The aim of this study was to analyze the influence of non-surgical applications of diode laser (940 nm) on the cell proliferation and mRNA expressions of type I collagen and <em>growth</em> <em>factors</em> in human gingival <em>fibroblasts</em> (GF). Gingival <em>fibroblasts</em> were isolated from human gingival connective tissue of systemically healthy individuals. Cells were treated with different laser parameters as follows; (1) Infected pocket setting (power: 2 W, pulse interval: 1 ms, pulse length: 1 ms, <em>20</em> s/cm(2)); (2) Perio-pocket setting (power: 1.5 W, pulse interval: <em>20</em> ms, pulse length: <em>20</em> ms, <em>20</em> s/cm(2)); and (3) Biostimulation setting (power: 0.3 W in continuous wave, <em>20</em> s/cm(2)). Proliferation of GF was evaluated after different laser applications using a real-time cell analyzer. Total RNA was isolated on day 2 and cDNA synthesis was performed. Type I collagen, insulin-like <em>growth</em> <em>factor</em> (IGF), vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and transforming <em>growth</em> <em>factor</em>-beta (TGF-β) mRNA expressions were determined with quantitative RT-PCR. In a proliferation experiment, no significant differences were observed in the different laser applications when compared to the control group. Statistically significant increases in IGF, VEGF, and TGF-β mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). A significant increase in collagen type I mRNA expression was noted in only biostimulation set-up of diode laser (p < 0.05). The results of this study demonstrate that non-surgical laser applications modulate behavior of gingival <em>fibroblasts</em> inducing <em>growth</em> <em>factors</em> mRNA expressions and these applications can be used to improve periodontal wound healing.

Mitophagy, which is a conserved cellular process for selectively removing damaged or unwanted mitochondria, is critical for mitochondrial quality control and the maintenance of normal cellular physiology. However, the precise mechanisms underlying mitophagy remain largely unknown. Prior studies on mitophagy focused on the events in the mitochondrial outer membrane. PHB2 (prohibitin 2), which is a highly conserved membrane scaffold protein, was recently identified as a novel inner membrane mitophagy receptor that mediates mitophagy. Here, we report a new signaling pathway for PHB2-mediated mitophagy. Upon mitochondrial membrane depolarization or misfolded protein aggregation, PHB2 depletion destabilizes PINK1 in the mitochondria, which blocks the mitochondrial recruitment of PRKN/Parkin, ubiquitin and OPTN (optineurin), leading to an inhibition of mitophagy. In addition, PHB2 overexpression directly induces PRKN recruitment to the mitochondria. Moreover, PHB2-mediated mitophagy is dependent on the mitochondrial inner membrane protease PARL, which interacts with PHB2 and is activated upon PHB2 depletion. Furthermore, PGAM5, which is processed by PARL, participates in PHB2-mediated PINK1 stabilization. Finally, a ligand of PHB proteins that we synthesized, called FL3, was found to strongly inhibit PHB2-mediated mitophagy and to effectively block cancer cell <em>growth</em> and energy production at nanomolar concentrations. Thus, our findings reveal that the PHB2-PARL-PGAM5-PINK1 axis is a novel pathway of PHB2-mediated mitophagy and that targeting PHB2 with the chemical compound FL3 is a promising strategy for cancer therapy. <b>Abbreviations</b>: AIFM1: apoptosis inducing <em>factor</em> mitochondria associated 1; ATP5F1A/ATP5A1: ATP synthase F1 subunit alpha; BAF: bafilomycin A₁; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chemical reagent carbonyl cyanide m-chlorophenyl hydrazine; FL3: flavaglines compound 3; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryo <em>fibroblasts</em>; MPP: mitochondrial-processing peptidase; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; MTS: mitochondrial targeting sequence; OA: oligomycin and antimycin A; OPTN: optineurin; OTC: ornithine carbamoyltransferase; PARL: presenilin associated rhomboid like; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB: prohibitin; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Roc-A: rocaglamide A; TOMM<em>20</em>: translocase of outer mitochondrial membrane <em>20</em>; TUBB: tubulin beta class I.

Parkinson's disease (PD), one of the most common human neurodegenerative disorders, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Our recent case-control association study of 268 SNPs in 121 candidate genes identified alpha-synuclein (SNCA) as a susceptibility gene for sporadic PD (P = 1.7 x 10(-11)). We also replicated the association of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF<em>20</em>) with PD (P = 0.0089). To find other susceptibility genes, we added 34 SNPs to the previous screen. Of 302 SNPs in a total 137 genes, but excluding SNCA, SNPs in NDUFV2, FGF2, CALB1 and B2M showed significant association (P < 0.01; 882 cases and 938 control subjects). We replicated the association analysis for these SNPs in a second independent sample set (521 cases and 1,003 control subjects). One SNP, rs1805874 in calbindin 1 (CALB1), showed significance in both analyses (P = 7.1 x 10(-5); recessive model). When the analysis was stratified relative to the SNCA genotype, the odds ratio of CALB1 tended to increase according to the number of protective alleles in SNCA. In contrast, FGF<em>20</em> was significant only in the subgroup of SNCA homozygote of risk allele. CALB1 is a calcium-binding protein that widely is expressed in neurons. A relative sparing of CALB1-positive dopaminergic neurons is observed in PD brains, compared with CALB1-negative neurons. Our genetic analysis suggests that CALB1 is associated with PD independently of SNCA, and that FGF<em>20</em> is associated with PD synergistically with SNCA.

<em>Fibroblast</em> migration is directed by gradients of platelet-derived <em>growth</em> <em>factor</em> (PDGF) during wound healing. As in other chemotactic systems, it has been shown recently that localized stimulation of intracellular phosphoinositide (PI) 3-kinase activity and production of 3' PI lipids in the plasma membrane are important events in the signaling of spatially biased motility processes. In turn, 3' PI localization depends on the effective diffusion coefficient, D, and turnover rate constant, k, of these lipids. Here we present a systematic and direct comparison of mathematical model calculations and experimental measurements to estimate the values of the effective 3' PI diffusion coefficient, D, turnover rate constant, k, and other parameters in individual <em>fibroblasts</em> stimulated uniformly with PDGF. In the context of our uniform stimulation model, the values of D and k in each cell were typically estimated within 10-<em>20</em>% or less, and the mean values across all of the cells analyzed were D = 0.37 +/- 0.25 microm2/s and k = 1.18 +/- 0.54 min(-1). In addition, we report that 3' PI turnover is not affected by PDGF receptor signaling in our cells, allowing us to focus our attention on the regulation of 3' PI production as this system is studied further.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has recently been shown to be a mitogen for keratinocytes. This observation has now been extended in a porcine model of epidermal wound healing. A single application of recombinant human bFGF given at the time of injury to healthy animals accelerated the rate of epithelialization by <em>20</em>%; multiple applications gave no greater effect than the single application. Histologic analysis of biopsies of these partial-thickness wounds taken during bFGF-mediated healing supported the assessment of an enhanced rate of epithelialization and an earlier onset of dermal healing. Because no histologic abnormalities were observed, bFGF induced an acceleration of what appears to be the normal healing process.

Following the introduction of cholinesterase inhibitors in 1986 and a <em>20</em>-yr long period of successful clinical application in mild, moderate and severe patients, the treatment of AD has turned to modify the course of pathological processes thought to comprise the disease. Several active and passive vaccines are presently under investigation for efficacy, reducing amyloid-beta in the brain of patients with mild-moderately advanced disease. Three large international immunization trials are in progress in US and Europe on mild-moderate AD patients. Among these, the most advanced trial in time is the humanized antibody trial. In addition, drugs aiming to reduce tau phosphorylation (GSK3 inhibitors) are about to enter clinical phases of development. Due to intrinsic difficulties, the developments of gamma-and beta-secretase inhibitors have not yet reached clinical stages. Only one anti-amyloid-aggregation, an aminoglycan compound, and one anti-APO-E approach with rosiglitazone are currently in clinical testing. Stem-cell therapy and gene-replacing therapy remain experimental and far from clinical application. Based on experimental evidence that NGF (nerve <em>growth</em> <em>factor</em>) treatment could provide prolonged protection of the central cholinergic system, i.c.v. infusion of NGF, with genetically modified <em>fibroblasts</em> or gene therapy are under current investigation. NGF treatment could probably double the clinical effect of ChEIs in time. Given the level of scientific and clinical activity it is reasonable to expect that within the next five to ten years a new therapy for AD will, by blocking disease progression, both produce long term stabilization of at least 5 years in patients with AD and prevent or delay emergence in persons at risk for AD.

nur77 is an immediate-early gene inducible by nerve <em>growth</em> <em>factor</em> or membrane depolarization in the rat pheochromocytoma cell line PC12 and by serum <em>growth</em> <em>factors</em> in <em>fibroblasts</em>. The nur77-encoded protein is a member of the steroid/thyroid hormone receptor superfamily and can act as a potent transcription activator. The induction of nur77 in PC12 cells is rapid and transient, with kinetics similar to those of the c-fos protooncogene. Induction does not require de novo protein synthesis. Whereas transcriptional activation of c-fos by nerve <em>growth</em> <em>factor</em> in PC12 cells requires a <em>20</em>-base pair serum response element in its promoter, there is no such sequence in the nur77 promoter. To understand the mechanism for the activation of nur77, we have analyzed the inducibility of a series of transfected nur77 minigenes using an S1 nuclease protection assay. We identified the sequence 22-86 nucleotides upstream of the transcription start site as necessary and sufficient for nur77 induction by nerve <em>growth</em> <em>factor</em> and membrane depolarization in PC12 cells. Sequences farther upstream enhance the induction. Analysis of base substitution mutations allowed us to identify three sequence elements within this region that are essential for induction. These sequence elements include two copies of an AP1-like element and a GC-rich sequence. Unlike transcriptional activation of c-fos, the sequence requirements for the activation of nur77 by nerve <em>growth</em> <em>factor</em> and membrane depolarization cannot be readily separated. Taken together, our data suggest that activation of nur77 and c-fos by nerve <em>growth</em> <em>factor</em> occurs through different mechanisms in PC12 cells.

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, <em>20</em>00). 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 (<em>20</em> 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 (15 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.

OBJECTIVE

To study the corneal biocompatibility of bevacizumab on various cultured human corneal cells.

METHODS

Cell cultures of corneal keratinocytes (CKs), corneal fibroblasts (CFs), and corneal endothelial cells (CECs) were harvested from human donor eyes and exposed to various concentrations of bevacizumab (0.25-5.0 mg/mL). Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at days 1 and 4 after exposure. For cytotoxicity testing, confluent cells were cultured in serum-depleted medium, and the MTT test was performed after 24 hours of incubation. Expression of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR1 and VEGFR2), keratan sulphate (KS), and cytokeratin-3 (AE5) was studied by immunohistochemistry. Live/dead viability/cytotoxicity assay was performed and analyzed by fluorescence microscopy after 24 hours of incubation. Cell morphology was assessed with a phase-contrast microscope after 7 days of exposure with different concentrations of bevacizumab (0.25-5.0 mg/mL), and signs of cellular damage were assessed.

RESULTS

No cytotoxic effect of bevacizumab on CKs, CFs, and CECs could be observed when used at a concentration of 5.0 mg/mL or lower. Bevacizumab-treated cells showed no signs of cellular damage compared with the control. CKs, CFs, and CECs stained positively for VEGF, VEGFR1, and VEGFR2. CKs and CECs stained positively for AE5, whereas CFs were immunopositive for KS.

CONCLUSIONS

Bevacizumab is not toxic to corneal cells of human origin in vitro at doses usually used for treatment of corneal neovascularization, which is 20-fold higher than that used for intravitreal application.

Nuclear hormone receptor coregulator (NRC) is a 2,063-amino-acid coregulator of nuclear hormone receptors and other transcription <em>factors</em> (e.g., c-Fos, c-Jun, and NF-kappaB). We and others have generated C57BL/6-129S6 hybrid (C57/129) NRC(+/-) mice that appear outwardly normal and grow and reproduce. In contrast, homozygous deletion of the NRC gene is embryonic lethal. NRC(-/-) embryos are always smaller than NRC(+/+) embryos, and NRC(-/-) embryos die between 8.5 and 12.5 days postcoitus (dpc), suggesting that NRC has a pleotrophic effect on <em>growth</em>. To study this, we derived mouse embryonic <em>fibroblasts</em> (MEFs) from 12.5-dpc embryos, which revealed that NRC(-/-) MEFs exhibit a high rate of apoptosis. Furthermore, a small interfering RNA that targets mouse NRC leads to enhanced apoptosis of wild-type MEFs. The finding that C57/129 NRC(+/-) mice exhibit no apparent phenotype prompted us to develop 129S6 NRC(+/-) mice, since the phenotype(s) of certain gene deletions may be strain dependent. In contrast with C57/129 NRC(+/-) females, <em>20</em>% of 129S6 NRC(+/-) females are infertile while 80% are hypofertile. The 129S6 NRC(+/-) males produce offspring when crossed with wild-type 129S6 females, although fertility is reduced. The 129S6 NRC(+/-) mice tend to be stunted in their <em>growth</em> compared with their wild-type littermates and exhibit increased postnatal mortality. Lastly, both C57/129 NRC(+/-) and 129S6 NRC(+/-) mice exhibit a spontaneous wound healing defect, indicating that NRC plays an important role in that process. Our findings reveal that NRC is a coregulator that controls many cellular and physiologic processes ranging from <em>growth</em> and development to reproduction and wound repair.

Ultraviolet (UV) light is one of the major <em>factors</em> implicated in the pathogenesis of pterygium. The mechanism by which UV light induces this disease remains elusive. The aim of this study was to evaluate the effects of UVB irradiation on the expression of <em>growth</em> <em>factors</em> in cultured pterygium epithelial cells and to demonstrate their distribution within pterygium. We cultured pterygial epithelial cells from pterygium explants and these cells were exposed to <em>20</em> mJ/cm(2) of UVB. Total RNA was extracted at 0, 6, and 12 hours after irradiation. (32)P-labeled cDNA was synthesized and analyzed using microarray technology to determine the differential expression of 268 <em>growth</em> <em>factor</em> and cytokine related genes. Semiquantitative reverse transcriptase-polymerase chain reaction was used to corroborate this data. Conditioned media derived from cells exposed to UVB irradiation was analyzed for protein expression by enzyme-linked immunosorbent assay. Immunohistochemistry was used to evaluate the distribution of heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em> (HB-EGF) in pterygium tissue. Analysis of the hybridization signals revealed that the genes encoding HB-EGF, <em>fibroblast</em> <em>growth</em> <em>factor</em> 3, and cytotoxic trail ligand receptor were consistently elevated at 6 and 12 hours after UVB treatment. HB-EGF mRNA was elevated 6.8-fold at 6 hours after irradiation and was augmented in culture supernatants after the same treatment. Furthermore, HB-EGF reactivity was identified in the epithelium and vasculature of pterygium by immunohistochemistry. HB-EGF was present in normal limbal epithelium, although it was not induced in cultured limbal epithelial cells by UV irradiation. HB-EGF is a potent mitogen, localized in pterygium tissue, and significantly induced by UVB in pterygium-derived epithelial cells. We postulate that this <em>growth</em> <em>factor</em> is a major driving force in the development of pterygia and a means by which UV irradiation causes the pathogenesis of pterygium.

We show here using synchronized Swiss mouse 3T3 <em>fibroblasts</em> that p70 S6 kinase (p70S6k) and mitogen-activated protein kinases (p42mapk/p44mapk) are not only activated at the G0/G1 boundary, but also in cells progressing from M into G1. p70S6k activity increases <em>20</em>-fold in G1 cells released from G0. Throughout G1, S, and G2 it decreases constantly, so that during M phase low kinase activity is measured. The kinase is reactivated 10-fold when cells released from a nocodazole-induced metaphase block enter G1 of the next cell cycle. p42mapk/p44mapk in G0 cells are activated transiently early in G1 and are reactivated late in mitosis after nocodazole release. p70S6k activity is dependent on permanent signaling from <em>growth</em> <em>factors</em> at all stages of the cell cycle. Immunofluorescence studies showed that p70S6k and its isoform p85S6k become concentrated in localized spots in the nucleus at certain stages in the cell cycle. Cell cycle-dependent changes in p70S6k activity are associated with alterations in the phosphorylation state of the protein. However, examination of the regulation of a p70S6k mutant in which the four carboxyl-terminal phosphorylation sites are changed to acidic amino acids suggests that a mechanism independent of these phosphorylation sites controls the activity of the enzyme during the cell cycle.

Platelet-derived <em>growth</em> <em>factor</em>-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured <em>fibroblasts</em> with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with <em>20</em> microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.

Osteoporosis is a skeletal disorder characterized by low bone mass and deterioration of bone microarchitecture resulting in bone fragility, which impairs fixation of the implants. Zoledronic acid (ZOL) is a potential inhibitor of osteoclast-mediated bone resorption and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a <em>growth</em> <em>factor</em> that stimulates osteoblast-mediated bone formation, and these drugs could enhance fixation of implants under osteoporotic conditions. In this study, 40 ovariectomized (OVX) rats were randomly divided into 4 groups (n=10 for each group) and underwent bilateral tibiae implantation using hydroxyapatite (HA)-coated titanium implant: Control group (distilled water immersing before implantation), ZOL group (1 mg/ml of ZOL immersing), bFGF group (<em>20</em> microg/ml of bFGF immersing), and ZOL+bFGF group (1 mg/ml of ZOL and <em>20</em> microg/ml of bFGF immersing). At 3 months after implantation, all animal were sacrificed and the tibiae were harvested for histology, micro-CT examinations and biomechanical testing. Bone area and contact, determined by histomorphometric analysis, were 2.7-fold and 1.8-fold in the ZOL-treated implants, 1.9-fold and 1.8-fold in the bFGF-treated implants, 3.6-fold and 2.3-fold in the both-treated implants compared with controls (p<0.01). Such significant effects were further confirmed by microstructure parameters, the bone volume ratio and the percentage osteointegration were significantly increased by ZOL treatment (3.0-fold and 1.8-fold), bFGF treatment (1.2-fold and 1.9-fold) and ZOL+bFGF treatment (3.3-fold and 2.7-fold) (p<0.001). In addition, push-out test showed that the maximum force and the corresponding interfacial shear strength of the implants treated by ZOL, bFGF and ZOL+bFGF was 8.4-fold and 8.6-fold, 3.8-fold and 3.7-fold, 10.8-fold and 10.7-fold of the control levels, respectively (p<0.05). The combined treatment was better than either treatment alone for force, but was not different from ZOL alone for interfacial strength. The significant correlation between biomechanical and micro-CT parameters demonstrates the role of microstructure assessments in predicting mechanical fixation of implants (p<0.01). Our study suggests that locally applied ZOL or bFGF may improve implant fixation in the ovariectomized rats, and that combined treatment has more beneficial effects on osseointegration, peri-implant bone formation and maximum force than either intervention alone.

We generated transgenic (TG) mice overexpressing <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 protein (22- to 34-fold) in the heart. Chronic FGF-2 overexpression revealed no significant effect on heart weight-to-body weight ratio or expression of cardiac differentiation markers. There was, however, a significant <em>20</em>% increase in capillary density. Although there was no change in FGF receptor-1 expression, relative levels of phosphorylated c-Jun NH(2)-terminal kinase and p38 kinase as well as of membrane-associated protein kinase C (PKC)-alpha and total PKC-epsilon were increased in FGF-2-TG mouse hearts. An isolated mouse heart model of ischemia-reperfusion injury was used to assess the potential of increased endogenous FGF-2 for cardioprotection. A significant 34-45% increase in myocyte viability, reflected in a decrease in lactate dehydrogenase released into the perfusate, was observed in FGF-2 overexpressing mice and non-TG mice treated exogenously with FGF-2. In conclusion, FGF-2 overexpression causes augmentation of signal transduction pathways and increased resistance to ischemic injury. Thus, stimulation of endogenous FGF-2 expression offers a potential mechanism to enhance cardioprotection.

Accumulating evidence suggests the involvement of chondroitin sulfate (CS) and dermatan sulfate (DS) hybrid chains in the brain's development and critical roles for oversulfated disaccharides and IdoUA residues in the <em>growth</em> <em>factor</em>-binding and neuritogenic activities of these chains. In the pursuit of sources of CS/DS with unique structures, neuritogenic activity, and therapeutic potential, two novel CS/DS preparations were isolated from shark liver by anion exchange chromatography. The major (80%) low sulfated and minor (<em>20</em>%) highly sulfated fractions had an average molecular mass of 3.8-38.9 and 75.7 kDa, respectively. Digestion with various chondroitinases (CSases) revealed a large panel of disaccharides with either GlcUA or IdoUA scattered along the polysaccharide chains in both of the fractions. The higher M(r) fraction, richer in IdoUA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate) and GlcUAbeta/IdoUAalpha1-3GalNAc(4,6-O-disulfate) units, exerted greater neurite out<em>growth</em>-promoting (NOP) activity and better promoted the binding of various heparin-binding <em>growth</em> <em>factors</em>, including pleiotrophin (PTN), midkine, recombinant human heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em>, VEGF(165), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, <em>fibroblast</em> <em>growth</em> <em>factor</em>-7, and hepatocyte <em>growth</em> <em>factor</em> (HGF). These activities were largely abolished by digestion with CSase ABC or B but only moderately affected by a mixture of CSases AC-I and AC-II. In addition, the NOP activity of the larger fraction was markedly reduced by desulfation with alkali, suggesting a role for the 2-O-sulfate of IdoUA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate). The NOP activity of the higher molecular weight fraction and that of the embryonic pig brain-derived CS/DS fraction were also sup pressed to a large extent by antibodies against HGF, PTN, and their individual receptors cMet and anaplastic lymphoma kinase, revealing the involvement of the HGF and PTN signaling pathways in the activity.

DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial-mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days <em>20</em>-34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming <em>growth</em> <em>factor</em>-β (TGFβ), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into <em>fibroblast</em>-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8 h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.

Despite numerous studies on the effects of gonadotropins on ovarian cells in tissue culture, the <em>factors</em> controlling the proliferation of granulosa cells in vitro remain unknown. We have examined the effect of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and epidermal <em>growth</em> <em>factor</em> (EGF) on granulosa cell proliferation in vitro in an attempt to clarify their possible roles in the control of ovarian development. FGF and EGF both stimulate DNA synthesis in resting populations of granulosa cells. The half-maximal response forthis effect with FGF was observed at 4 X 10(-11)M and with EGF at 1.5 X 10(-13)M. Autoradiography demonstrated that the whole cell population initiated DNA synthesis in the presence of either EGF or FGF, thus precluding an additive effect of the two mitogens. When cells were maintained at low density (100 cells/cm2) in the presence of low serum (1%) they divided with a doubling time of 48-72 h, but addition of either EGF or FGF accelerated their proliferation. The doubling time observed in the presence of FGF was 16 h versus <em>20</em> h with EGF and the final cell density reached in the presence of EGF or FGF was <em>20</em> times that of cells maintained in the presence of 1% calf serum alone. In the presence of 10% serum, granulosa cells had a doubling time of 24 h and the final density reached was similar to that observed in 1% serum with EGF and FGF. Addition of EGF or FGF to 10% serum resulted in a final density 3 to 4-fold higher than that observed with 10% serum alone. The ultrastructure of the granulosa cells grown in the presence of EGF or FGF was similar to that of cells maintained in the absence of added mitogens. The only marked difference was that cells grown in the presence of FGF or EGF had a high lipid granule content while cells grown in their absence had a low lipid granule content. The effect of various concentrations of FGF and EGF on the proliferation of granulosa cells has been analyzed. The minimal effective dose of EGF was 3 X 10(-14)M and saturation was observed at 3 X 10(-11)M, with a half-maximal response at 6 X 10(-13)M. With FGF the minimal dose stimulating proliferation was 1.5 X 10(-12)M and saturation was achieved at 1.5 X 10(-10)M, with a half-maximal response at 3 X 10(-11)M. Our results show that EGF and FGF are the most potent mitogens ever observed and are mitogenic for granulosa cells at 300 to 3000-fold lower concentrations than for other cell types which have been studied, such as <em>fibroblasts</em> or lens epithelial cells.

<em>Factor</em> H (FH) of the complement system acts as a regulatory co<em>factor</em> for the <em>factor</em> I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of <em>20</em> short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-16 to SCR-<em>20</em> is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-<em>20</em> substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-<em>20</em>. As this surface coincides with conserved basic residues in the C-terminal SCR-<em>20</em> domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>-heparin complex. A tenth substitution and another conserved basic residue in SCR-19 are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney.

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day 15-<em>20</em>), 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 <em>20</em>, and decreased dramatically after birth. Angiotensin II-induced <em>growth</em> in Agtr2(+) VSMC (embryonic day <em>20</em>) 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-<em>20</em> min. Activation by T4 of STAT3 was maximal at 30 min (15+/-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.

The aim of this investigation was to determine the effect of <em>growth</em> <em>factor</em> treatment on ovine meniscal chondrocyte (OMC) proliferation in vitro and on the production of matrix proteins by OMCs grown within a polyglycolic acid (PGA) scaffold. Analysis of 72-h monolayer cultures using the mean transit time (MTT) assay revealed a greater increase in OMC numbers in the presence of platelet-derived <em>growth</em> <em>factor</em> (PDGF)-AB, PDGF-BB, insulin-like <em>growth</em> <em>factor</em> (IGF)-I, transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) than in untreated controls. In contrast, IGF-II and bone morphogenetic protein-2 had no effect on OMC proliferation at the concentrations tested. The <em>growth</em> <em>factors</em> that elicited the greatest proliferative response (PDGF-AB, PDGF-BB, TGF-beta1, and IGF-I) were subsequently tested for their ability to enhance OMC proliferation and differentiation within PGA scaffolds. Biochemical analysis revealed less glycosaminoglycan (GAG) production in the presence of all <em>growth</em> <em>factors</em> tested compared to untreated control samples. In contrast, all of the <em>growth</em> <em>factors</em> increased collagen type I production by OMCs within the scaffolds at day <em>20</em>, and all except PDGF-BB resulted in an increase at day 39, when compared to appropriate control samples. With the exception of IGF-I, none of the <em>growth</em> <em>factors</em> tested had any significant effect on collagen type II production. Histological staining of sections from OMC-PGA scaffolds did not reveal any difference in GAG or collagen production between the treatment groups. However, immunohistochemical analysis demonstrated an increase in collagen type I expression and a decrease in collagen type II at day 39 in all <em>growth</em> <em>factor</em>treated constructs, concomitant with a high infiltration of cells. This suggests that PDGF-AB, PDGF-BB, TGF-beta1, and IGF-1 may be useful in future tissue engineering studies for promoting meniscal cell proliferation and differentiation within scaffolds.

Somatomedins/insulin-like <em>growth</em> <em>factors</em> (Sm/IGFs) are present in blood and in extracts from multiple tissues of the human fetus and induce the proliferation of cultured human fetal cells. To identify the cellular location of immunoreactive Sm/IGF in human fetal tissues, we have performed immunocytochemistry in tissues from prostaglandin-induced human fetal abortuses of 12 to <em>20</em> wk in gestation. Every tissue studied except the cerebral cortex contains Sm/IGF immunoreactive cells. Cells staining positively include hepatocytes, hepatic hemopoietic cells, columnar epithelia of the pulmonary airways, intestine and kidney tubules, adrenal cortical cells, dermal cells, skeletal and cardiac muscle fibers, and pancreatic islet and acinar cells. Immunostaining was specific for Sm/IGFs, but because of the cross-reactivity of the antibodies it was not possible to determine whether the immunoreactivity represented Sm-C/IGF I, IGF II, or both. Liver contained the greatest proportion of immunoreactive cells, while the thymus and spleen had only a few immunostained cells. With the possible exception of dermal and some adrenal cortical cells, the immunoreactive cells do not appear to be the primary sites of Sm/IGF synthesis, because parallel in situ hybridization histochemical studies using Sm/IGF oligodeoxyribonucleotide probes show that Sm/IGF mRNAs are localized predominantly to <em>fibroblasts</em> and mesenchymal cells. Therefore the immunoreactive cells identified in this study may define sites of action of Sm/IGFs.

Microarray gene expression profiling revealed fibromodulin (FMOD) is among differentially expressed genes in leiomyoma (L) and myometrium. Using realtime PCR, western blotting and immunohistochemistry, we validated the expression of FMOD in paired leiomyoma and myometrium (N = <em>20</em>) during the menstrual cycle, from women who received gonadotropin-releasing hormone analogue (GnRHa) therapy (N = 7) and in leiomyoma and myometrial (M) smooth muscle cells (SMC) due to transforming <em>growth</em> <em>factor</em> (TGF)-beta and GnRHa treatment. The results indicated that FMOD is expressed at significantly higher levels in leiomyoma as compared to myometrium from proliferative phase (two- to three-folds; P < 0.05), but not the secretory phase of the menstrual cycle, whereas GnRHa therapy reduced FMOD expression to levels detected in myometrium from proliferative phase (P = 0.05). By using western blotting and immunohistochemistry immunoreactive FMOD was detected in leiomyoma and myometrial tissue-extract and in LSMC and MSMC, connective tissue <em>fibroblasts</em> and arterial walls. In a time- and cell-dependent manner, TGF-beta1 (2.5 ng/ml) increased the expression of FMOD in MSMC, whereas GnRHa (0.1 microM) inhibited that in MSMC and LSMC (P < 0.05). The effect of TGF-beta and GnRHa on FMOD expression was reversed following pretreatment of LSMC and MSMC with Smad3 SiRNA and U0126 (MEK1/2 inhibitor), respectively. In summary, menstrual cycle-dependent expression of FMOD and suppression following GnRHa therapy in leiomyoma and myometrium, as well as differential regulation by TGF-beta and GnRHa in vitro suggests that FMOD, a key regulator of tissue organization, plays a critical role in leiomyoma fibrotic characteristics.