet-derived <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, indicating that Agtr2(-) cells exhibit a generalized exaggerated <em>growth</em> phenotype. We studied the mechanism responsible for this phenotype and observed that extracellular signal-regulated kinase (ERK) activity was higher in Agtr2(-) VSMC at baseline and also in response to serum. ERK kinase inhibitor PD98059 inhibited both <em>growth</em> and ERK phosphorylation dose-dependently, while the regression lines between <em>growth</em> and ERK phosphorylation were identical in Agtr2(+) and Agtr2(-) VSMC, suggesting that increased ERK activity in Agtr2(-) VSMC is pivotal in the <em>growth</em> enhancement. Furthermore, the difference in ERK phosphorylation between Agtr2(+) and Agtr2(-) was abolished by vanadate but not by okadaic acid, implicating tyrosine phosphatase in the difference in ERK activity. These results suggest that the AT2 receptor expression during the fetal vasculogenesis influences the <em>growth</em> phenotype of VSMC via the modulation of ERK cascade.

We have examined the effects of l-thyroxine (T4) on the activation of signal transducer and activator of transcription 3 (STAT3) and on the STAT3-dependent induction of c-Fos expression by epidermal <em>growth</em> <em>factor</em> (EGF). T4, at a physiological concentration of 100 nM, caused tyrosine phosphorylation and nuclear translocation (i.e. activation) of STAT3 in HeLa cells in as little as 10-20 min. Activation by T4 of STAT3 was maximal at 30 min (<em>15</em>+/-4-fold enhancement; mean+/-S.E.M.) in 18 experiments. This effect was reproduced by T4-agarose (100 nM) and blocked by CGP 41251, genistein, PD 98059 and geldanamycin, inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK) kinase and Raf-1 respectively. Tyrosine-phosphorylated MAPK also appeared in nuclear fractions within 10 min of treatment with T4. In the nuclear fraction of T4-treated cells, MAPK immunoprecipitate also contained STAT3. The actions of T4 were similar in HeLa and CV-1 cells, which lack thyroid hormone receptor (TR), and in TR-replete skin <em>fibroblasts</em> (BG-9). T4 also potentiated the EGF-induced nuclear translocation of activated STAT1alpha and STAT3 and enhanced the EGF-stimulated expression of c-Fos. Hormone potentiation of EGF-induced signal transduction and c-Fos expression was inhibited by CGP 41251, geldanamycin and PD 98059. Therefore the non-genomically induced activation by T4 of STAT3, and the potentiation of EGF by T4, require activities of PKC, PTK and an intact MAPK pathway.

Treatment of BC3H1 myocytes or 3T3-L1 <em>fibroblasts</em> with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42-kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from <em>growth</em> <em>factor</em>-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within <em>15</em> min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by <em>growth</em> <em>factors</em> in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase.

The biosynthesis, phosphorylation, and degradation of the epidermal <em>growth</em> <em>factor</em> (EGF) receptor were examined in normal human <em>fibroblasts</em>. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally <em>growing</em> and quiescent cells, and treatment with EGF or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a two- to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from <em>15</em> h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.

BACKGROUND

Preimplantation cross-talk between a functional blastocyst and the endometrium is critical for successful blastocyst implantation. This interaction is mediated in part by endometrial cytokines/growth factors secreted by glandular epithelium into the uterine cavity. Recent evidence suggests that blastocyst-derived hCG may influence the endometrial milieu in conception cycles thereby enhancing receptivity and implantation success. This study investigated the effect of hCG on the secretory profile of a select cohort of 44 cytokines/growth factors from primary human endometrial epithelial cells (hEECs). These factors included those with both known and unknown roles during receptivity and implantation. The expression of one previously unknown hCG-regulated factor, fibroblast growth factor 2 (FGF2), in human endometrium and its effects on hEEC function were further examined.

METHODS

hEECs isolated from endometrial biopsies collected from fertile cycling women (n = 15) were treated ± recombinant hCG (0.2-20 IU/ml) for 48 h and conditioned media was quantitatively analysed using Luminex™ multiplex technology. FGF2 was further investigated by immunohistochemistry, western blot and cell-adhesion assays.

RESULTS

Of 44 cytokines/growth factors examined, 39 were produced by hEECs with a distinct profile. hCG (2 IU/ml) significantly increased the production of six factors, including those with known roles in receptivity and trophoblast function (interleukin-11), blastocyst migration and adhesion (CXCL10), blastocyst development (granulocyte macrophage colony-stimulating factor) and one unknown with respect to receptivity and implantation (FGF2). Up-regulation of known hCG-regulated proteins, vascular endothelial growth factor and leukaemia inhibitory factor, validated this study. Immunoreactive epithelial FGF2 increased across the menstrual cycle, being highest in secretory and first trimester pregnancy endometrium in vivo. FGF2 (100 ng/ml) stimulated phosphorylation of ERK1/2 in hEEC with no effect on ERK1/2 abundance and stimulated hEEC adhesion to fibronectin and collagen IV (components of blastocyst/trophectoderm extracellular matrix).

CONCLUSIONS

These findings clearly support roles for hCG and FGF2 in the blastocyst-endometrial cross-talk important for endometrial receptivity and blastocyst implantation.