Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat <em>fibroblasts</em>. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and <em>20</em>% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of <em>fibroblasts</em>, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) <em>fibroblast</em>-conditioned media; 3) exogenous decorin; or 4) treatment with individual <em>growth</em> <em>factors</em> known to be involved in angiogenesis. The mechanism(s) by which <em>fibroblasts</em> induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of <em>fibroblasts</em> for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) regulate many important developmental and homeostatic physiological events. The FGF superfamily contains several families. In this review, we present recent findings on the two FGFs of the nematode Caenorhabditis elegans from both functional and phylogenic points of view. C. elegans has a single FGFR (EGL-15) with two functionally exclusive isoforms, and two FGFs (LET-756 and EGL-17), which play distinct roles: an essential function for the former, and guidance of the migrating sex myoblasts for the latter. Regulation of homeostasis by control of the fluid balance could be the basis for the essential function of LET-756. Phylogenetic and functional studies suggest that LET-756, like vertebrate FGF9, -16, and -<em>20</em>, belongs to the FGF9 family, whereas EGL-17, like vertebrate FGF8, -17, and -18, could be included in the FGF8 family.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) is a potent mitogen that can induce angiogenesis in vivo. We have recently reported a marked increase of basic FGF in the pericardial fluid of patients with severe coronary stenosis and an increase in vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in the pericardial fluid of patients with severe myocardial ischemia. The purpose of this study was to evaluate whether acidic FGF levels in the pericardial fluid are associated with severe myocardial ischemia. Immediately after incision of the pericardium in 48 patients during open-heart surgery, 3-5ml of pericardial fluid was obtained. Concentrations of basic FGF and VEGF in the pericardial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). The ELISA system for human acidic FGF was newly developed using a rabbit antibovine acidic FGF antibody. The patients were divided into three groups (group A: 13 patients undergoing emergency coronary artery bypass grafting (CABG) for unstable angina; group B: 17 patients undergoing elective CABG for stable angina; group C: 18 patients undergoing nonischemic open-heart surgery). The VEGF level in the pericardial fluid in group A was 68 +/- 59pg/ml, which was significantly higher than 33 +/- 9 pg/ml in group B and 31 +/- <em>20</em> pg/ml in group C (P < 0.05). The concentrations of basic FGF in the pericardial fluid in groups A and B were 722 +/- 601 and 773 +/- 763pg/ml, respectively, significantly higher than 263 +/- 349pg/ml in group C. The pericardial acidic FGF level in group A was 4,291 +/- 2,336 pg/ml, which was also significantly higher than 2,386 +/- 1,048 pg/ml in group B and 2,589 +/- 990 pg/ml in group C (P < 0.05). The acidic FGF level correlated well with the level of VEGF (r = 0.61, P < 0.0001). It is concluded that the level of acidic FGF in pericardial fluid is associated with severe myocardial ischemia. This result indicates that the release of acidic FGF from the myocardial tissue into pericardial fluid is closely related to severe myocardial ischemia.

Transforming <em>growth</em> <em>factor</em> beta (TGF beta) is an extremely potent chemoattractant for macrophages, mononuclear leukocytes, and <em>fibroblasts</em>. It also acts as a potent stimulant for collagen and fibronectin synthesis and inhibits epithelial cell <em>growth</em>. TGF beta plays an important role in healing many types of wounds, but its role in peritoneal adhesion formation is not known. These studies were performed to determine if TGF beta could affect postoperative wound healing in a rat model. In the first experiment, <em>20</em> rats were divided into two groups and received either 2 micrograms TGF beta or control diluent IP daily for 5 days after surgical injury to the uterine horns. The severity of the adhesions were graded 2 weeks postoperatively using a score of 0-3. The TGF beta group showed a higher adhesion score at 2 weeks compared to control, 2.9 +/- 0.34 and 1.6 +/- 0.61, respectively (P less than 0.001). On H&E stained sections of the adhesions, there was an increase in the number of both inflammatory cells and <em>fibroblasts</em> in the TGF beta-treated animals. A comparison trial of bone-derived TGF beta (a gift from Collagen Corporation, Palo Alto, CA) versus recombinant TGF beta (a gift from Oncogen, Seattle, WA) versus control using the same protocol as above showed that both sources of TGF beta were more effective in promoting postoperative adhesions when compared to controls, and there was no difference between TGF beta groups, 3.0 +/- 0 for both TGF beta groups, and 2.2 +/- 0.91 for control (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to stimulate wound healing. However, consistent delivery of bFGF has been problematic. We studied the stability of bFGF incorporated into a chitosan film as a delivery vehicle for providing sustained release of bFGF. The therapeutic effect of this system on wound healing in genetically diabetic mice was determined as a model for treating clinically impaired wound healing. A chitosan film was prepared by freeze-drying hydroxypropylchitosan (a water-soluble derivative of chitosan) acetate buffer solution. <em>Growth</em> <em>factor</em> was incorporated into films before drying by mixing bFGF solution with the hydroxypropylchitosan solution. bFGF activity remained stable for 21 days at 5 degrees C, and 86.2% of activity remained with storage at 25 degrees C. Full-thickness wounds were created on the backs of diabetic mice, and chitosan film or bFGF-chitosan film was applied to the wound. The wound was smaller in after 5 days in both groups, but the wound was smaller on day <em>20</em> only in the bFGF-chitosan group. Proliferation of <em>fibroblasts</em> and an increase in the number of capillaries were observed in both groups, but granulation tissue was more abundant in the bFGF-chitosan group. These results suggest that chitosan itself facilitates wound repair and that bFGF incorporated into chitosan film is a stabile delivery vehicle for accelerating wound healing.

OBJECTIVE

Fibroblast growth factor 23 is a phosphate- and vitamin D-regulating hormone. The objective of this study was to determine the effect of ergocalciferol administration on fibroblast growth factor 23 levels in healthy vitamin D-deficient subjects.

METHODS

In this 12-week trial conducted in a clinical research center, 18- to 45-year-old subjects (n=90) with 25-hydroxyvitamin D levels ≤20 ng/ml (by chemiluminescent immunoassay) were randomized to weekly ergocalciferol treatment of 50,000 international units or placebo, while consuming a self-selected diet. Changes in fibroblast growth factor 23, 25-hydroxyvitamin D (by liquid chromatography/tandem mass spectroscopy), 1,25-dihydroxyvitamin D, parathyroid hormone, and serum phosphate were measured.

RESULTS

Mean 25-hydroxyvitamin D (P<0.0001), 1,25-dihydroxyvitamin D (P=0.01), and fibroblast growth factor 23 (P=0.003) increased in the treatment versus placebo group. In the treatment group, 25-hydroxyvitamin D increased from 18 ± 7 to 40 ± 12 ng/ml at week 4 (P<0.0001) and remained stable at 43 ± 12 ng/ml at week 12 (P<0.0001); 1,25-dihydroxyvitamin D increased from 42 ± 17 to 52 ± 18 pg/ml at week 4 (P<0.001) and then remained stable, and fibroblast growth factor 23 increased from 43 ± 17 to 60 ± 33 pg/ml at week 8 (P=0.001) and 74 ± 42 pg/ml at week 12 (P<0.0001). Urinary phosphate excretion increased within the treatment group, but parathyroid hormone and serum phosphate were unchanged.

CONCLUSIONS

Ergocalciferol administration increases circulating fibroblast growth factor 23. When measuring fibroblast growth factor 23, concurrent 25-hydroxyvitamin D measurements should be obtained, because vitamin D deficiency may lower circulating fibroblast growth factor 23 levels.

OBJECTIVE

To investigate the clotting and fibrinolytic activities in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to examine the role of thrombin in synovial hyperplasia.

METHODS

We measured the amounts of thrombin-antithrombin-III complex (TAT), antithrombin-III (AT-III), thrombin, plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI), and plasmin-alpha 2-antiplasmin complex (PAP) in SF of <em>20</em> patients with RA and 16 patients with osteoarthritis (OA). The proliferative response of synovial <em>fibroblast</em>-like cells to thrombin was measured using [3H] thymidine incorporation. Expression of platelet derived <em>growth</em> <em>factors</em> (PDGF) in conditioned medium was analyzed using a Western blot method, and expression of the mRNA of PDGF and their receptors was analyzed by reverse transcription polymerase chain reaction.

RESULTS

The amounts of clotting factors (TAT, AT-III) and fibrinolytic factors (plasminogen, alpha 2-PI, and PAP) were significantly higher in the patients with RA than in patients with OA (p < 0.01). Moreover, SF thrombin concentrations of patients with RA correlated significantly with erythrocyte sedimentation rates (rs = 0.751, p < 0.01) and serum C-reactive protein concentrations (rs = 0.531, p < 0.05). Thrombin exhibits mitogenic activity toward synovial fibroblast-like cells in vitro, and this mitogenic activity is associated with an increase in the expression of mRNA of both PDGF-alpha receptor and PDGF-beta-receptor.

CONCLUSIONS

The high levels of thrombin activity in the SF of patients with RA and strong mitogenic activity of thrombin toward the synovial fibroblast-like cells suggest that thrombin plays an important role in the pathogenesis of RA.

All lepidopteran baculovirus genomes sequenced to date encode a viral <em>fibroblast</em> <em>growth</em> <em>factor</em> homolog (vfgf), suggesting that vfgf may play an important role in the infection cycle of lepidopteran baculoviruses. Here, we describe the characterization of a Bombyx mori nucleopolyhedrovirus (BmNPV) mutant lacking functional vfgf. We constructed a vfgf deletion mutant (BmFGFD) and characterized it in BmN cells and B. mori larvae. We observed that budded virus (BV) production was reduced in BmFGFD-infected BmN cells and B. mori larvae. The larval bioassays also revealed that deletion of vfgf did not reduce the infectivity; however, the mutant virus did take <em>20</em> h longer to kill B. mori larvae than wild-type BmNPV, when tested either by BV injection or by polyhedrin-inclusion body ingestion. These results suggest that BmNPV vfgf is involved in efficient virus production in BmN cells and B. mori larvae.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) is an important stimulator of angiogenesis that has been implicated in neoplastic progression. Attempts to neutralize or modulate FGF-2 have met with some success in controlling neovascularity and tumor <em>growth</em>. In the present study, two peptides: one corresponding to the heparin binding domain and the other to the receptor binding domain of FGF-2, exerted dose-dependent inhibition of FGF-2-stimulated human umbilical vein endothelial cell proliferation (IC(50)=70 and <em>20</em> microg/ml, respectively). The identification of these functional regions suggested that targeting these domains might be an approach for the modulation of FGF-2 function. To investigate this possibility, we vaccinated mice with either the heparin binding domain peptide or the receptor binding domain peptide of FGF-2 in a liposome/adjuvant format, and analyzed the effect of vaccination on FGF-2-driven angiogenesis, tumor development and immune status. Mice vaccinated with the heparin binding domain peptide generated a specific antibody response to FGF-2, blocked neovascularization in a gelfoam sponge model of angiogenesis, and inhibited experimental metastasis by >90% in two tumor models: the B16BL6 melanoma and the Lewis lung carcinoma. These effects were not observed in mice treated with the receptor binding domain peptide conjugated to liposomes or liposomes lacking conjugated peptide. These data suggest that a heparin binding domain peptide of FGF-2, when presented to a host in a liposomal adjuvant formulation, can ultimately lead to inhibition of angiogenesis and tumor <em>growth</em>.

After the debridement of the entire corneal epithelium of the rabbit eye, epithelial cells of conjunctival origin cover the denuded corneal surface. Under such experimental conditions, the rate of wound healing is considerably delayed and total regeneration is completed within 15 to <em>20</em> days, allowing evaluation of various drugs, such as the <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>. Both acidic and basic FGF were administered topically on totally de-epithelialized rabbit eye, at three different concentrations of 1.5 and 10 Stimulation Units/50 microliters, 3 times per day. A dose-response effect was observed and in each case, acidic FGF was found to be much more potent than bFGF in increasing the rate of wound healing of the cornea. These results are correlated with a new purification procedure, avoiding acid treatment of the tissue extract. The systemic diffusion of FGF allows the contralateral eye cells to be also stimulated for mitosis and migration and to heal faster than the control eyes.

Activation of the nuclear receptors constitutive androstane receptor, pregnane X receptor, and peroxisome proliferator-activated receptor alpha results in hepatomegaly, and these nuclear receptors are implicated in the regulation of liver regeneration. Retinoid X receptor (RXR)alpha is an essential partner of these nuclear receptors. Therefore, we studied the role of hepatocyte RXRalpha in liver regeneration using partial hepatectomy model. The results showed that hepatocyte RXRalpha deficiency caused an approximately <em>20</em>-hour delay in hepatocyte proliferation after partial hepatectomy. Several pathways, including <em>growth</em> <em>factors</em> and the circadian cell cycle, were impaired due to hepatocyte RXRalpha deficiency. In addition, the expression patterns of hepatocyte <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2, platelet-derived <em>growth</em> <em>factor</em>, and transforming <em>growth</em> <em>factor</em> alpha were altered due to lack of RXRalpha. Furthermore, the peroxisome proliferator-activated receptor alpha/brain and muscle Arnt-like protein 1/Rev-erbalpha/P21 pathway was compromised, and Cry1/Cry2 and Wee1/Per1 expression was deregulated in regenerating RXRalpha-null livers. Accordingly, the expression and regulation of cyclin D1/Cyclin- dependent Kinase (Cdk)4, cyclin E1/Cdk2, cyclin A2/Cdk2, and cyclin B1/Cdk1 after partial hepatectomy were altered in regenerating RXRalpha-null livers. Hepatocyte RXRalpha deficiency also affected the basal, as well as regeneration-induced cyclin E1 expression levels. Activation of RXRalpha by retinoic acids increased the cyclin E1 promoter activity indicating retinoic acid-mediated signaling positively controls cyclin E1 gene expression. As many of these observed changes were not documented in the regenerating livers of other nuclear receptor knockout mice, these observed effects may be hepatocyte RXRalpha specific.

To examine whether angiogenesis and myocardial salvage occur, 30 micrograms basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and 3 mg heparin sulfate (HS) were injected through the right atrium into the pericardial cavity by a thin needle-tipped catheter in a canine model of acute myocardial infarction. One month later infarcted weight/left ventricle weight was 24% +/- 5.2%, 25% +/- 4.0%, 18% +/- 2.4%, and 10% +/- 1.8% (mean + SE) in saline solution, HS, bFGF alone, and bFGF plus HS groups, respectively. Vascular number in the infarcted area of the outer layer was 13 +/- 3.3, <em>20</em> +/- 2.2, 47 +/- 8.3, and 136 +/- 26.3/<em>20</em>0 x <em>20</em>0 microns2 in saline solution, HS, bFGF alone, and bFGF plus HS groups, respectively. Thus the vascular number was the largest in the bFGF plus HS group. The vascular number was larger in the subepicardial than in the subendocardial infarcted areas. Vessels directed from the epicardium toward the subepicardial infarcted area were also observed. The transcatheter intrapericardial injection of bFGF plus HS caused angiogenesis and myocardial salvage. This method might bring about a selective therapeutic and preventive modality of myocardial infarction irrespective of coronary anatomy and contraindications for coronary interventions and surgery.

Progressive renal fibrosis is considered to be the final common pathway leading to chronic renal insufficiency. In this study, the authors examined some of the cellular and molecular mechanisms regulating the renal accumulation of extracellular matrix (ECM) proteins using rats with puromycin amino-nucleoside (PAN) nephrosis as an acute model system. Puromycin aminonucleoside rats developed reversible nephrotic syndrome accompanied by an interstitial infiltrate of monocytes. The number of interstitial <em>fibroblasts</em> expressing ST4 antigen did not increase. During the first 4 days, steady-state mRNA levels for all genes examined remained at or below control levels. At 1 week, nephrotic syndrome and interstitial inflammation were established, and a period of renal cell proliferation occurred, identified by increased histone mRNA levels and localized by tritiated thymine autoradiography to tubular epithelial cells and occasional interstitial cells. Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) steady-state mRNA levels were increased eightfold, but returned to control levels by 3 weeks. At week 1, there was a 10- to <em>20</em>-fold increase in kidney steady-state mRNA levels for genes encoding interstitial matrix proteins collagen I and fibronectin and basement membrane collagen IV. By in situ hybridization, alpha 1(I) procollagen mRNA was localized to interstitial cells. Immunofluorescence microscopy demonstrated focal accumulation of ECM proteins in the tubulointerstitial compartment at 2 and 3 weeks, but by 6 weeks, kidney immunohistology was normal again. Steady-state mRNA levels for the matrix degrading metalloproteinase stromelysin remained at control values, whereas the levels for interstitial collagenase were normal at week 1 and increased twofold to threefold at 2 and 3 weeks. Steady-state mRNA levels for the tissue inhibitor of metalloproteinases (TIMP) increased fivefold at 1 week and returned to baseline values over the next 2 weeks. The results of this study suggest that tubulointerstitial ECM accumulation occurs in rats with acute PAN nephrosis because of the activation of genes encoding several matrix proteins and inhibition of matrix degradation mediated by TIMP. These events are reversed during the phase of recovery from nephrotic syndrome. Increased mRNA levels for TGF-beta, possibly originating from inflammatory interstitial monocytes, are likely to be one of the mediators of the molecular events observed.

We previously showed that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent mitogen for human bone marrow (BM) stromal cells and significantly delays their senescence. In the present study, we demonstrated that low concentrations of bFGF (0.2 to 2 ng/mL) enhance myelopoiesis in long-term human BM culture. Addition of bFGF to long-term BM cultures resulted in an increase in (a) the number of nonadherent cells (sixfold), particularly those of the neutrophil granulocyte series; (b) the number of nonadherent granulocyte colony-stimulating <em>factor</em> (G-CSF)- and granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF)-responsive progenitor cells; (c) the number of adherent foci of hematopoietic cells (10-fold); and (d) the number of progenitor cells in the adherent stromal cell layer. These effects were not noted with higher concentrations of bFGF (<em>20</em> ng/mL). Thus, low concentrations of bFGF effectively augment myelopoiesis in human long-term BM cultures, and bFGF may therefore be a regulator of the hematopoietic system in vitro and in vivo.

BACKGROUND

Previously, we found angiogenesis measured as microvessel density (MVD) to be associated with both pathological stage and clinical outcome after radical prostatectomy (RP). In addition, we have shown that Vascular Endothelial Growth Factor (VEGF) is one of the important inducers of angiogenesis in prostate cancer (PC). The aim of this study was to investigate the expression of additional angiogenic factors, namely basic Fibroblast Growth Factor (hFGF) and the c-met receptor of Hepatocyte Growth Factor/Scatter Factor (HGF/SF) in PC.

METHODS

Ninety-eight paraffin-embedded RP specimens and 20 adjacent normal prostatic tissues were evaluated for factor VIII staining and microvessel counting. Expression of VEGF (n=55), bFGF (n=65) and c-met (n=66) was studied by immunohistochemistry. Results were correlated with pathological grade and stage, MVD and clinical outcome.

RESULTS

While adjacent benign tissue in RP specimens generally showed low MVD, VEGF-, bFGF- and c-met-expression, this was different in PC. All angiogenesis inducers were associated with stage while c-met as well as VEGF expression were associated with grade. Tumor progression was associated with grade and MVD. There was a clear correlation between VEGF and c-met expression and MVD.

CONCLUSIONS

VEGF and c-met expression increase with tumor stage and grade, while bFGF expression increases only with tumor stage. In addition to VEGF, c-met seems to be important and clinically relevant to the induction of angiogenesis in PC. Both VEGF and c-met appear to influence tumor progression, mainly through their effect on MVD.

A recurrent point mutation in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) gene that converts proline 250 into arginine is commonly associated with coronal craniosynostosis and has allowed definition of a new syndrome on a molecular basis. Sixty-two patients with sporadic or familial forms of coronal craniosynostosis were investigated for the P250R FGFR3 mutation. It was identified in <em>20</em> probands originating from 27 unrelated families (74%), while only 6/35 sporadic cases (17%) harboured the mutation. In both familial and sporadic cases, females were significantly more severely affected than males. Hence, while 68% of females carrying the P250R mutation showed brachycephaly, only 35% of males had the same phenotype. In the most severe forms of the disease, the association of bicoronal craniosynostosis with hypertelorism and marked bulging of the temporal fossae were common hallmarks that might be helpful for clinical diagnosis. Taken together, these results indicate that the P250R FGFR3 mutation is mostly familial and is associated with a more severe phenotype in females than in males. The sex related severity of the condition points to the possible implication of modifier genes in this syndrome.

The objective of this study was to begin to examine the cellular and biophysical effects on human retinal glial cells of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), which is endogenous to the retina and likely to play a role in retinal pathobiology. Experiments were performed on cultured glial cells derived from the adult postmortem retina. A proliferative response to bFGF established a sensitivity of the retinal glia to this <em>growth</em> <em>factor</em>. The possibility that bFGF alters calcium currents was assessed using the whole-cell recording configuration of the patch-clamp technique to analyze inward currents carried by barium. Two types of voltage-gated calcium channels could be expressed by the glial cells. One, similar to the T-type current described in various kinds of cells, had a low threshold of activation, a transient response, and an insensitivity to the dihydropyridine nifedipine. The other type of inward current, which closely resembles the L-type calcium current found in other cells, had a high threshold, had a long-lasting response, and was inhibited by nifedipine. When continuous whole-cell recordings were made from retinal glial cells, the L-type calcium current increased significantly within <em>20</em> min after exposure of the cells to bFGF. The physiological significance of this modulatory effect remains uncertain, though the observation that nifedipine inhibits both the L-type calcium current and the bFGF-induced proliferation is consistent with the hypothesis that dihydropyridine-sensitive channels may play a role in modulating the mitogenic response of retinal glial cells to this <em>growth</em> <em>factor</em>.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, 15, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to 15 dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by <em>20</em> min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by 15 dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal <em>growth</em> <em>factor</em> in human dermal <em>fibroblasts</em> have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of <em>fibroblasts</em> with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining <em>20</em>% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.

A genetic association between the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF<em>20</em>) gene and Parkinson's disease has been found by the pedigree disequilibrium test. This association, however, was not replicated by a case-control association study. In order to clarify the association between the FGF<em>20</em> gene and Parkinson's disease, we attempted to replicate this association by a case-control association study using a large number of Japanese samples (1388 patients and 1891 controls). rs1721100 exhibited a significant difference in allele C versus G (P=0.0089), and in genotype CC+CG versus GG (P=0.0053). Haplotype association analysis showed that haplotype 2 was the protective haplotype for Parkinson's disease (permutation-P=0.0075). These results suggest that the FGF<em>20</em> gene is a susceptibility gene for Parkinson's disease in the Japanese population.

The objective of this study was to develop and evaluate a hydrogel vehicle for sustained release of <em>growth</em> <em>factors</em> for wound healing applications. Hydrogels were fabricated using ultraviolet photo-crosslinking of acrylamide-functionalized nondegradable poly(vinyl alcohol) (PVA). Protein permeability was initially assessed using trypsin inhibitor (TI), a 21 000 MW model protein drug. TI permeability was altered by changing the solids content of the gel and by adding hydrophilic PVA fillers. As the PVA content increased from 10% to <em>20</em>%, protein flux decreased, with no TI permeating through <em>20</em>% PVA hydrogels. Further increase in model drug release was achieved by incorporating hydrophilic PVA fillers into the hydrogel. As filler molecular weight increased, TI flux increased. The mechanism for this is most likely an alteration in protein/gel interactions and transient variations in water content. The percent protein released was also altered by varying protein loading concentration. Release studies conducted using <em>growth</em> <em>factor</em> in vehicles with hydrophilic filler showed sustained release of platelet-derived <em>growth</em> <em>factor</em> (PDGF-beta,beta) for up to 3 days compared with less than 24 hours in the controls. In vitro bioactivity was demonstrated by doubling of normal human dermal <em>fibroblast</em> numbers when exposed to <em>growth</em> <em>factor</em>-loaded vehicle compared to control. The release vehicle developed in this study uses a rapid and simple fabrication method, and protein release can be tailored by modifying solid content, incorporating biocompatible hydrophilic fillers, and varying protein loading concentration.

Loss of heterozygosity at 10q26 was mapped using microsatellite markers in <em>20</em> osteosarcomas. A four-megabase region centered on marker D10S587 was affected by allelic loss in 60 percent of osteosarcomas. The most frequently lost marker was D10S1723. Around 15 known genes are found in this region. The gene immediately adjacent to D10S1723 encodes BUB3, an element of the spindle assembly mitotic checkpoint. Loss of BUB3 function could contribute to chromosomal instability. The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) gene is located 2 Mb from the BUB3 gene and has the potential for a role in cancer. Inherited mutations of the FGFR2 gene result in skeletal dysplasias. FGFR2 alterations have also been implicated in gastric cancer. Human genome project data were used to design primers for amplifying FGFR2 in 18 genomic segments and BUB3 in 7 genomic segments. In each case, the segments encompassed coding exons and flanking intron sequences. The primers were used to search for mutations by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Several shifted bands were detected in the BUB3 exon 3 fragment. Sequencing resolved the BUB3 exon 3 fragment shifts into polymorphisms in intron 2. No mutations of BUB3 or FGFR2 were detected. It remains possible that BUB3 or FGFR2 hemizygosity alone contributes to osteosarcoma, or that one of the genes is cryptically inactivated by a higher-order modification or mutation outside the coding region. There may also be a yet undiscovered tumor suppressor gene in this region.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) are both recognized as stimulators of migration and angiogenesis during the progression of melanoma. However, the timepoints during tumour progression at which the expression of these angiogenic <em>factors</em> is most essential is still controversial. Using immunohistochemical analyses, melanoma cells were found to express bFGF in 18 out of 19 primary tumours and in 13 out of <em>20</em> metastases. Eleven of the 19 primary tumours and 15 of the <em>20</em> metastases were found to contain VEGF-positive melanoma cells; five of the 19 patients showed no VEGF-expressing melanoma cells at all. This indicates that VEGF expression may be a later event in the progression of melanoma than bFGF expression. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of the melanoma cell lines showed that all cell lines were positive for both bFGF and VEGF mRNA. CD31-positive endothelial cells were primarily seen in the metastases (17 out of <em>20</em>). Only four of the primary tumours contained CD31-positive cells, but these tumours expressed bFGF as well as VEGF, indicating that both angiogenic <em>factors</em> may be important for the formation of vessels in tumours.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) is a heparin-binding polypeptide that acts as a neurotrophic <em>factor</em> for certain central and peripheral neurons. Acidic FGF was injected stereotaxically into the striatum of young (2-month-old) and aging (12-month-old) C57BL/6 mice that were treated 1 week before with systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP treatment (4 x <em>20</em> mg/kg, i.p. given 12 h apart) reduced tyrosine hydroxylase (TH)-immunoreactive (IR) fibers in the striatum and reduced dopamine (DA) concentration to 32% of the controls in young and <em>20</em>% of the controls in aging mouse brain 5 weeks after administration. Although the DA concentration recovered to 43% of the controls in young mice following stereotaxic injection of aFGF 5 weeks after MPTP treatment, aging mice with such treatment did not show a significant recovery of DA concentration. Computerized image analysis of TH-IR fibers in the striatum also showed significant recovery in young mice treated with aFGF, while aging mice did not show a significant recovery. We conclude that treatment of MPTP-depleted young mice with aFGF results in partial recovery in the nigrostriatal DA system but such benefits decline with age.