<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>/19 (FGF<em>15</em>/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF<em>15</em>/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF<em>15</em>/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration.

Fgf<em>15</em>-/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF<em>15</em>/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH.

Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf<em>15</em>-/- mice. Hepatic expression of Pparγ2 was elevated in Fgf<em>15</em>-/- mice, being reversed by FGF19 treatment. PA induced FGF<em>15</em>/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH.

FGF<em>15</em>/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.

<AbstractText>Skeletal muscle is a plastic tissue that adapts to changes in exercise, nutrition, and stress by secreting myokines and myometabolites. These muscle-secreted <em>factors</em> have autocrine, paracrine, and endocrine effects, contributing to whole body homeostasis. Muscle dysfunction in aging sarcopenia, cancer cachexia, and diabetes is tightly correlated with the disruption of the physiological homeostasis at the whole body level. The expression levels of the myokine <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) are very low in normal healthy muscles. However, fasting, ER stress, mitochondrial myopathies, and metabolic disorders induce its release from muscles. Although our understanding of the systemic effects of muscle-derived FGF21 is exponentially increasing, the direct contribution of FGF21 to muscle function has not been investigated yet.</AbstractText><AbstractText>Muscle-specific FGF21 knockout mice were generated to investigate the consequences of FGF21 deletion concerning skeletal muscle mass and force. To identify the mechanisms underlying FGF21-dependent adaptations in skeletal muscle during starvation, the study was performed on muscles collected from both fed and fasted adult mice. In vivo overexpression of FGF21 was performed in skeletal muscle to assess whether FGF21 is sufficient per se to induce muscle atrophy.</AbstractText><AbstractText>We show that FGF21 does not contribute to muscle homeostasis in basal conditions in terms of fibre type distribution, fibre size, and muscle force. In contrast, FGF21 is required for fasting-induced muscle atrophy and weakness. The mass of isolated muscles from control-fasted mice was reduced by <em>15</em>-25% (P < 0.05) compared with fed control mice. FGF21-null muscles, however, were significantly protected from muscle loss and weakness during fasting. Such important protection is due to the maintenance of protein synthesis rate in knockout muscles during fasting compared with a 70% reduction in control-fasted muscles (P < 0.01), together with a significant reduction of the mitophagy flux via the regulation of the mitochondrial protein Bnip3. The contribution of FGF21 to the atrophy programme was supported by in vivo FGF21 overexpression in muscles, which was sufficient to induce autophagy and muscle loss by <em>15</em>% (P < 0.05). Bnip3 inhibition protected against FGF21-dependent muscle wasting in adult animals (P < 0.05).</AbstractText><AbstractText>FGF21 is a novel player in the regulation of muscle mass that requires the mitophagy protein Bnip3.</AbstractText>

Bovine aortic endothelial cells (BAEC) can be isolated in large numbers without major contamination by other cells and maintained in culture with a limited life span for about 100 population doublings. In order to study phenotypic changes of BAEC during long-term culture, stocks of different passages of BAEC were established and their morphological, migratory, and proliferative properties analyzed. Early-passage BAEC (passages 5-<em>15</em>) rapidly produce dense, cobblestone-like monolayers. Their <em>growth</em> beyond the monolayer configuration is characterized by the formation of an irregular network of spindle-shaped, crisscrossing BAEC <em>growing</em> either on top or beneath the monolayer, and by the assembly of elongated BAEC into well-differentiated capillary-like tubes. In contrast, senescent BAEC (passages 35-45) form perfect cobblestone monolayers that contain several, often multinucleated giant cells and a few capillary-like tubes but not the crisscrossing networks of their early-passage counterparts. The rates of BAEC migration and proliferation gradually decline during in vitro senescence. This decline is neutralized by exogenous basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) which elevates the migratory and proliferative capacities of early-passage and senescent BAEC to uniformly high levels. Northern blot analysis shows a gradual decline in bFGF message and an increase in laminin message during in vitro BAEC senescence. The present study supports the concept of autocrine <em>growth</em> regulation of BAEC and associates a decreased bFGF message with decreased rates of migration and proliferation as well as loss of the crisscrossing BAEC morphotype in senescent cultures.

OBJECTIVE

Focal adhesion kinase (FAK) is involved in processes integral to angiogenesis, such as cell growth, survival, and migration. FAK is activated by angiogenic growth factors, such as insulin-like growth factor (IGF)-I, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The study was conducted to determine whether overexpression of FAK or FAK-related nonkinase (FRNK), an inhibitor of FAK, could influence human retinal endothelial cell (HREC) migration and in vivo angiogenesis.

METHODS

Migration in response to a combination of growth factors was examined in transfected HRECs overexpressing FAK or FRNK. The effect of FAK or FRNK overexpression on preretinal neovascularization was examined in a mouse model of oxygen-induced retinopathy.

RESULTS

Overexpression of FAK in HRECs resulted in a 102% +/- 13% increase (P = 1.4 x 10(-4)) in cell migration, whereas overexpression of FRNK resulted in a 20% +/- 8% decrease (P = 0.01). Overexpression of FAK in mouse eyes led to formation of numerous large vascular tufts resembling glomeruli and a 57% +/- 7% increase in preretinal neovascularization (P = 3 x 10(-9)), whereas FRNK resulted in a 55% +/- 15% reduction (P = 5 x 10(-5)).

CONCLUSIONS

Modulating the FAK/FRNK system may provide a novel approach to inhibiting pathologic retinal angiogenesis.

Therapeutic targeting of late-stage breast cancer is limited by an inadequate understanding of how tumor cell signaling evolves during metastatic progression and by the currently available small molecule inhibitors capable of targeting these processes. Herein, we demonstrate that both β3 integrin and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-1 (FGFR1) are part of an epithelial-mesenchymal transition (EMT) program that is required to facilitate metastatic out<em>growth</em> in response to <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2). Mechanistically, β3 integrin physically disrupts an interaction between FGFR1 and E-cadherin, leading to a dramatic redistribution of FGFR1 subcellular localization, enhanced FGF2 signaling and increased three-dimensional (3D) out<em>growth</em> of metastatic breast cancer cells. This ability of β3 integrin to drive FGFR signaling requires the enzymatic activity of focal adhesion kinase (FAK). Consistent with these mechanistic data, we demonstrate that FGFR, β3 integrin, and FAK constitute a molecular signature capable of predicting decreased survival of patients with the basal-like subtype of breast cancer. Importantly, covalent targeting of a conserved cysteine in the P-loop of FGFR1-4 with our newly developed small molecule, FIIN-4, more effectively blocks 3D metastatic out<em>growth</em> as compared with currently available FGFR inhibitors. In vivo application of FIIN-4 potently inhibited the <em>growth</em> of metastatic, patient-derived breast cancer xenografts and murine-derived metastases <em>growing</em> within the pulmonary microenvironment. Overall, the current studies demonstrate that FGFR1 works in concert with other EMT effector molecules to drive aberrant downstream signaling, and that these events can be effectively targeted using our novel therapeutics for the treatment of the most aggressive forms of breast cancer. Mol Cancer Ther; <em>15</em>(9); 2096-106. ©2016 AACR.

OBJECTIVE

Stem cell-based therapies are being considered for various neurologic diseases, such as epilepsy. Recent studies have suggested that some effects of transplanted stem cells are due to bystander effects that modulate the host environment, rather than direct effects of cell replacement. The extract from human adipose stem cells (ASCs) that secrete multiple growth factors including cytokines and chemokines may be a potential source of bystander effects for the treatment of epilepsy, in which inflammation is thought to play an important role. Here, we investigated the effects of a cytosolic extract of human ASCs (ASCs-E) in a mouse model of epilepsy.

METHODS

Human ASCs-E, boiled ASCs-E, or fibroblast-extract (fibroblast-E) was intraperitoneally administrated to C57BL/6 mice 15 min before pilocarpine-induced status epilepticus (SE) or during chronic epileptic stage. Blood-brain barrier (BBB) leakage was evaluated by measuring Evans blue dye extravasation. Spontaneous recurrent seizure (SRS) was investigated by long-term video-electroencephalography (EEG) monitoring. The mice performed elevated plus maze, open-field, light/dark transition, and novel object recognition tasks.

RESULTS

Acute application of human ASCs-E before SE led to earlier attenuation of seizure spike activities after treatment with diazepam, reduction of BBB leakage, and inhibition of the development of epilepsy. Human ASCs-E treatment (for 7 days) during the chronic epileptic stage suppressed SRS and reduced abnormal epileptic behavioral phenotypes. However, neither boiled ASCs-E nor fibroblast-E had any effects in the experimental epilepsy model.

CONCLUSIONS

Our results demonstrate that human ASCs-E prevents or inhibits epileptogenesis and SRS in mice. They also suggest a stem cell-based, noninvasive therapy for the treatment of epilepsy.

OBJECTIVE

The concept of neovascularization in response to tissue ischemia was recently extended by the finding of postnatal vasculogenesis through circulating endothelial progenitor cells (EPCs). The aim of this study was to assess the role of acute ischemia for EPC mobilization in patients with peripheral arterial occlusive disease (PAOD) and in healthy volunteers.

METHODS

The number of circulating EPCs was analyzed by flow cytometry in PAOD patients (n = 23) with exercise-induced limb ischemia for up to 72 h after a maximal treadmill test and in healthy volunteers (n = 17) who underwent a <em>15</em>-min suprasystolic occlusion of one lower extremity to induce limb ischemia. Plasma concentrations of vascular endothelial <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, tumor necrosis <em>factor</em>-α, and granulocyte macrophage-colony stimulating <em>factor</em> were determined by ELISA.

RESULTS

EPCs (CD 34 pos/KDRpos) increased significantly in both PAOD patients from 82 ± 20 to 256 ± 52 (P < 0.05) and healthy volunteers from 144 ± 39 to 590 ± 61 cells per 1 million events (P < 0.05) in response to induced ischemia, with a maximum after 24 h and returned to baseline within 72 h. The relative increase in EPC numbers was significantly lower in patients with PAOD as compared with healthy volunteers (P < 0.05). Plasma levels of vascular endothelial growth factor increased from 27.4 ± 3.1 to 126.4 ± 12 pg/ml in patients with PAOD (P < 0.05) and from 30.7 ± 6.1 to 134.1 ± 12.4 pg/ml in healthy volunteers (P < 0.05).

CONCLUSIONS

Both patients with symptomatic PAOD and healthy volunteers respond to a single episode of limb ischemia with a time-dependent increase in circulating EPCs. The increase of EPC numbers in response to ischemia is reduced when vascular disease is present, underlining the reduced vasculogenic potential of patients with PAOD.

OBJECTIVE

Approximately <em>15</em>% of patients with multiple myeloma (MM) exhibit a t(4;14) translocation, which often results in constitutive activation of the receptor tyrosine kinase (RTK) <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3). This study evaluated the efficacy and safety of dovitinib, an RTK inhibitor with in vitro inhibitory activity against FGFR, in patients with relapsed or refractory MM with or without t(4;14) translocation.

METHODS

Adult patients with relapsed or refractory MM who had received ≥2 prior regimens were enrolled in this multicenter, 2-stage, phase 2 trial. Patients were grouped based on their t(4;14) status. Dovitinib (500 mg/day orally) was administered on a 5-days-on/2-days-off schedule. The primary endpoint was overall response rate by local investigator review (per International Myeloma Working Group criteria). In non-responding patients, treatment could continue with the addition of low-dose dexamethasone.

RESULTS

In total, 43 patients (median age, 63 years) were enrolled (13 t(4;14) positive, 26 t(4;14) negative, and 4 t(4;14) status non-interpretable). Patients had received a median of 5 prior regimens. Median duration of treatment was 8.7 weeks in the t(4;14)-positive group and 3.7 weeks in the t(4;14)-negative group. None of the patients on dovitinib had objective responses. The stable disease rate was 61.5% in the t(4;14)-positive group and 34.6% in the t(4;14)-negative group. Overall, 39 patients (90.7%) had adverse events suspected to be related to study drug, most commonly diarrhea (60.5%), nausea (58.1%), vomiting (46.5%), and fatigue (32.6%).

CONCLUSIONS

Dovitinib showed no single-agent activity in relapsed or refractory MM but may stabilize disease in some t(4;14)-positive patients.

BACKGROUND

Irritable bowel syndrome (IBS) physiopathology is multifactorial and roles for both microbiota and bile acid (BA) modifications have been proposed. We investigated role of dysbiosis, transit pattern and BA metabolism in IBS.

METHODS

Clinical data, serum, and stool samples were collected in <em>15</em> healthy subjects (HS), 16 diarrhea-predominant (IBS-D) and <em>15</em> constipation-predominant IBS (IBS-C). Fecal microbiota composition was analyzed by real-time PCR. Sera and fecal BA profiles, 7α-C4 levels, and in vitro BA transformation activity by fecal microbiota were measured by mass spectrometry. Serum <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> 19 (FGF19) was assayed by ELISA.

UNASSIGNED

Dysbiosis was present in IBS patients with an increase in Escherichia coli in IBS-D patients (p = 0.03), and an increase in Bacteroides (p = 0.01) and Bifidobacterium (p = 0.04) in IBS-C patients. Sera primary and amino-conjugated BA were increased in IBS-D (63.5 ± 5.5%, p = 0.01 and 78.9 ± 6.3%, p = 0.03) and IBS-C patients (55.9 ± 5.5%, p = 0.04 and 65.3 ± 6.5%, p = 0.005) compared to HS (37.0 ± 5.8% and 56.7 ± 8.1%). Serum 7α-C4 and FGF19 levels were not different among all three groups. Fecal primary BA were increased in IBS-D patients compared to HS, including chenodeoxycholic acid which has laxative properties (25.6 ± 8.5% vs 3.5 ± 0.6%, p = 0.005). Bile acid deconjugation activity was decreased in IBS-D (p = 0.0001) and IBS-C (p = 0.003) feces. Abdominal pain was positively correlated with serum (R = 0.635, p < 0.001) and fecal (R = 0.391, p = 0.024) primary BA.

CONCLUSIONS

Different sera and fecal BA profiles in IBS patients could be secondary to dysbiosis and further differences between IBS-C and IBS-D could explain stool patterns. This study opens new fields in IBS physiopathology and suggests that modification of BA profiles could have therapeutic potential.

The role of 25-hydroxyvitamin D [25(OH)D] and <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) in chronic kidney disease-mineral and bone disorder (CKD-MBD) remains elusive in predialysis CKD patients. From the fact that FGF-23 suppresses bone mineralization in vitro and that 1alpha-hydroxylase is present in parathyroid cells and osteoblasts, they may be associated with bone mass or serum parathyroid hormone (PTH) level. In this cross-sectional observational study, we investigated the potential associations of 25(OH)D or FGF-23 with 1-84 PTH and bone mineral density (BMD) in the femoral neck (FN) and lumbar spine (LS) of 325 non-diabetic patients. All patients had stages 3-5 CKD and had never been treated with bisphosphonate, estrogen, or vitamin D. We measured bone-specific alkaline phosphatase (bone ALP), intact FGF-23 and 1-84 PTH in a third generation assay, and performed a multiple regression analysis for 1-84 PTH and BMD Z-score. In our cohort, 80.1% had 25(OH)D levels less than 30 ng/mL, and 4.1% had levels less than <em>15</em> ng/mL. A univariate analysis indicated a negative association for 25(OH)D with 1-84 PTH and bone ALP. A multivariate analysis showed that the significant determinants for 1-84 PTH were 25(OH)D, estimated glomerular filtration rate (eGFR), corrected calcium, serum calcitriol and phosphate. Intriguingly, the three former parameters had negative associations with 1-84 PTH while calcitriol had a positive association. While further adjustment of FGF-23 extinguished the positive association of phosphate and 1-84 PTH, there was absolutely no increase in the R2. With regard to the BMD Z-score, 25(OH)D and the body mass index were the significant common independent positive determinants for both FN and LS, whereas bone ALP was the negative determinant even though there was no correlation noted for 1-84 PTH, calcitriol, or FGF-23 with BMD. In addition, eGFR positively contributed to the Z-score only in FN. Therefore, despite a positive correlation between 25(OH)D and calcitriol, their contribution to the CKD-MBD appears to be different. Since the significant associations for 25(OH)D with 1-84 PTH and BMD were independent of serum calcitriol and bone ALP, this might imply that 25(OH)D has a direct effect on the parathyroid gland and bone.

Cellular and molecular mechanisms responsible for the observed vulnerary effects of recombinant human platelet-derived <em>growth</em> <em>factor</em> BB (rP-DGF-BB) in man have not been elucidated. In a double-blinded trial, patients having chronic pressure ulcers were treated topically with either rPDGF-BB or placebo for 28 days. To explore how rPDGF-BB may induce chronic wounds to heal, biopsies were taken from the ulcers of a cohort of 20 patients from the trial and evaluated in a blinded fashion by light microscopy for 1), <em>fibroblast</em> content, 2) neovessel formation, and 3), collagen deposition. Electron microscopy also was used to assess <em>fibroblast</em> activation and collagen deposition. Before initiation of therapy most wounds had few <em>fibroblasts</em> and most of those present were not activated. When mean scores for the total active treatment phase (days 8, <em>15</em>, and 29) for rPDGF-BB-treated ulcers were compared with the scores for placebo-treated ulcers, <em>fibroblast</em> content was significantly higher for the rPDGF-BB-treated ulcers (P = 0.03, Kruskal-Wallis test). More significant differences in <em>fibroblast</em> and neovessel content were observed when six nonhealing wounds were eliminated from the analysis (three placebo, three treatment). Thus, in all healing wounds, rPDGF-BB therapy significantly increased <em>fibroblast</em> (P = 0.0007) and neovessel (P = 0.02) content. These results were correlated with increased collagen fibrillogenesis by <em>fibroblasts</em> from healing rPDGF-BB-treated wounds, as assessed by intracellular procollagen type I immunostaining, and by electron microscopy, and were concordant with clinical measurements (eg, area of ulcer opening and ulcer volume) which showed greater healing in rPDGF-BB-treated wounds. These results suggest induction of <em>fibroblast</em> proliferation and differentiation is one mechanism by which rPDGF-BB can accelerate wound healing and that rPDGF-BB can augment healing responses within a majority of, but not all, nonhealing chronic pressure ulcers in man.

Hypercholesterolemic (HC) rabbits exhibit suppressed compensatory vascular <em>growth</em> after restriction of arterial supply. However, neovascularization is commonly found in atheromas containing inflammatory cells. We used an in vitro model to determine the effects of hypercholesterolemia on angiogenesis in the absence or presence of inflammatory cells. HC rabbit aortic explants (1 mm2) with or without (n = 90 each) lesion-forming inflammatory cells were cultured in a collagen matrix with serum-free medium. Explant-derived endothelial cell <em>growth</em> was organized into capillary-like microtubes (CLM) that could be videomicroscopically quantified. CLM <em>growth</em> from lesion-free HC explants was significantly reduced to 13 +/- 4% of the value in explants (n = 90) from normocholesterolemic (NC, n = <em>15</em>) rabbits (P < .001). In contrast, in lesion-containing HC explants, the matrix was invaded by foam cells, and CLM <em>growth</em> was not inhibited. Immunoassayable basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, in pg/mL) in the culture medium was significantly lower in lesion-free HC (< 5) than NC explants (11 +/- 2, P < .01) or HC explants with lesions (14 +/- 3). In addition, CLM <em>growth</em> was reduced in NC explants incubated with oxidized LDL (ox-LDL, 50-100 micrograms/mL). Exogenous bFGF (10 ng/mL) reversed the inhibitory effects of hypercholesterolemia and ox-LDL, whereas bFGF-neutralizing antibody (10 micrograms/mL) abolished CLM <em>growth</em> in all groups. In cultured rabbit aortic endothelial cells, ox-LDL reduced DNA synthesis, but this inhibition was reversed by bFGF. We conclude that hypercholesterolemia and ox-LDL inhibit angiogenesis like endothelial <em>growth</em> because of a suppressed availability of endogenous bFGF. Retained responsiveness to exogenous bFGF suggests that inducing bFGF expression at targeted sites may improve collateral <em>growth</em> in hyperlipidemic arterial disease.

Hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) is considered to be a mesenchymal-derived <em>factor</em> that acts via a dual system receptor, consisting of the MET receptor and proteoglycans present on adjacent epithelial cells. Surprisingly, HGS/SF stimulated the migration of rat mammary (Rama) 27 <em>fibroblasts</em>, although it failed to stimulate their proliferation. HGF/SF stimulated a transient activation of mitogen-activated protein kinases p44 and p42 (p42/44(MAPK)), with a maximum level of dual phosphorylation of p42/44(MAPK) occurring 10-<em>15</em> min after the addition of the <em>growth</em> <em>factor</em>, which was followed by a rapid decrease to near basal levels after 20 min. Interestingly, a second phase of p42/44(MAPK) dual phosphorylation was observed at later times (3 h to 10 h). PD098059, a specific inhibitor of MEK-1, prevented the dual phosphorylation of p42/44(MAPK) and also the phosphorylation of p90(RSK) (ribosomal subunit S6 kinase), which mirrored the kinetics of p42/44(MAPK) phosphorylation. Moreover, PD098059 prevented the HGF/SF-induced migration of Rama 27 cells. HGF/SF also induced an early increase in the phosphorylation of protein kinase B/Akt. Akt phosphorylation was elevated <em>15</em> min after the addition of HGF/SF and then declined to basal levels by 30 min. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), prevented the increase in Akt phosphorylation and abolished HGF/SF-induced migration of <em>fibroblasts</em>. PD098059 also inhibited the stimulation of Akt phosphorylation by HGF/SF and wortmannin similarly inhibited the stimulation of p42/44(MAPK) dual phosphorylation. These results suggest that HGF/SF-induced motility depends on both the transient dual phosphorylation of p42/44(MAPK) and the activation of PtdIns3K in Rama 27 <em>fibroblasts</em> and that these pathways are mutually dependent.

The differentiation of rat lens epithelial cells to fibre cells can be mimicked using lens epithelial explants, which differentiate in vitro when exposed to <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). A previous study demonstrated that FGF is required only for initiation of differentiation: once induced by FGF, differentiation can be maintained by insulin (as assessed by following the accumulation of fibre-cell specific crystallins). The aim of this investigation was to determine whether insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1) can also maintain differentiation and to include a cellular analysis of explants undergoing insulin-or IGF-maintained differentiation in vitro. Measurement of the accumulation of alpha-, beta- and gamma-crystallins showed that IGF-1, like insulin, can replace FGF-2 in directing the pulses of alpha-, beta- and gamma-crystallin gene expression once differentiation is initiated by FGF-2. Cells in both the peripheral and the central region of the explants responded. Immunolocalization of alpha, beta- and gamma-crystallins in these explants showed that a <em>15</em> min pulse of FGF-2 triggered the differentiation of only a few cells, whereas a 12 hr pulse primed virtually all the cells for differentiation. This indicates that in explants, individual cells differ in the rate at which they can respond to FGF-2.

The level of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in cerebrospinal fluid (CF) was measured by an EIA in <em>15</em> patients with moyamoya disease, in 11 patients with atherosclerotic occlusive disease, and in 8 patients with spinal disc disease. In the moyamoya patients, bFGF was found in 13 out of <em>15</em> CF samples with a mean value of 101 pg/ml. However, bFGF was detected in only 4 out of 11 patients with atherosclerotic disease with a mean of 8 pg/ml. In all the patients with disc disease, bFGF was not detected. The elevated level of bFGF may play a crucial role in the pathogenesis of moyamoya disease.

The interaction of somatomedin (Sm) with <em>growth</em> plate chondrocytes (GPCs) is believed to be the primary stimulus of skeletal <em>growth</em>. Using techniques designed to disrupt as little as possible the phenotypic characteristics of GPCs, we have been able to obtain 3-4 x 10(8) viable cells from the major physes of one newborn calf. The availability of these cells plus essentially pure Sm-C/insulin-like <em>growth</em> <em>factor</em> I, the most GH-dependent Sm, has now made possible detailed studies of the interaction of this radiolabeled peptide with the GPC receptor and of the subsequent processing of this hormone by these cells. The enzymatic methods required to free GPCs from their matrix led to loss of receptors, followed by rapid receptor regeneration by de novo synthesis in suspension cultures. Binding of [125I]iodo-Sm-C to GPCs was time dependent and saturable, with optima at <em>15</em> C and pH 7.8. At 37 C, binding peaked at 90 min and declined thereafter. Multiplication-stimulating activity, insulin, and nerve <em>growth</em> <em>factor</em> were less potent than unlabeled Sm-C in competition with [125I]iodo-Sm-C for its receptor. Human GH, epidermal <em>growth</em> <em>factor</em>, and <em>fibroblast</em> <em>growth</em> <em>factor</em> failed to show competition even at 10(-6) M. Analysis of the fate of [125I]iodo-Sm-C bound to GPCs at 37 C provided evidence that this hormone is internalized and extruded from the cell in a partially degraded form. Scatchard analysis of [125I]iodo-Sm-C binding to GPCs and to chondrocytes isolated from articular cartilage revealed similar Ka values, but reproducibly 2-6 times more receptors on <em>growth</em> plate than on articular chondrocytes.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR)-3 gene encodes a receptor tyrosine kinase that is frequently mutated in non-muscle invasive bladder cancer (NMIBC). A sensitive and quantitative assay using peptide nucleic acid-mediated real-time PCR was developed for detecting FGFR3 mutations in the urine samples and evaluated as a molecular marker for detecting intravesical recurrence of NMIBC in patients undergoing transurethral resection of bladder tumor. FGFR3 mutation was examined in tumor tissues and serially taken pre- and postoperative urine sediments in 45 NMIBC patients with a median follow up of 32 months. FGFR3 mutations were detected in 53.3% (24/45) of primary tumor tissues, among which intravesical recurrence developed in 37.5% (9/24) of cases. FGFR3 mutation in the primary tumor was not a significant prognostic indicator for recurrence, while the proportion of FGFR3 mutation (i.e. tumor cellularity was>>or=11%) in the preoperative urine sediments was a significant indicator for recurrence in patients with FGFR3 mutations in the primary tumors. FGFR3 mutations were detected in 78% (7/9) of postoperative urine samples from recurrent cases with FGFR3 mutations in the tumor, while no mutations were detected in the urine of <em>15</em> non-recurrent cases. Urine cytology was negative in all cases with FGFR3 mutations in the primary tumors, while the sensitivity of cytological examination was as high as 56% (5/9) in cases showing wild-type FGFR3 in the primary tumors. Urine FGFR3 mutation assay and cytological examination may be available in the future as complementary diagnostic modalities in postoperative management of NMIBC.

IFN regulatory <em>factor</em>-1 (IRF-1) regulates the IFN system, inhibits cell <em>growth</em>, and has tumor-suppressor activities. p21 is a universal cyclin-dependent kinase inhibitor, the induction of which depends on both p53 and IRF-1 in mouse embryonic <em>fibroblasts</em>. The expression of p21 in hepatocellular carcinomas (HCCs) is regulated by wild-type p53. We examined the expressions of IRF-1 and p21 in 32 HCCs by quantitative reverse transcription-PCR and the mutation p53 gene in 32 HCCs by single-strand conformation polymorphism and direct sequencing. The expression of IRF-1 mRNA in <em>15</em> of 32 HCCs was lower than that in adjacent noncancerous tissue. IRF-1 mRNA expression was reduced in 0 of 3 specimens of well-differentiated HCC, 9 of 21 (42%) specimens of moderately differentiated HCC, and 6 of 8 (75%) specimens of poorly differentiated HCC. IRF-1 mRNA expression was significantly lower in tumors with portal thrombus than in those without portal thrombus (P = 0.003). p53 mutations were detected in 7 of 32 HCCS: p21 expression was reduced in 6 of the 7 (86%) HCCs with p53 mutations. In contrast, p21 expression was reduced in 13 of 25 (52%) HCCs with wild-type p53. IRF-1 expression was reduced in 7 of 13 (53%) HCCs with both wild-type p53 and reduced expression of p21. These results suggest that IRF-1 may be a tumor-suppressor gene for HCC and that IRF-1 is related to p21 expression in HCC with wild-type p53.

BACKGROUND

Imatinib is an inhibitor of platelet-derived growth factor receptors. We have reported that treatment with imatinib inhibited bleomycin-induced pulmonary fibrosis in mice. However, late treatment with imatinib had no effect.

OBJECTIVE

To clarify why imatinib had no antifibrotic effect when its administration was delayed, we focused on alpha(1)-acid glycoprotein (AGP), because it was reported to bind imatinib and mediate drug resistance.

METHODS

The concentration of AGP in serum of mice and patients with idiopathic pulmonary fibrosis was measured by radial immunodiffusion testing. The effects of AGP in vitro were evaluated by assaying the growth of lung fibroblasts. We examined the combined effects of erythromycin (EM) or clarithromycin (CAM) on bleomycin-induced pulmonary fibrosis in mice.

RESULTS

Addition of AGP abrogated imatinib-mediated inhibition of the growth of fibroblasts. However, treatment with EM or CAM restored the growth-inhibitory effects of imatinib. The elevated level of AGP was detected in serum and lung homogenates in bleomycin-exposed mice and reached a plateau on Day 14. Imatinib alone did not ameliorate pulmonary fibrosis when treatment was started on Day 15, whereas coadministration of imatinib and EM or CAM significantly reduced the fibrogenesis via inhibition of the growth of fibroblasts in vivo. Serum levels of AGP were higher in patients with idiopathic pulmonary fibrosis than in healthy subjects.

CONCLUSIONS

AGP is an important regulatory factor modulating the ability of imatinib to prevent pulmonary fibrosis in mice, and combined therapy with imatinib and EM or CAM might be useful for treatment of pulmonary fibrosis.

The type beta transforming <em>growth</em> <em>factors</em> (TGF-beta s) are a family of potent cytokines with diverse effects on proliferation, differentiation, turnover of extracellular matrix components, oncogene expression, and other aspects of cellular phenotype. Unlike lung <em>fibroblasts</em> of certain species, unstimulated human lung <em>fibroblast</em> lines produce little or no TGF-beta in culture. However, TGF-beta has been reported to autoregulate its own production in certain human tumor cells and in rodent cell lines. To test whether this phenomenon is operative in <em>fibroblasts</em> from normal human lung tissue, confluent cultures of IMR90 normal fetal lung <em>fibroblasts</em> were exposed to TGF-beta. Cultures were exposed briefly to purified TGF-beta 1 under serum-free conditions and secretion of newly synthesized TGF-beta over the ensuing 72 h was determined by immunoblotting and bioassays made specific with the use of neutralizing antibodies. Steady-state levels of mRNA for TGF-beta 1 were detected by Northern and slot blot hybridization analysis of total cellular RNA. The 2.5 kb TGF-beta 1 mRNA species rose within 1.5 h of exposure of IMR90 cells to TGF-beta 1 and reached maximal levels after 16 h. Increased levels of TGF-beta were detected in conditioned medium 9 h after the start of the exposure. Thereafter, TGF-beta continued to accumulate at an elevated rate (90 +/- 7 versus < or = <em>15</em> pg/10(6) cells/h in uninduced cells) for up to 72 h. As little as 1 ng/ml TGF-beta 1 auto-induced TGF-beta secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

During acute lung injury, there is an outpouring of <em>growth</em> <em>factors</em> into the alveolar space that drive local repair and fibrosis. During the remodeling that follows the instillation of bleomycin via the trachea into the adult rat, at least two platelet-derived <em>growth</em> <em>factor</em> (PDGF)-like peptides are released sequentially into lung lining fluid. Groups of four to five animals were killed at 3, 6, <em>15</em>, and 26 days after exposure to bleomycin and lungs lavaged with isotonic saline. PDGF-like peptides in epithelial lining fluid (ELF) were partially purified by cation exchange chromatography and concentrated. Isolated peptides were analyzed by immunoblotting to determine their molecular weight and immunologic identity. Western blots were probed with polyclonal antibodies to PDGF-BB and PDGF-AA. PDGF-like peptides of two distinct size classes (38-40 kD and 29 kD) were present in alveolar fluid from all rats with lung injury induced by bleomycin. No PDGF-like peptides were found in comparably prepared ELF from control animals. The 38-40 kD peptide was detected only with anti-PDGF-BB antibody; the 29 kD peptide was detected only with anti-PDGF-AA antibody. The presence of these two peptides varied independently with time after exposure to bleomycin. The 38-40 kD peptide was at peak levels at 3 to 6 days. In contrast, the 29 kD peptide was present at all times following injury but with far less variation over time. In parallel with these immunoassays for PDGF-like molecules, there was abundant <em>growth</em>-promoting activity for <em>fibroblasts</em> present in concentrated ELF during the course of injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Using tissue engineering, a complex of neural stem cells (NSCs) and collagen type I was transplanted for the therapy of cerebral ischemic injury. NSCs from E14 d rats were dissociated and cultured by neurosphere formation in serum-free medium in the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), then seeded onto collagen to measure cell adhesive ability. BrdU was added to the culture medium to label the NSCs. Wistar rats (n=100) were subjected to 2-hour middle cerebral artery occlusion. After 24 hours of reperfusion, rats were assigned randomly to five groups: NSCs-collagen repair group, NSCs repair group, unseeded collagen repair group, MCAO medium group, and sham group. Neurological, immunohistological and electronic microscope assessments were performed to examine the effects of these treatments. Scanning electronic microscopy showed that NSCs assemble in the pores of collagen. At 3, 7, <em>15</em>, and 30 d after transplantation of the NSC-collagen complex, some of the engrafted NSCs survive, differentiate and form synapses in the brains of rats subjected to cerebral ischemia. Six d after transplantation of the NSC-collagen complex into the brains of ischemic rats, the collagen began to degrade; 30 d after transplantation, the collagen had degraded completely. The implantation of NSCs and type I collagen facilitated the structural and functional recovery of neural tissue following ischemic injury.

Previously we have reported changes in <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) in conditioned medium (CM) derived from rat mammary tumours undergoing remission. We have used a similar approach to assay for the presence of FGFs in human breast tissue and cell lines. The majority of cancer tissues (35/50), benign tissues (8/9) and all cancer adjacent normal tissues (20/20) released heat labile, NR6 transforming activity which coeluted from heparin with acidic FGF (aFGF) at 0.9-1.1 M NaCl and was neutralised by antibodies to aFGF. The conclusion that the majority of breast cancers contain active aFGF was supported by immunoblotting. The CM of a minority (<em>15</em>/50) of cancers and one benign tissue had highly transforming activity for NR6 cells, and was mitogenic for a breast cancer cell line, was heat labile, and strongly heparin binding, eluting at 1.5-2.0 M salt. It was not immunoreactive with antibodies to aFGF, basic FGF (bFGF) or Kaposi's FGF (kFGF) and its activity was reduced by the presence of aFGF, suggesting competition for the same receptor. Very little aFGF was observed in the CM of these tumours, and neither aFGF nor other FGF activity was detected in CM of breast cell lines.

Granulation-tissue myo<em>fibroblasts</em> are important in wound contraction, disappearing in normal scars but persisting in hypertrophic scars. While transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1) is implicated in excessive scarring and induces the accumulation of myo<em>fibroblasts</em>, the role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) on scarring remains unreported. Four linear full-thickness incisions, each 20 mm long, were made dorsally on 25 adult male Hooded Lister rats. In each animal, one wound was unmanipulated (control), one was injected with TGF-beta 1 (positive control), one with vehicle alone and one with FGF-2 (test). The wounds were injected daily for 5 days. Animals were sacrificed in groups of five on days 5, 10, <em>15</em>, 20 and 30 after wounding. Wounds were subjected to tensiometry. Sections were stained for collagen fibres, immunostained for myo<em>fibroblasts</em> and studied under light microscopy and electron microscopy. Myo<em>fibroblasts</em> were present in the granulation tissue on day 5, reached their maximum number on day 10 and disappeared from all wounds by day 30. Treatment with FGF-2 inhibited this transient phenotypic change of granulation-tissue <em>fibroblasts</em> into myo<em>fibroblasts</em>, relative to controls (<em>15</em>.23% versus 58.71%, <em>15</em>.23% versus 54.71% and <em>15</em>.23% versus 53.<em>15</em>%; P<0.003 on day 10, paired t-test). Test collagen-fibre orientation resembled that of the normal dermis, in contrast to that of the control wounds. Test wound breaking strength was unreduced. These results suggest a possible anti-scarring effect of FGF-2 during wound healing.