Human corneal endothelial cells (HCEC) were isolated by means of enzymatic treatment of excised corneas. The corneas were incubated for 1.5 hr together with a high concentration of collagenase (0.5%), followed by a long-term incubation (up to 16 hr) using a low concentration of the enzyme (0.04%). Endothelial cells were enriched against contaminating <em>fibroblasts</em> by using a selective L-valine-free medium which inhibited <em>fibroblast</em> <em>growth</em> during the first passages. Subcultures of HCEC were passaged for more than <em>20</em> generations without showing signs of senescence. Laminin and chondroitin sulfate functioned as a substrate for HCEC, promoting proliferation and allowing the cells to grow in monolayer formation. The inclusion of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) as well as chondroitin sulfate in the medium led to an additional increase in the rate of proliferation.

OBJECTIVE

The primary objective of this study was to determine whether intracoronary administration of the adenoviral gene for fibroblast growth factor (Ad5FGF-4) can improve myocardial perfusion compared with placebo.

BACKGROUND

Animal studies and observational clinical studies have shown improvement in perfusion of the ischemic myocardium using genes encoding angiogenic growth factors; however, randomized, double-blind data in humans are lacking.

METHODS

We performed a randomized, double-blind, placebo-controlled trial of intracoronary injection of 10(10) adenoviral particles containing a gene encoding fibroblast growth factor (Ad5FGF-4) to determine the effect on myocardial perfusion. Fifty-two patients with stable angina and reversible ischemia comprising >9% of the left ventricle on adenosine single-photon emission computed tomography (SPECT) imaging were randomized to gene therapy (n = 35) or placebo (n = 17). Clinical follow-up was performed, and 51 (98%) patients underwent a second adenosine SPECT scan after 8 weeks.

RESULTS

Overall (n = 52), the mean total perfusion defect size at baseline was 32.4% of the left ventricle, with 20% reversible ischemia and 12.5% scar. At eight weeks, Ad5FGF-4 injection resulted in a significant reduction of ischemic defect size (4.2% absolute, 21% relative; p < 0.001) and placebo-treated patients had no improvement (p = 0.32). Although the change in reversible perfusion defect size between Ad5FGF-4 and placebo was not significant (4.2% vs. 1.6%, p = 0.14), when a single outlier was excluded a significant difference was observed (4.2% vs. 0.8%, p < 0.05). Ad5FGF-4 was well tolerated and did not result in any permanent adverse sequelae.

CONCLUSIONS

Intracoronary injection of Ad5FGF-4 showed an encouraging trend for improved myocardial perfusion; however, further studies of therapeutic angiogenesis with Ad5FGF-4 will be necessary.

Signal transducer and activator of transcription 3 (Stat3) is a key mediator in the development of many cancers. For <em>20</em> years, it has been assumed that Stat3 mediates its biological activities as a nuclear localized transcription <em>factor</em> activated by many cytokines. However, recent studies from this laboratory and others indicate that Stat3 has an independent function in the mitochondria (mitoStat3) where it controls the activity of the electron transport chain (ETC) and mediates Ras-induced transformation of mouse embryo <em>fibroblasts</em>. The actions of mitoStat3 in controlling respiration and Ras transformation are mediated by the phosphorylation state of serine 727. To address the role of mitoStat3 in the pathogenesis of cells that are transformed, we used 4T1 breast cancer cells, which form tumors that metastasize in immunocompetent mice. Substitution of Ser-727 for an alanine or aspartate in Stat3 that has a mitochondrial localization sequence, MLS-Stat3, has profound effects on tumor <em>growth</em>, complex I activity of the ETC, and accumulation of reactive oxygen species (ROS). Cells expressing MLS-Stat3(S727A) display slower tumor <em>growth</em>, decreased complex I activity of the ETC, and increased ROS accumulation under hypoxia compared with cells expressing MLS-Stat3. In contrast, cells expressing MLS-Stat3(S727D) show enhanced tumor <em>growth</em> and complex I activity and decreased production of ROS. These results highlight the importance of serine 727 of mitoStat3 in breast cancer and suggest a novel role for mitoStat3 in regulation of ROS concentrations through its action on the ETC.

The binding interactions for the three primary reactants of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the co<em>factor</em> heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<=>>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<=>>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<=>>bFGF/HS, K3 = 470 (+/- <em>20</em>) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed <em>growth</em> <em>factor</em> and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Our preliminary study demonstrated that 70% ethanol Cortidis Rhizoma extracts (CR) had a hypoglycemic action in diabetic animal models. We determined whether CR fractions acted as anti-diabetic agent, and a subsequent investigation of the action mechanism of the major compound, berberine ([C(<em>20</em>)H(18)NO(4)](+)), was carried out in vitro. The <em>20</em>, 40 and 60% methanol fractions from the XAD-4 column contained the most insulin sensitizing activities in 3T3-L1 adipocytes. The common major peak in these fractions was berberine. Treatment with 50 microM berberine plus differentiation inducers significantly reduced triglyceride accumulation by decreased differentiation of 3T3-L1 <em>fibroblasts</em> to adipocytes and triglyceride synthesis. Significant insulin sensitizing activity was observed in 3T3-L1 adipocytes which were given 50 microM berberine plus 0.2 nM insulin to reach a glucose uptake level increased by 10 nM of insulin alone. This was associated with increased glucose transporter-4 translocation into the plasma membrane via enhancing insulin signaling pathways and the insulin receptor substrate-1-phosphoinositide 3 Kinase-Akt. Berberine also increased glucose-stimulated insulin secretion and proliferation in Min6 cells via an enhanced insulin/insulin-like <em>growth</em> <em>factor</em>-1 signaling cascade. Data suggested that berberine can act as an effective insulin sensitizing and insulinotropic agent. Therefore, berberine can be used as anti-diabetic agent for obese diabetic patients.

The regulation of fibronectin (FN) biosynthesis by dexamethasone (a synthetic glucocorticoid), forskolin (an activator of adenylate cyclase), and transforming <em>growth</em> <em>factor</em> beta (TGF-beta) was examined in six human cell lines. Dexamethasone treatment produced the largest increase in FN biosynthesis in the fibrosarcoma cell line, HT-1080 (approximately 45-fold). This seems to result from a dexamethasone-mediated increase in FN mRNA stability which increases the message half-life from approximately 11 to 26 h. The relative instability of FN mRNA in the fibrosarcoma (t1/2 11 h) compared to normal <em>fibroblasts</em> (70 h) appears to result from the particular transformed phenotype of the HT-1080 cells. Forskolin and TGF-beta increase the rate of FN gene transcription in most of the cell lines. These effects (four- to six-fold) occur rapidly and do not require protein synthesis in the responsive cell lines which include normal <em>fibroblasts</em>. However, in the fibrosarcoma (HT-1080), a surprisingly large induction (<em>20</em>-30-fold) is observed and this induction is different from that in the normal <em>fibroblasts</em> and the other cell lines in that both protein synthesis and a lag period are required. Synergism is seen with dexamethasone and either forskolin or TGF-beta in HT-1080 cells increasing the rate of FN biosynthesis approximately <em>20</em>0-fold to a level similar to normal <em>fibroblasts</em>. This seems to result from a combination of FN mRNA stabilization (dexamethasone) and increased transcription (forskolin and TGF-beta).

BACKGROUND

Anti-angiogenic treatment is believed to have at least cystostatic effects in highly vascularized tumours like pancreatic cancer. In this study, the treatment effects of the angiogenesis inhibitor Cilengitide and gemcitabine were compared with gemcitabine alone in patients with advanced unresectable pancreatic cancer.

METHODS

A multi-national, open-label, controlled, randomized, parallel-group, phase II pilot study was conducted in <em>20</em> centers in 7 countries. Cilengitide was administered at 600 mg/m2 twice weekly for 4 weeks per cycle and gemcitabine at 1000 mg/m2 for 3 weeks followed by a week of rest per cycle. The planned treatment period was 6 four-week cycles. The primary endpoint of the study was overall survival and the secondary endpoints were progression-free survival (PFS), response rate, quality of life (QoL), effects on biological markers of disease (CA 19.9) and angiogenesis (vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>), and safety. An ancillary study investigated the pharmacokinetics of both drugs in a subset of patients.

RESULTS

Eighty-nine patients were randomized. The median overall survival was 6.7 months for Cilengitide and gemcitabine and 7.7 months for gemcitabine alone. The median PFS times were 3.6 months and 3.8 months, respectively. The overall response rates were 17% and 14%, and the tumor growth control rates were 54% and 56%, respectively. Changes in the levels of CA 19.9 went in line with the clinical course of the disease, but no apparent relationships were seen with the biological markers of angiogenesis. QoL and safety evaluations were comparable between treatment groups. Pharmacokinetic studies showed no influence of gemcitabine on the pharmacokinetic parameters of Cilengitide and vice versa.

CONCLUSIONS

There were no clinically important differences observed regarding efficacy, safety and QoL between the groups. The observations lay in the range of other clinical studies in this setting. The combination regimen was well tolerated with no adverse effects on the safety, tolerability and pharmacokinetics of either agent.

The novel hematopoietic <em>growth</em> <em>factor</em> FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell <em>factor</em>), and FMS. the receptor for M-CSF (macrophage colony- stimulating <em>factor</em>). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (IL-3), PIXY-321 (an IL-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced <em>growth</em> of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-7, IL-11, IL-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL-stimulated leukemia cell <em>growth</em> underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean <em>20</em>%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (22-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic <em>growth</em> <em>factor</em>- dependent cell lines increased their proliferation upon incubation with FL, whereas all <em>growth</em> <em>factor</em>-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M-CSF, IL-3, PIXY- 321 or SCF and FL with IL-3 or IL-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>), GM-CSF, IL-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming <em>growth</em> <em>factor</em>-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine.

p190 RhoGAP is a 190-kDa protein that stably associates with p1<em>20</em> RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal <em>growth</em> <em>factor</em> (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what <em>factors</em> regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine <em>fibroblasts</em>. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p1<em>20</em> in C3H10T1/2 cells revealed that p190 could bind to p1<em>20</em> in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to <em>20</em>% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p1<em>20</em>. These studies describe a specific mechanism by which c-Src can regulate p190-p1<em>20</em> association and also document a significant role for p-Tyr-independent means of p190-p1<em>20</em> binding.

BACKGROUND

Patients with kidney disease have disordered bone and mineral metabolism, including elevated serum concentrations of fibroblast growth factor-23 (FGF23). These elevated concentrations are associated with cardiovascular and all-cause mortality. The objective was to determine the effects of the calcimimetic cinacalcet (versus placebo) on reducing serum FGF23 and whether changes in FGF23 are associated with death and cardiovascular events.

RESULTS

This was a secondary analysis of a randomized clinical trial comparing cinacalcet to placebo in addition to conventional therapy (phosphate binders/vitamin D) in patients receiving hemodialysis with secondary hyperparathyroidism (intact parathyroid hormone ≥300 pg/mL). The primary study end point was time to death or a first nonfatal cardiovascular event (myocardial infarction, hospitalization for angina, heart failure, or a peripheral vascular event). This analysis included 2985 patients (77% of randomized) with serum samples at baseline and 2602 patients (67%) with samples at both baseline and week 20. The results demonstrated that a significantly larger proportion of patients randomized to cinacalcet had ≥30% (68% versus 28%) reductions in FGF23. Among patients randomized to cinacalcet, a ≥30% reduction in FGF23 between baseline and week 20 was associated with a nominally significant reduction in the primary composite end point (relative hazard, 0.82; 95% confidence interval, 0.69-0.98), cardiovascular mortality (relative hazard, 0.66; 95% confidence interval, 0.50-0.87), sudden cardiac death (relative hazard, 0.57; 95% confidence interval, 0.37-0.86), and heart failure (relative hazard, 0.69; 95% confidence interval, 0.48-0.99).

CONCLUSIONS

Treatment with cinacalcet significantly lowers serum FGF23. Treatment-induced reductions in serum FGF23 are associated with lower rates of cardiovascular death and major cardiovascular events.

BACKGROUND

URL: http://www.clinicaltrials.gov. Unique identifier: NCT00345839.

Recent findings point to low oxygen tension (hypoxia) as an important mechanism for the expression of several eukaryotic genes. We have previously shown that hypoxia (2% O2), when compared to standard oxygen tension (<em>20</em>% O2), upregulates the mRNA levels of the human alpha1(I) (COL1A1) procollagen gene and transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) in human dermal <em>fibroblasts</em>. In this report, we determined the effect of hypoxia on collagen synthesis and transcription. Exposure of human dermal <em>fibroblasts</em> to hypoxia for 24-72 h led to a threefold, dose-dependent increase in collagenous protein (P < 0.0001; r = 0.9794) and to enhanced type I procollagen deposition, as shown by direct immunofluorescence. Transient transfections with a series of luciferase- and CAT-promoter constructs of the human COL1A1 gene (spanning from -2.5 kb to +113 bp) showed that hypoxia increases the transcriptional activity of constructs having 5' endpoints between -804 bp and -107 bp, with loss of stimulation at -84 bp. Maximal increase in promoter activity in hypoxia was observed between -190 and -174 bp of the proximal promoter, once a cKrox repressor site (-199 to -224 bp) was deleted. Upregulation of COL1A1 mRNA levels in hypoxia was blocked by a TGF-beta1 anti-sense oligonucleotide, and failed to occur in <em>fibroblasts</em> from TGF-beta1 knock-out mice. Co-transfection and overexpression with a Smad7 construct abrogated the increase in COL1A1 promoter activity observed in hypoxia. Upregulated transcriptional activity of the TGF-beta1 promoter in hypoxia was found to be maximal between -453 and -175 bp from the transcriptional start site. Since hypoxia is a critical feature of the early phases of wound repair, we conclude that it may act as a potent physiologic stimulus for collagen synthesis. TGF-beta1 appears to be a critical component of this response.

Cytochrome P450 enzymes of the 4A family (CYP4A) convert arachidonic acid to <em>20</em>-hydroxyeicosatetraenoic acid (<em>20</em>-HETE) in blood vessels of several vascular beds. The present study examined the effects of inhibiting the formation of <em>20</em>-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016) on the mitogenic response of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in human umbilical vein endothelial cells (HUVECs) in vitro, and on <em>growth</em> <em>factor</em>-induced angiogenesis in the cornea of rats in vivo. HET0016 (10 micromol/L and <em>20</em> microg, respectively) abolished the mitogenic response to VEGF in HUVECs and the angiogenic response to VEGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and epidermal <em>growth</em> <em>factor</em> in vivo by 80 to 90% (P < 0.001). Dibromododecenyl methylsulfonimide (DDMS), a structurally and mechanistically different inhibitor of <em>20</em>-HETE synthesis, also abolished angiogenic responses when tested with VEGF. Additionally, administration of the stable <em>20</em>-HETE agonist, <em>20</em>-hydroxyeicosa-6(Z) 15(Z)-dienoic acid (WIT003) induced mitogenesis in HUVECs and angiogenesis in the rat cornea in vivo. We studied the ability of HET0016 to alter the angiogenic response in the rat cornea to human glioblastoma cancer cells (U251). When administered locally into the cornea, HET0016 (<em>20</em> microg) reduced the angiogenic response to U251 cancer cells by 70%. These results suggest that a product of CYP4A product, possibly <em>20</em>-HETE, plays a critical role in the regulation of angiogenesis and may provide a useful target for reduction of pathological angiogenesis.

We recently reported <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 4<em>20</em> lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in <em>20</em>% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

Hyp mice possess a mutation that inactivates the phosphate-regulating gene, which is homologous to the endopeptidases of the X-chromosome (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hypophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a co<em>factor</em> of FGF23 signaling). We generated Hyp and klotho double-mutant mice (Hyp/klotho(-/-)). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho(-/-) double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho(-/-) mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1-34 down-regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho(-/-) mice. Moreover, in contrast to the Hyp mice, the Hyp/klotho(-/-) mice showed significantly higher serum levels of 1,25-dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho(-/-) mice were smaller in size, showed features of generalized tissue atrophy, and generally died by 15-<em>20</em> wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in diseases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeutically modulated by manipulating the effects of klotho.

The expression of the gene encoding the facilitated glucose transporter (GT) protein was studied in <em>fibroblast</em> cell lines. Addition of 15% calf serum to confluent BALB/c3T3, NIH3T3, or Rat-2 cells rapidly induced a 5-10-fold increase in GT mRNA, as determined by hybridization of size-fractionated total RNA to a rat brain GT cDNA. The rise in GT mRNA was maximal at 3-4 h after stimulation, and then returned to basal values by 16 h. The serum-stimulated increase in GT mRNA was not blocked by the protein synthesis inhibitors cycloheximide (10 micrograms/ml) or anisomycin (100 microM). In BALB/c3T3 cells, <em>fibroblast</em> <em>growth</em> <em>factor</em> (100 ng/ml), platelet-derived <em>growth</em> <em>factor</em> (5 units/ml), and epidermal <em>growth</em> <em>factor</em> (40 ng/ml) stimulated GT mRNA accumulation, although, when added individually, none of these <em>growth</em> <em>factors</em> increased DNA synthesis. The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates the enzyme protein kinase C, also caused GT mRNA accumulation in BALB/c3T3 and NIH3T3 cells. Prolonged pretreatment of cells with TPA abolished the response to TPA but not <em>fibroblast</em> <em>growth</em> <em>factor</em>. The involvement of GT gene transcription was assessed by the nuclear run-on technique. Treatment of NIH3T3 cells with serum increased transcription at least 10-<em>20</em>-fold by 30 min and returned to near basal levels by 2 h. This rapid activation paralleled that of the c-fos gene, but preceded the increase in c-myc gene transcription. These data indicate the following: 1) serum <em>growth</em> <em>factors</em> increase glucose transporter mRNA levels by a process not requiring intermediary new protein synthesis and clearly dissociable from mitogenesis, 2) the changes in GT mRNA are preceded by a rapid and transient activation of GT gene transcription, and 3) there exist protein kinase C-dependent and independent pathways for regulation of GT gene expression.

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic <em>factor</em> (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-<em>fibroblast</em> <em>growth</em> <em>factor</em>. One group of animals received fibrin glue with acidic-<em>fibroblast</em> <em>growth</em> <em>factor</em> only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord-implant interfaces. With BDNF foam, up to <em>20</em>% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord-implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly(D,L-lactic acid) foams, with or without axonal <em>growth</em>-promoting <em>factors</em>, seeded with Schwann cells to enhance the axonal regeneration and myelination response.

The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum <em>growth</em> <em>factors</em> correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent <em>fibroblasts</em> cultured in vitro. Young <em>fibroblasts</em> cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent <em>fibroblasts</em> constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent <em>fibroblasts</em> constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of <em>growth</em> inhibition, since young cells <em>growth</em> arrested by density-dependent <em>growth</em> inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than <em>20</em>-fold elevation of activity in trypsin-activated extracts from senescent versus young <em>fibroblasts</em> when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young <em>fibroblasts</em> cultured in the presence of a low serum concentration suggests that aging <em>fibroblasts</em> may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.

As normal cells progress to malignancy they must acquire an angiogenic phenotype that will enable them to attract the blood vessels necessary to support their progressive <em>growth</em>. Here we define the mechanism by which human <em>fibroblasts</em> cultured from Li Fraumeni patients and progressing to tumorigenicity in vitro become angiogenic. Initially cells were anti-angiogenic due to the secretion of high levels of inhibitory thrombospondin that overrode the modest amounts of the major inducer, vascular endothelial cell <em>growth</em> <em>factor</em> (VEGF), that were also produced. Cells became fully angiogenic in two steps, the first dependent on the loss of both alleles of wild-type p53 which caused a drop of at least <em>20</em>-fold in secreted thrombospondin and a fourfold increase in secreted VEGF. Angiogenic activity increased again upon transformation by activated ras due to a further twofold increase in VEGF. Changes in relative levels of VEGF mRNA were sufficient to account for changes in secreted protein levels and in overall angiogenic activity. These studies demonstrate that an angiogenic phenotype able to support tumorigenicity can arise in a step-wise fashion in response to both oncogene activation and tumor suppressor gene loss and involve both a decrease in the secretion of inhibitors and the sequential ratcheting up of the secretion of inducers of angiogenesis.

A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF<em>20</em>) is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells) within the organ of Corti during a specific developmental time. In the absence of FGF<em>20</em>, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells) and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf<em>20</em> knockout mice suggest that FGF<em>20</em> may also be a deafness-associated gene in humans.

Recent studies have suggested that cancer cells behave as metabolic parasites, by inducing oxidative stress in adjacent normal <em>fibroblasts</em>. More specifically, oncogenic mutations in cancer cells lead to ROS production and the "secretion" of hydrogen peroxide species. Oxidative stress in stromal <em>fibroblasts</em> then induces their metabolic conversion into cancer-associated <em>fibroblasts</em>. Such oxidative stress drives the onset of autophagy, mitophagy, and aerobic glycolysis in <em>fibroblasts</em>, resulting in the local production of high-energy mitochondrial fuels (such as L-lactate, ketone bodies, and glutamine). These recycled nutrients are then transferred to cancer cells, where they are efficiently burned via oxidative mitochondrial metabolism (OXPHOS). We have termed this new energy-transfer mechanism "Two-Compartment Tumor Metabolism", to reflect that the production and consumption of nutrients (L-lactate and other catabolites) is highly compartmentalized. Thus, high-energy onco-catabolites are produced by the tumor stroma. Here, we used a genetic approach to stringently test this energy-transfer hypothesis. First, we generated hTERT-immortalized <em>fibroblasts</em> which were genetically re-programmed towards catabolic metabolism. Metabolic re-programming towards glycolytic metabolism was achieved by the recombinant over-expression of MFF (mitochondrial fission <em>factor</em>). MFF over-expression results in extensive mitochondrial fragmentation, driving mitochondrial dysfunction. Our results directly show that MFF<em>fibroblasts</em> undergo oxidative stress, with increased ROS production, and the onset of autophagy and mitophagy, both catabolic processes. Mechanistically, oxidative stress induces autophagy via NF-kB activation, also providing a link with inflammation. As a consequence MFF-<em>fibroblasts</em> showed intracellular ATP depletion and the extracellular secretion of L-lactate, a critical onco-catabolite. MFF-<em>fibroblasts</em> also showed signs of myofibroblast differentiation, with the expression of SMA and calponin. Importantly, MFF-<em>fibroblasts</em> signficantly promoted early tumor <em>growth</em> (up to 6.5-fold), despite a <em>20</em>% overall reduction in angiogenesis. Thus, catabolic metabolism in cancer-associated <em>fibroblasts</em> may be a critical event during tumor intiation, allowing accelerated tumor <em>growth</em>, especially prior to the onset of neoangiogenesis.

Fibrillin-1 (FBN1) contains 47 epidermal <em>growth</em> <em>factor</em> (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant <em>fibroblast</em> strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in <em>20</em>/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.

The chemotactic activities of three different isoforms of platelet-derived <em>growth</em> <em>factor</em> (PDGF) on <em>fibroblasts</em>, monocytes, and granulocytes of human origin were investigated. PDGF-AB and PDGF-BB induced strong, dose-dependent responses in both <em>fibroblasts</em> and monocytes, whereas PDGF-AA did not stimulate chemotaxis of these cell types. Instead, PDGF-AA inhibited the chemotactic activity of PDGF-AB and PDGF-BB on <em>fibroblasts</em> and monocytes. However, PDGF-AA was not able to block monocyte chemotaxis induced by FMLP. In contrast, in granulocytes, dose-dependent chemotactic responses were obtained with all three isoforms of PDGF. All isoforms gave maximal responses at concentrations between 5 and <em>20</em> ng/ml. At higher concentrations the migration was reduced. Reduction and alkylation of the PDGF molecule, which leads to loss of the mitogenic activity, also caused a loss of the chemotactic activities for all three cell types. The data suggest that the various isoforms of PDGF stimulate and inhibit chemotaxis in an isoform- and cell type-specific manner.

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to <em>20</em>-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two <em>growth</em> <em>factors</em> of the FGF family.

Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal <em>fibroblasts</em>, and proliferation ([(3)H]thymidine) and collagen synthesis ([(3)H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dose-dependent manner. The activity of the eosinophil fibrogenic <em>factor</em>(s) remained unaltered when heated (56 degrees C, 30 min). Supernatants of cultured eosinophils (<em>20</em> min or 18 hr) also enhanced lung <em>fibroblast</em> proliferation, indicating that the preformed mitogenic <em>factor</em>(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung <em>fibroblasts</em> while increasing it in dermal <em>fibroblasts</em>. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not influence either <em>fibroblast</em> matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung <em>fibroblasts</em> in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming <em>growth</em> <em>factor</em> beta (TGF-beta) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-beta neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal <em>fibroblasts</em> and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-beta. In conclusion, we have shown that eosinophils act as direct modulatory cells in <em>fibroblast</em> proliferation, collagen synthesis, and lattice contraction, in part, through TGF-beta. These data corroborate the importance of eosinophils in skin and lung fibrosis.