The balanced proliferation and cell cycle exit of neural progenitors, by generating the appropriate amount of postmitotic progeny at the correct time and in the proper location, is required for the establishment of the highly ordered structure of the adult brain. Little is known about the extrinsic signals regulating these processes, particularly in the midbrain. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (Fgf) <em>15</em>, the mouse ortholog of FGF19 and member of an atypical Fgf subfamily, is prominently expressed in the dorsolateral midbrain of the midgestational mouse embryo. In the absence of Fgf<em>15</em>, dorsal midbrain neural progenitors fail to exit the cell cycle and to generate the proper amount of postmitotic neurons. We show here that this is due to the altered expression of inhibitory/neurogenic and proneural/neuronal differentiation helix-loop-helix transcription <em>factor</em> (TF) genes. The expression of Id1, Id3, and Hes5 was strongly increased and ectopically expanded, whereas the expression of Ascl1 (Mash1), Neurog1 (Ngn1) and Neurog2 (Ngn2) was strongly decreased and transcription of Neurod1 (NeuroD) was completely abolished in the dorsolateral midbrain of Fgf<em>15</em>(-/-) mice. These abnormalities were not caused by the mis-expression of cell cycle regulatory proteins such as cyclin-dependent kinase inhibitors or retinoblastoma proteins. Furthermore, human FGF19 promotes cell cycle exit of murine dorsal neural progenitors in vitro. Therefore, our data suggest that Fgf<em>15</em> is a crucial signaling molecule regulating the postmitotic transition of dorsal neural progenitors and thus the initiation and proper progression of dorsal midbrain neurogenesis in the mouse, by controlling the expression of neurogenic and proneural TFs.

We have previously reported increased expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1 and FGF-2) in benign and malignant human thyroid neoplasia. To determine the role of these <em>factors</em> in thyroid hyperplasia we have examined their expression in multinodular goiter and compared findings with those in normal thyroid tissue. Because the effects of FGF-1 and FGF-2 are predominantly mediated through the FGF receptor-1 (FGFR-1), its expression has also been examined. Immunocytochemistry was performed on sections from multinodular goiters (n = 18) and normal thyroid (n = 7). Cytoplasmic staining for FGF-1, FGF-2, and FGFR-1 was scored on a scale of 0 (no staining) to 3 (heavy staining) and expressed as a percentage of total cells stained. Confocal microscopy of immunofluorescent staining for FGF-1, FGF-2, and FGFR-1 in sections of multinodular goiter (n = 3) and normal thyroid (n = 3) provided quantitation of immunostaining. FGF-1 expression was significantly increased in multinodular goiter when compared with normal. A mean of 74% of follicular cells in multinodular goiter compared with 9% of follicular cells in normal thyroid expressed FGF-1 (P < 0.0001). When expression of FGF-2 was examined, 77% of the follicular cells in multinodular goiter compared with 5% in normal thyroids were immunopositive (P < 0.0001). Confocal microscopy revealed that the intensity was 160 times greater in follicular cells in sections of multinodular goiters when compared with normal. When expression of FGFR-1 was analyzed, 89% of the follicular cells in multinodular goiter stained positively, compared with <em>15</em>% of follicular cells in sections of normal thyroid. Confocal microscopy revealed a 6-fold increase in intensity of FGFR-1 expression in follicular cells of multinodular goiter (P < 0.05). In addition, there was significant nuclear expression of FGFR-1 in multinodular goiter contrasting with negligible expression in normal thyroid. These data show that enhanced expression of FGF-1, FGF-2, and FGFR-1 accompany thyroid hyperplasia and are not exclusively associated with the neoplastic state. These <em>factors</em> may be involved in the pathogenesis of uncontrolled thyroid <em>growth</em> observed in these conditions.

Bone marrow abnormalities are frequently observed in HIV-1-infected individuals. Infection of marrow mesenchymal stem cells (MSCs) may abrogate their <em>growth</em> properties and hematopoietic supportive functions. To delineate the cell type infected, and <em>factors</em> responsible for the deleterious effects, human bone marrow cells were exposed to HIV-1 in vitro. By week 4, the ability of MSCs to form colonies of purely <em>fibroblasts</em> (CFU-F) and mixed colonies of <em>fibroblasts</em> and adipocytes (CFU-FA) was suppressed by 23 +/- 5 and 55 +/- 7%, respectively. The p24 concentration in culture supernatants steadily declined from 170 ng/ml in the inoculum to 134 +/- 30, 35 +/- <em>15</em>, 2.3 +/- 3, and <0.02 ng/ml at the end of week 1, 2, 3, and 4, respectively. However, even at week 4, coculturing with MT-4 lymphocytes for 1 week dramatically increased p24 levels. Polymerase chain reaction (PCR) amplification, using HIV-1-specific primers, and in situ hybridization with an HIV-1 cDNA probe demonstrated the presence of virus-specific nucleic acids within stromal colonies. Coimmunostaining with antibody to CD83 implicated the presence of HIV-1 within dendritic progenitor cells. Immunostaining with HIV-1 Tat antibody demonstrated the presence of Tat protein and reverse transcriptase (RT)-PCR assays showed increased (160-220%) mRNA levels for inflammatory cytokines (tumor necrosis <em>factor</em> alpha [TNF-alpha], interleukin 1beta [IL-1beta], IL-6, and macrophage inflammatory protein 1alpha [MIP-1alpha]). A concentration-dependent decrease in CFU-STROs was observed on incubation with either Tat protein (1-100 ng/ml) or with TNF-alpha or IL-1beta (0.025-25 ng/ml). These results suggest that HIV-1 infection of stromal cells may produce inhibitory <em>factors</em> that suppress the clonogenic potential of MSCs.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that eliminates aberrant mRNAs containing premature termination codons (PTCs). NMD inhibits the production of aberrant proteins that still retain, at least in part, wild-type function as well as dominant-negative peptides. Therefore, the selective inhibition of NMD has the potential to ameliorate NMD-exacerbated mutant phenotypes. However, we do not have sufficient knowledge of how to effectively suppress NMD with minimum cytotoxic effects. In this study, we aimed to identify NMD-related <em>factors</em> that can be targeted to efficiently inhibit NMD without causing significant cytotoxicity to restore the levels of truncated but partially functional proteins. We evaluated the knockdown of <em>15</em> NMD components in Ullrich congenital muscular dystrophy <em>fibroblasts</em>, which have a homozygous frameshift mutation causing a PTC in the collagen type VI α 2 gene. Of the <em>15</em> NMD <em>factors</em> tested, knockdown of SMG-8 produced the best effect for restoring defective mRNA and protein levels without affecting cell <em>growth</em>, cell-cycle progression, or endoplasmic reticulum stress. The efficacy of SMG-8 knockdown to improve the mutant phenotype was confirmed using another cell line, from a cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy patient who carries a PTC-containing mutation in HtrA serine peptidase 1. Our results suggest that SMG-8 is an appropriate target for inhibiting NMD to improve NMD-exacerbated mutant phenotypes. NMD inhibition by knockdown of SMG-8 may also be useful to induce synergy in combining the use of read-through drugs for patients with nonsense mutation-associated diseases.

BACKGROUND

Platelet-rich plasma (PRP) contains multiple growth factors and has been shown to enhance fat graft survival after lipotransfer. However, the molecular mechanisms mediating this effect remain unknown. Adipose-derived stem cells (ASCs) play an important role in fat graft survival and are a likely target for PRP-mediated effects. This study seeks to investigate the impact of PRP on ASC proliferation and adipogenic differentiation.

METHODS

Human ASCs were isolated using our laboratory protocol. The experiments were divided into four arms: (1) ASCs cultured in general culture medium alone; (2) ASCs in general culture medium + 5%, 10%, 15%, or 20% PRP; (3) ASCs cultured in adipogenic differentiation medium alone; (4) ASCs cultured in adipogenic medium + 5%, 10%, 15%, or 20% PRP. Cell proliferation was analyzed and comparative m-RNA expression of adipogenic genes was assessed by quantitative PCR. Protein expression was determined by western blot.

RESULTS

PRP significantly enhanced proliferation of ASCs, even in the presence of antiproliferative, proadipogenic media. In contrast, PRP inhibited adipogenic differentiation in adipogenic media, evidenced by decreased intracellular lipid accumulation and reduced adipogenic gene expression (PPAR-γ and FABP4). Inhibition appears to occur through downregulation of bone morphogenetic protein receptor IA (BMPRIA) and fibroblast growth factor receptor 1 (FGFR1). Interestingly, PRP elicited these effects across the entire range of doses studied.

CONCLUSIONS

PRP appears to modulate ASC function primarily by enhancing cell proliferation. The consequences of its impact on adipogenesis are less clear. Enhanced proliferation initially might set the stage for more robust regeneration and adipogenesis at later time points, providing an important target for ongoing research.

Bile acids regulate lipid and carbohydrate metabolism by interaction with membrane or intracellular proteins including the nuclear farnesoid X receptor (FXR). Postprandial activation of ileal FXR leads to secretion of fibroblast growth factor 19 (FGF-19), a gut hormone that may be implicated in postprandial glucose metabolism.

To describe postprandial plasma concentrations of 12 individual bile acids and FGF-19 in patients with type 2 diabetes (T2D) and healthy controls.

Descriptive study, performed at the Center for Diabetes Research, Gentofte Hospital, Hellerup, Denmark.

Fifteen patients with T2D and 15 healthy matched controls with normal glucose tolerance.

A 75-g oral glucose tolerance test and three isocaloric and isovolemic liquid meals with low, medium, and high fat content, respectively.

Bile acid and FGF-19 concentrations.

Postprandial total bile acid concentrations increased with increasing meal fat content (P < .05), peaked after 1-2 hours, and were higher in T2D patients vs controls (oral glucose tolerance test, low and medium fat meals, P < .05; high fat meal, P = .30). Differences reflected mainly unconjugated and glycine-conjugated forms of deoxycholic acid (DCA) and to a lesser extent cholic acid (CA) and ursodeoxycholic acid (UDCA), whereas chenodeoxycholic acid (CDCA) concentrations were comparable in the two groups. FGF-19 concentrations tended to be lower in T2D patients vs controls, but differences were not statistically significant due to considerable variation.

Postprandial plasma patterns of bile acids with FXR agonistic properties (CDCA, DCA, and CA) and FXR antagonistic properties (UDCA) in T2D patients support the notion of a "T2D-bile acid-FGF-19" phenotype with possible pathophysiological implications.

Cholangiocarcinoma is the most common aggressive biliary tract malignancy with dismal prognosis. Though surgical resection of the primary tumors yields better prognosis, majority of patients present at advanced, inoperable stages rendering systemic therapy as the only option. A significant progress has been made in understanding the cholangiocarcinoma tumorigenesis and molecular markers over the last decade, which opens doors to precision medicine in this dismal cancer. Intrahepatic cholangiocarcinomas are most likely to harbor mutations in isocitrate dehydrogenase genes (IDH1, IDH2), <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR1, FGFR2, FGFR3), Eph receptor 2 (EPHA2), and BAP1 (gene involved in chromatin remodeling) genes, whereas ARID1B, ELF3, PBRM1, cAMP dependent protein kinase (PRKACA, and PRKACB) genetic mutations were implicated more commonly in distal and perihilar subtypes. Genomic studies have shown that FGFR2 aberrations are implicated in approximately <em>15</em>% of intrahepatic cholangiocarcinomas, which make FGFR2 aberrations (Achilles heel) as potential novel targets in the management of cholangiocarcinoma. The current review comprehensively focuses on the role of FGFR2 inhibition either alone or in combination with other targeted therapy that act on down-stream and alternate kinase pathways in cholangiocarcinoma.

Lenvatinib significantly prolonged progression-free survival (PFS) versus placebo in the phase III Study of (E7080) LEnvatinib in differentiated Cancer of the Thyroid (SELECT) of patients with radioiodine-refractory differentiated thyroid cancer. This exploratory analysis investigated potential predictive biomarkers of lenvatinib efficacy and target engagement.

Circulating cytokine/angiogenic factors (CAFs) in blood samples collected at baseline and throughout treatment were analysed from patients randomised to receive lenvatinib or placebo from August 5, 2011 to October 4, 2012. For CAF biomarker analyses, patients were dichotomised by baseline levels. Tumour tissues were analysed for BRAF and NRAS/KRAS/HRAS mutations.

Tumours and CAFs were analysed from 183/392 (47%) and 387/392 (99%) patients, respectively. Lenvatinib PFS benefit was maintained in all assessments. For lenvatinib-treated patients, interaction-term analyses revealed that low baseline Ang2 level was predictive of tumour shrinkage (Pinteraction = 0.016) and PFS (Pinteraction = 0.018). Vascular endothelial growth factor and fibroblast growth factor 23 (FGF23) were significantly upregulated with lenvatinib, and FGF23 upregulation on cycle 1/day 15 was associated with longer PFS. In mutation analyses, no significant differences in clinical outcomes were observed. BRAFWT may be a negative prognostic factor for PFS in placebo-treated patients with papillary thyroid cancer (P = 0.019).

The lenvatinib PFS benefit was maintained regardless of baseline CAF or BRAF/RAS status. Baseline Ang2 was predictive of PFS in a subgroup of lenvatinib-treated patients, indicating that Ang2 may be predictive of lenvatinib sensitivity. BRAFWT may be a poor prognostic factor in patients with radioiodine-refractory papillary thyroid cancer. Improved PFS associated with upregulated FGF23 suggests that lenvatinib-induced FGF receptor inhibition contributes to lenvatinib efficacy. Trial registration ID of the main study, SELECT: ClinicalTrials.gov: NCT01321554.

Uterine leiomyomas are benign tumors in the smooth muscle layer of the uterus. The most common histological type is the "usual leiomyoma", characterized by overexpression of ECM proteins, whereas the "cellular type" has higher cellular content. Our objective is to investigate the involvement of inflammatory and reparative processes in leiomyoma pathobiology. Using a morphological approach, we investigate the presence of inflammatory cells. Next, we determine the localization of the ECM, the presence/absence of fibrotic cells via α-sma and desmin and the immunohistochemical profile of the mesenchymal cells with respect to CD34. Finally, we explore the effect of inflammatory mediators (TNF-α, IL-1β, IL-6, IL-<em>15</em>, GM-CSF and IFN-γ) on pro-fibrotic <em>factor</em> activin A mRNA expression in vitro. Higher numbers of macrophages were found inside and close to leiomyomas as compared to the more distant myometrium. Cellular leiomyomas showed more macrophages and mast cells than the "usual type". Inside the fibroid tissue, we found cells positive for α-sma, but negative for desmin and a large amount of collagen surrounding the nodule, suggestive of myo<em>fibroblasts</em> producing ECM. In the myometrium and leiomyomas of the "usual type", we identified numerous CD34+ <em>fibroblasts</em>, which are known to give rise to myo<em>fibroblasts</em> upon loss of CD34 expression. In leiomyomas of the "cellular type", stromal <em>fibroblasts</em> were CD34-negative. Finally, we found that TNF-α increased activin A mRNA in myometrial and leiomyoma cells. In conclusion, this study demonstrates the presence of inflammatory cells in uterine leiomyomas, which may contribute to excessive ECM production, tissue remodeling and leiomyoma <em>growth</em>.

OBJECTIVE

Some targeted anticancer agents are associated with serious ventricular tachyarrhythmias, which may be predicted by electrocardiographic evaluation of drug-related QT prolongation. We studied the effects of nintedanib (BIBF 1120; an oral, triple angiokinase inhibitor targeting vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptors) on the QT interval in patients with renal cell carcinoma (RCC) participating in an open-label phase II trial.

METHODS

Treatment-naïve, adult patients with unresectable/metastatic, clear cell RCC received nintedanib 200 mg twice daily. QT intervals were evaluated at baseline (day -1), on day 1 (after the first dose), and on day 15 (steady state) by 12-lead electrocardiograms (ECGs) performed in triplicate. Pharmacokinetic sampling was also undertaken.

RESULTS

Among 64 evaluable patients, the upper limits of the 2-sided 90 % confidence intervals for the adjusted mean time-matched changes in QTcF interval (corrected for heart rate by Fridericia's method) from baseline to day 1 and 15 (primary ECG endpoint) were well below the regulatory threshold of 10 ms at all times. No relationship between nintedanib exposure and change from baseline in QTcF was seen. Nintedanib was generally well tolerated with no drug-related cardiovascular adverse events.

CONCLUSIONS

Nintedanib administered at 200 mg twice daily was not associated with clinically relevant QT prolongation.

Mitogenesis induced by most polypeptide <em>growth</em> <em>factors</em> is mediated by either G-protein- or tyrosine kinase-linked receptor pathways. Thrombin, a potent mitogen for vascular smooth muscle cells, activates a G-protein-coupled receptor but also requires tyrosine kinase activity for its mitogenic effect (Weiss, R. H., and Nuccitelli, R. (1992) J. Biol. Chem. 267, 5608-5613). Since this <em>growth</em> <em>factor</em> elicits a synergistic effect on DNA synthesis when applied to cells concurrently with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), we suspected that the two <em>growth</em> <em>factors</em> have points of convergence in their signaling pathways. We now show that when 1 unit/ml thrombin is removed after an incubation period of from 4 h to <em>15</em> min prior to <em>15</em> ng/ml bFGF addition, its synergistic effect with bFGF on mitogenesis in vascular smooth muscle cells is preserved. Furthermore, appearance of bFGF in the cellular lysate is maximal 1 h after the addition of 1 unit/ml thrombin. While monoclonal antibody to bFGF inhibits thrombin's mitogenic effect by 63% at 30 micrograms/ml, it lacks an inhibitory effect on platelet-derived <em>growth</em> <em>factor</em>-BB-induced mitogenesis. The inhibitory effect of bFGF antibody on thrombin's <em>growth</em> is completely reversed by the addition of bFGF. These data demonstrate that the presence of bFGF is essential for thrombin to exert its full mitogenic effect in vascular smooth muscle cells, providing an example of a system where a tyrosine kinase-linked <em>growth</em> <em>factor</em> receptor system can act as an essential intermediary in the mitogenic signaling pathway of a G-protein-coupled receptor.

Skeletal muscle has been emerging as a research field since the past 2 decades. Contraction of a muscle, which acts as a secretory organ, stimulates production, secretion, and expression of cytokines or other muscle fiber-derived peptides, i.e., myokines. Exercise-induced myokines influence crosstalk between different organs in an autocrine, endocrine, or paracrine fashion. Myokines are recently recognized as potential candidates for treating metabolic diseases through their ability to stimulate AMP-activated protein kinase signaling, increase glucose uptake, and improve lipolysis. Myokines may have positive effects on metabolic disorders, type 2 diabetes, or obesity. Numerous studies on myokines suggested that myokines offer a potential treatment option for preventing metabolic diseases. This review summarizes the current understanding of the positive effects of exercise-induced myokines, such as interleukin-<em>15</em>, brain-derived neurotrophic <em>factor</em>, leukemia inhibitory <em>factor</em>, irisin, <em>fibroblast</em> <em>growth</em> <em>factor</em> 21, and secreted protein acidic and rich in cysteine, on metabolic diseases.

Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na⁺/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine.

The response of endothelial cells (EC) to high radiation doses leads to damage of normal tissue or tumor. The precise mechanisms of the endothelial-tissue linkage are still largely unknown. We investigated the possible involvement of a bystander effect, secondary to endothelial damage, in tissue response to radiation. Proliferating human intestinal epithelial T84 cells were grown in a non-contact co-culture with confluent primary human microvascular EC (HMVEC-L). The bystander response in unirradiated T84 cells co-cultured with irradiated EC was studied by evaluating cell <em>growth</em>, cell death and epithelial morphology. Twenty-four hours after exposure of EC to <em>15</em> Gy, unirradiated T84 cells showed a decreased cell number (29%) and percentage in mitosis (66%) as well as increased apoptosis (1.5-fold) and cell surface area (1.5-fold), highlighting the involvement of bystander effects on T84 cells after irradiation of EC. Furthermore, the responses of T84 cells were amplified when EC and T84 cells were irradiated together, indicating that the bystander response in T84 cells adds further to direct radiation damage. As opposed to direct irradiation, the T84 cell bystander response did not involve the cell cycle-related protein p21(Waf1) (CDKN1A) and pro-apoptosis protein BAX. The bystander effect was specific to EC since the irradiation of human colon <em>fibroblasts</em> did not induce bystander responses in unirradiated T84 cells. These results strengthen previous in vivo evidence of the role of EC in tissue damage by radiation. In addition, this study provides a suitable and useful model to identify soluble <em>factors</em> involved in bystander effects secondary to endothelial damage. Modulating such <em>factors</em> may have important clinical implications.

The gut microbiota were shown to play critical roles in the development of atherosclerosis, but the detailed mechanism is limited. The purpose of this study is to clarify the influence of gut microbiota on atherogenesis via lipid metabolism and systemic inflammation. Germ-free or conventionally raised (Conv) ApoE-deficient (ApoE-/-) mice were fed chow diet and euthanized at 20 weeks of age. We found that the lack of gut microbiota in ApoE-/- mice caused a significant increase in the plasma and hepatic cholesterol levels compared with Conv ApoE-/- mice. The absence of gut microbiota changed the bile acid composition in the ileum, which was associated with activation of the enterohepatic <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 axis, and reduction of cholesterol 7α-hydroxylase and hepatic bile acid synthesis, resulting in the accumulation of liver cholesterol content. However, we found that the lack of microbiota caused a significant reduction in atherosclerotic lesion formation compared with Conv ApoE-/- mice, which might be associated with the attenuation of lipopolysaccharide-mediated inflammatory responses. Our findings indicated that the gut microbiota affected both hypercholesterolemia and atherogenesis in mice.

OBJECTIVE

Human hepatoma-derived growth factor, purified from the conditioned medium of hepatoma-derived cell line, HuH-7, stimulates the growth of Swiss 3T3 fibroblasts and HuH-7 cells. To evaluate the role of hepatoma-derived growth factor on the growth of hepatoma cells, we investigated the effects of recombinant hepatoma-derived growth factor protein and hepatoma-derived growth factor antisense oligonucleotides on the proliferation of several hepatoma cell lines.

METHODS

We examined the effects of hepatoma-derived growth factor antisense oligonucleotides on the growth of hepatoma cells by cell growth assay.

RESULTS

Hepatoma-derived growth factor stimulated the proliferation of some hepatoma cells (HuH-7, HLF, HepG2, AH66tc cells) about 15-70% over than the control. Hepatoma-derived growth factor antisense oligonucleotides, phosphorothioate-linked or encapsulated in liposome, can inhibit the growth of hepatoma cells. The ID50 of hepatoma-derived growth factor antisense phosphorothioate oligonucleotides for HuH-7 cells, in which hepatoma-derived growth factor expression was abundant, was 3 microM by the assay of cell proliferation and [3H]-thymidine incorporation. Their ID50 for AH66tc cells, on which the effects of exogenous hepatoma-derived growth factor were weak, was higher than 10 microM. To omit the toxic effects due to phosphorothioate modification of oligonucleotides and keep the cellular uptake more without their destruction in the culture medium, we used oligonucleotides encapsulated in cationic liposome. Hepatoma-derived growth factor antisense oligonucleotides encapsulated in liposome suppressed the growth of hepatoma cells effectively (ID50:2.0 microM).

CONCLUSIONS

These findings suggest that hepatoma-derived growth factor is one of important autocrine, and/or intracrine factors for hepatoma cells, and that hepatoma-derived growth factor anti-sense oligonucleotides may be useful for human hepatocellular carcinoma as an anti-cancer agent.

Using a discovery/replication approach, we investigated associations between a multiplex panel of 80 circulating proteins associated with cardiovascular pathology or inflammation, and eGFR decline per year and CKD incidence.

We used two cohorts, the Prospective Investigation of the Vasculature in Uppsala Seniors Study (PIVUS; n=687, mean age of 70 years, 51% women) and the Uppsala Longitudinal Study of Adult Men (ULSAM; n=360 men, mean age of 78 years), with 5-year follow-up data on eGFR. There were 231 and 206 incident cases of CKD during follow-up in the PIVUS and ULSAM studies, respectively. Proteomic profiling of 80 proteins was assessed by a multiplex assay (proximity extension assay). The assay uses two antibodies for each protein and a PCR step to achieve a high-specific binding and the possibility to measure multiple proteins in parallel, but gives no absolute concentrations.

In the discovery cohort from the PIVUS Study, 28 plasma proteins were significantly associated with eGFR decline per year, taking into account the multiple testing. Twenty of these proteins were significantly associated with eGFR decline per year in the replication cohort from the ULSAM Study after adjustment for age, sex, cardiovascular risk <em>factors</em>, medications, and urinary albumin-to-creatinine ratio (in order of significance: TNF-related apoptosis-inducing ligand receptor 2*, CD40L receptor, TNF receptor 1*, placenta <em>growth</em> <em>factor</em>*, thrombomodulin*, urokinase plasminogen activator surface receptor*, <em>growth</em>/differentiation <em>factor</em> <em>15</em>*, macrophage colony-stimulating <em>factor</em> 1, fatty acid-binding protein*, cathepsin D, resistin, kallikrein 11*, C-C motif chemokine 3, proteinase-activated receptor 1*, cathepsin L, chitinase 3-like protein 1, TNF receptor 2*, <em>fibroblast</em> <em>growth</em> <em>factor</em> 23*, monocyte chemotactic protein 1, and kallikrein 6). Moreover, 11 of the proteins predicted CKD incidence (marked with * above). No protein consistently predicted eGFR decline per year independently of baseline eGFR in both cohorts.

Several circulating proteins involved in phosphate homeostasis, inflammation, apoptosis, extracellular matrix remodeling, angiogenesis, and endothelial dysfunction were associated with worsening kidney function. Multiplex proteomics appears to be a promising way of discovering novel aspects of kidney disease pathology.

We propose the acute, local suppression of transforming <em>growth</em> <em>factor</em> beta (TGF-ß), a major profibrotic cytokine, to reduce fibrosis around silicone implants. To this end, we prepared silicone implants that were able to release tranilast, a TGF-ß inhibitor, in a sustained manner for 5 days or <em>15</em> days. We performed histologic and immunohistochemical analyses for 12 weeks after the implantation of the implants in living rats. The capsule thicknesses and collagen densities significantly decreased compared with those around the non-treated silicone implants. Notably, early suppression of TGF-ß affected the fibrogenesis that actually occurs at the late stage of wound healing. This change may be ascribed to the decrease in monocyte recruitment mediated by early TGF-ß during the acute inflammatory reaction. Thus, a significant decrease in differentiated macrophages was observed along with a decrease in the quantity of TGF-ß and <em>fibroblasts</em> during the subsequent inflammation stage; these changes led to a diminished fibrotic capsule formation.

Limb bud outgrowth in chicken embryos is initiated during the third day of development by <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> 8 (FGF8) produced by the newly formed apical ectodermal ridge (AER). One of the earliest effects of this induction is a change in the properties of the limb field mesoderm leading to bulging of the limb buds from the body wall. Heintzelman et al. [Heintzelman, K.F., Phillips, H.M., Davis, G.S., 1978. Liquid-tissue behavior and differential cohesiveness during chick limb budding. J. Embryol. Exp. Morphol. 47, 1-<em>15</em>.] suggested that budding of the limbs is caused by a higher liquid-like cohesivity of limb bud tissue compared with flank. We sought additional evidence relevant to this hypothesis by performing direct measurements of the effective surface tension, a measure of relative tissue cohesivity, of 4-day embryonic chicken wing and leg bud mesenchymal tissue, and adjacent flank mesoderm. As predicted, the two types of limb tissues were 1.5- to 2-fold more cohesive than the flank tissue. These differences paralleled cell number and volume density differences: 4-day limb buds had 2- to 2.5-fold as many cells per unit area of tissue as surrounding flank, a difference also seen at 3 days, when limb budding begins. Exposure of flank tissue to exogenous FGF8 for 24 h increased its cell number and raised its cohesivity to limb-like values. Four-day flank tissue exhibited a novel and unique active rebound response to compression, which was suppressed by the drug latrunculin and therefore dependent on an intact actin cytoskeleton. Correspondingly, flank at this stage expressed high levels of alpha-smooth muscle actin (SMA) mRNA and protein and a dense network of microfilaments. Treatment of flank with FGF8 eliminated the rebound response. We term material properties of tissues, such as cohesivity and mechanical excitability, the "physical phenotype", and propose that changes thereof are driving forces of morphogenesis. Our results indicate that two independent aspects of the physical phenotype of flank mesoderm can be converted to a limb-like state in response to treatment with FGF8. The higher tissue cohesivity induced by this effect will cause the incipient limb bud to phase separate from the surrounding flank, while the active mechanical response of the flank could help ensure that the limb bud bulges out from, rather than becoming engulfed by, this less cohesive tissue.

Vitamin D deficiency (VDD) or insufficiency is recognized for its association with nonalcoholic steatohepatitis (NASH), whereas the underlying mechanism remains unknown. Using animal models, we found that vitamin D deficiency promoted the high-fat diet (HFD)-initiated simple steatosis into typical NASH, characterized by elevated hepatic inflammation and fat degeneration. The NASH derived from VDD + HFD was related to poor retention of bile acids in the liver and biliary tree, in line with downregulation of the ileal apical sodium-dependent bile acid cotransporter (iASBT). The impediment of hepatic bile acids by the VDD + HFD mice was related to increased expression of hepatic SREBP-1c and fatty acid synthase, suggesting that VDD may upregulate endogenous fatty acid synthesis into NASH through impaired enterohepatic circulation. Administration of 1,25(OH)2VD3 (calcitriol) corrected the NASH phenotypes in line with restoration of iASBT, promotion of bile filling in the biliary tree, suppression of hepatic lipogenesis, and inflammation. Moreover, administration of a bile acid-sequestering agent suppressed ileal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> expression, leading to increased iASBT expression to restore bile filling in the liver and biliary tree, which ameliorates steatosis and inflammation in the liver. These results suggest a novel mechanism for NASH development, by which VDD downregulates iASBT expression, resulting in a poor bile acid pool and elevation of hepatic lipogenesis and inflammation. In conclusion, vitamin D and bile acid sequestration may be explored as new strategies to treat or prevent NASH.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (<em>15</em>% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Keloids are abnormal fibrous <em>growths</em> of the dermis that develop only in response to wounding and represent a form of benign skin tumor. Previous studies have shown increased protein levels of TGF-beta in keloid tissue, suggesting a strong association with keloid formation leading us to examine mechanisms for why it is more highly expressed in keloids. Here, we use serum stimulation as an in vitro model to mimic a component of the wound microenvironment and examine differential gene expression in keloid human <em>fibroblasts</em> (KFs) vs. normal human <em>fibroblasts</em> (NFs). Transcription of TGF-beta2 was rapid and peaked between 1 and 6 h after serum stimulation in KFs vs. NFs. We confirmed increased TGF-beta activity in the conditioned medium from KFs, but not NFs. Inhibition of second messenger signaling pathways demonstrated that only the p38 MAPK inhibitor SB-203580 could block upregulation of TGF-beta2 following serum stimulation in KFs. Immunoblotting demonstrated that p38 MAPK was phosphorylated within <em>15</em> min and was maintained at a high level in KFs but not in NFs. The transcription <em>factors</em> activating transcription <em>factor</em>-2 and Elk-1 are activated by p38 MAPK, and also showed rapid and prolonged phosphorylation kinetics in KFs but not in NFs. In conclusion, increased TGF-beta2 transcription in response to serum stimulation in KFs appears to be mediated by the p38 MAPK pathway. This suggests the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the <em>fibroblasts</em> respond to wounding.

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated <em>growth</em>, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic <em>fibroblast</em> cell line expressing recombinant, membrane-bound porcine stem cell <em>factor</em> (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages <em>15</em> and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism.

To investigate the frequency and the prognostic impact of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) gene amplification in 526 curatively resected esophageal squamous cell carcinoma (ESCC). Using fluorescent in situ hybridization, high amplification was defined by an FGFR1/centromer 8 ratio is ≥ 2.0, or average number of FGFR1 signals/tumor cell nucleus ≥ 6.0, or percentage of tumor cells containing ≥ <em>15</em> FGFR1 signals or large cluster in ≥ 10%. Low amplification was defined by ≥ 5 FGFR1 signals in ≥ 50%. FGFR2 and FGFR3 mutations were assessed by direct sequencing in 388 cases and no mutation was detected. High and low amplification were detected in 8.6% and 1.1%, respectively. High FGFR1 amplification had significantly shorter disease-free survival (34.0 vs <em>15</em>8.5 months P=0.019) and overall survival (52.2 vs not reached P=0.022) than low/no amplification group. After adjusting for sex, smoking, stage, histology, and adjuvant treatment, high FGFR1 amplification had a greater risk of recurrence (adjusted hazard ratio [AHR], 1.6; P=0.029) and death (AHR, 1.53; P=0.050). High amplification was significantly higher in current smokers than former and never-smokers (Ptrend<0.001) and increased proportional to smoking dosage. High FGFR1 amplification is a frequent oncogenic alteration and an independent poor prognostic <em>factor</em> in resected ESCC.