BACKGROUND

Type 4 cardiorenal syndrome (CRS) refers to the cardiac injury induced by chronic kidney disease. We aimed to assess oxidative stress and cardiac injury in patients with type 4 CRS, determine whether the antioxidant apocynin attenuated cardiac injury in rats with type 4 CRS, and explore potential mechanisms.

RESULTS

A cross-sectional study was conducted among patients with type 4 CRS (n=<em>17</em>) and controls (n=16). Compared with controls, patients with type 4 CRS showed elevated oxidative stress, which was significantly correlated with cardiac hypertrophy and decreased ejection fraction. In vivo study, male Sprague-Dawley rats underwent 5/6 subtotal nephrectomy and sham surgery, followed with apocynin or vehicle treatment for 8 weeks. Eight weeks after surgery, the 5/6 subtotal nephrectomy rats mimicked type 4 CRS, showing increased serum creatinine, cardiac hypertrophy and fibrosis, and decreased ejection fraction compared with sham-operated animals. Cardiac malondialdehyde, NADPH oxidase activity, <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation increased significantly in the 5/6 subtotal nephrectomy rats. These changes were significantly attenuated by apocynin. In vitro study showed that apocynin reduced angiotensin II-induced NADPH oxidase-dependent oxidative stress, upregulation of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 and fibrosis biomarkers, and ERK1/2 phosphorylation in cardiac <em>fibroblasts</em>. Importantly, the ERK1/2 inhibitor U0126 reduced the upregulation of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 and fibrosis biomarkers in angiotensin II-treated <em>fibroblasts</em>.

CONCLUSIONS

Oxidative stress is a candidate mediator for type 4 CRS. Apocynin attenuated cardiac injury in type 4 CRS rats via inhibiting NADPH oxidase-dependent oxidative stress-activated ERK1/2 pathway and subsequent fibroblast growth factor-2 upregulation. Our study added evidence to the beneficial effect of apocynin in type 4 CRS.

The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1beta and tumor necrosis <em>factor</em> alpha induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a <em>17</em>- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming <em>growth</em> <em>factor</em> beta had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.

BACKGROUND

Tumor neovascularization is necessary for continued tumor growth and metastasis. During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface. Targeting these molecular markers would, in effect, allow for specific tumor targeting. Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs. These sequences were identified from in vivo phage peptide display libraries. The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature.

METHODS

FliTrx is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E. coli. Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor-derived endothelial cells (TDEC). MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor-containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature. FliTrx cells were incubated with MAGIC at 4 degrees C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets. The non-binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs. The bacterial population was then expanded and this "panning" process was carried out a total of five times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs.

RESULTS

Recurring peptide sequences were detected that were 3-mers (13 sequences) and 4-mers (4 sequences). Of the 3-mers, four repeated 3 times, whereas none of the 4-mers repeated more than twice. All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid. A tripeptide basic-basic-nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences). Two test peptides showed TDEC binding specificity, and both conformed to the basic-basic-nonpolar motif.

CONCLUSIONS

We report peptide sequences derived from panning an in vitro system designed to detect tumor-EC specific markers. These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.

Secreted proteins of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-<em>17</em> (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-<em>17</em>a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.

Interstitial tissue of the testis consists of Leydig cells, macrophages, lymphocytes, plasma cells, mast cells and <em>fibroblasts</em>. Previously we have reported that interleukin-1 (IL-1) inhibits Leydig cell androgen production. In the present study, the effect of IL-2 was investigated. Leydig cells (10(5) cells/ml) from adult Sprague-Dawley rats were cultured with or without IL-2 for 24 h. After medium changes, human CG (hCG), 8-bromo-cAMP, or forskolin was added with or without IL-2. Cultures were continued for an additional 24 h, and testosterone and cAMP levels were measured. IL-2 up to 100 U/ml had no effect on basal testosterone production. hCG-stimulated testosterone formation was inhibited in a dose-dependent manner by the addition of IL-2. IL-2 in a concentration of 100 U/ml decreased hCG-induced testosterone formation from 49.6 +/- 3.6 ng/ml (mean +/- SE) to 8.5 +/- 4.2 ng/ml. The hCG dose-response curve was shifted to the right by the addition of IL-2. Maximal testosterone production in response to hCG was reduced 40% in the presence of IL-2 (50 U/ml) without alteration of median effective dose (ED50). IL-2 also inhibited hCG-induced cAMP formation and 8-bromo cAMP- and forskolin-stimulated testosterone production. However, IL-2 did not alter the binding of [125I]hCG to purified Leydig cells. Furthermore, IL-2 significantly inhibited the conversion of 20-OH-cholesterol, 22-OH-cholesterol, pregnenolone, progesterone, <em>17</em> alpha-hydroxypregnenolone, and <em>17</em> alpha-hydroxyprogesterone to testosterone but did not alter the conversion of dehydroepiandrosterone and androstenedione to testosterone. Our results suggest that a T cell <em>growth</em> <em>factor</em>, IL-2, is a potent inhibitor of steroidogenesis. IL-2 may play a paracrine role in modulating Leydig cell function.

BACKGROUND

Fibroblast Growth Factors (FGF) and their receptors are well known for having major implications in cell signalling controlling embryonic development. Recently, a gene coding for a protein closely related to FGFRs (Fibroblast Growth Factor Receptors) called FGFR5 or FGFR-like 1 (FGFRL1), has been described in vertebrates. An orthologous gene was also found in the cephalochordate amphioxus, but no orthologous genes were found by the authors in other non-vertebrate species, even if a FGFRL1 gene was identified in the sea urchin genome, as well as a closely related gene, named nou-darake, in the planarian Dugesia japonica. These intriguing data of a deuterostome-specific gene that might be implicated in FGF signalling prompted us to search for putative FGFRL1 orthologues in the completely sequenced genomes of metazoans.

RESULTS

We found FGFRL1 genes in the cnidarian Nematostella vectensis as well as in many bilaterian species. Our analysis also shows that FGFRL1 orthologous genes are linked in the genome with other members of the FGF signalling pathway from cnidarians to bilaterians (distance < 10 Mb). To better understand the implication of FGFRL1 genes in chordate embryonic development, we have analyzed expression patterns of the amphioxus and the mouse genes by whole mount in situ hybridization. We show that some homologous expression territories can be defined, and we propose that FGFRL1 and FGF8/17/18 were already co-expressed in the pharyngeal endoderm in the ancestor of chordates.

CONCLUSIONS

Our work sheds light on the existence of a putative FGF signalling pathway actor present in the ancestor of probably all metazoans, the function of which has received little attention until now.

Synovial sarcoma is a soft tissue malignancy with a poor prognosis; many patients will die from this disease within 10 years of diagnosis, despite treatment. Gene expression profiling and immunohistochemistry studies have identified oncogenes that are highly expressed in synovial sarcoma. Included in this group are receptor tyrosine kinases such as epidermal <em>growth</em> <em>factor</em> receptor, insulin-like <em>growth</em> <em>factor</em> receptor 1, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3, KIT, and HER2. Inhibitors of these <em>growth</em>-promoting receptors are likely to inhibit proliferation of synovial sarcoma; however, the effect of receptor tyrosine kinase inhibitors on synovial sarcoma is largely unknown. We assessed the ability of the following receptor tyrosine kinase inhibitors to halt proliferation and induce apoptosis in synovial sarcoma monolayer and three dimensional spheroid in vitro models: gefitinib (Iressa), NVP-AEW541, imatinib mesylate (Gleevec), SU5402, PRO-001, trastuzumab (Herceptin), and <em>17</em>-allylamino-<em>17</em>-demethoxygeldanamycin (<em>17</em>-AAG). Gefitinib, NVP-AEW541, and imatinib inhibited proliferation only at relatively high concentrations, which are not clinically applicable. <em>17</em>-AAG, which destabilizes multiple receptor tyrosine kinases and other oncoproteins through heat shock protein 90 inhibition, prevented proliferation and induced apoptosis in synovial sarcoma monolayer models at concentrations achievable in human serum. <em>17</em>-AAG treatment was also associated with receptor tyrosine kinase degradation and induction of apoptosis in synovial sarcoma spheroid models. <em>17</em>-AAG was more effective than doxorubicin, particularly in the spheroid models. Here we provide in vitro evidence that <em>17</em>-AAG, a clinically applicable drug with known pharmacology and limited toxicity, inhibits synovial sarcoma proliferation by inducing apoptosis, and thus has potential as a systemic therapy for this disease.

Human sebaceous gland possesses all the steroidogenic enzymes required for androgen synthesis. It remains unclear whether the testosterone produced in situ mainly derives from circulating dehydroepiandrosterone (DHEA) or from de novo synthesis utilizing serum cholesterol. Using testosterone radioimmunoassay, we found that testosterone was barely detectable in the supernatant of cultured human SZ95 sebocytes when cholesterol was added alone, indicating a low basal expression of steroidogenic acute regulatory protein (StAR) in SZ95 cells. Human chorionic gonadotropin and <em>fibroblast</em> <em>growth</em> <em>factor</em>-9 were as potent as forskolin in activating StAR to enhance testosterone production, while interleukin-1 beta, dexamethasone, insulin and insulin-like <em>growth</em> <em>factor</em>-1 showed no stimulatory effect. A two-fold increase of testosterone production was observed in supplementation of DHEA as compared to pregnenolone, progesterone or <em>17</em> alpha-hydroxyprogesterone. Based on our findings, testosterone synthesized in cultured sebocytes derived mainly from DHEA and inhibition of 3beta-hydroxysteroid dehydrogenase and <em>17</em>beta-hydroxysteroid dehydrogenase may be a new target of androgen suppression for acne treatment.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and <em>17</em>-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.

OBJECTIVE

To assess the effect of crystalline triamcinolone acetonide on retinal endothelial cell proliferation in vivo and in vitro.

METHODS

For in vitro analysis, a sprouting assay was employed. Bovine retinal endothelial cells were stimulated with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and incubated with different concentrations of triamcinolone acetonide (0.05 mg/ml to 8 mg/ml). For in vivo analysis, a retinopathy of prematurity (ROP) model was used. 16 C57BL/J6 mice were exposed to 75% oxygen from postnatal day 7 to day 12. On day 12, triamcinolone acetonide was intravitreally injected into one eye ("study eye") and isotonic saline into the contralateral eye ("control eye"). On day <em>17</em>, the mice were sacrificed and the eyes removed for quantitative analysis of preretinal neovascularisation. Four non-exposed mice served as negative control.

RESULTS

The sprouting assay demonstrated a dose dependent inhibition of bovine retinal endothelial cell proliferation from 0.05 mg triamcinolone acetonide/ml (no inhibition) to 3 mg triamcinolone acetonide/ml (complete inhibition). Dosages of more than 2 mg/ml resulted in cytotoxic changes of endothelial cells. The ROP model demonstrated a significantly lower neovascular cell count of 58% in the study group compared to the control group (6.35 (SD 2.1) cells per histological section versus 14.9 (SD 5.3) cells; p<0.005).

CONCLUSIONS

Triamcinolone acetonide inhibits bFGF induced proliferation of retinal endothelial cells in vivo and in vitro. These findings contribute to understanding the mode of action and effects of triamcinolone acetonide on retinal neovascularisation.

OBJECTIVE

Antivascular endothelial growth factor receptor (VEGFR) agents improve hepatic fibrosis and portal hypertension in cirrhosis. Detail interactions among recruited/activated leucocytes, hepatic angiogenesis and fibrogenesis, splanchnic blood pooling, decreased hepatic veins to fluctuated splanchnic blood volume (hepatic venous dysregulation), portal hypertensive syndrome and ascites have never explored in cirrhosis. Our study used two anti-VEGFR agents - brivanib and sorafenib - to elucidate the relationship between above abnormalities of nonalcoholic steatohepatitis (NASH)-cirrhotic rats.

METHODS

NASH-cirrhotic rats received 2-week brivanib, sorafenib or vehicle (NASH-cirrhotic+briv, NASH-cirrhotic+soraf and NASH-cirrhotic rats) were included for various measurements.

RESULTS

In comparison with NASH-cirrhotic rats, significant decreased plasma VEGF, fibroblast growth factor, platelet-derived growth factor, hepatic tumour necrosis factor (TNFα), IL-1β, IL-6, IL-17 were accompanied by decreased leucocyte mass/activity ((99 m) Tc-phytate and (18) F-FDG SPECT/PET/CT scans), hepatic leucocytes recruitment/microvascular density (fluorescence-enhanced intravital microscopy) and hydroxyproline content, and increased hepatic blood flow in NASH-cirrhotic+briv and NASH-cirrhotic+soraf rats. In addition, increased hepatic microvasculatures compliance-related improved buffering effect of portal vein to acute mannitol infusion was associated with decreased circulating nitric oxide and aldosterone, plasma volume expansion (dye dilution method), splanchnic blood pooling ((99 m) Tc-RBC SPECT/PET/CT scans), peripheral hypotension, portal hypertension and ascites in brivanib and sorafenib-treated NASH-cirrhotic rats.

CONCLUSIONS

Besides antifibrotic, antiangiogenic and portal hypertensive effects, chronic antagonism of anti-VEGFR with brivanib and sorafenib improves hepatic blood flow, hepatic venous dysregulation, inhibits leucocytes recruitment/activation, splanchnic blood pooling and ascites formation in NASH-cirrhotic rats. Thus, brivanib and sorafenib might be ideal therapeutic agents in cirrhotic patients suffering from severe haemodynamic disarrangement and ascites.

We recently demonstrated that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) and platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF-BB) mainly activated extracellular signal-regulated kinase 2 (ERK2) in normal human osteoblastic (HOB) and bone marrow stromal (HBMS) cells by an "in-gel" MAP kinase assay, although both ERK1 and ERK2 proteins were present. In the present study, we examined whether ERK1 is also activated by <em>growth</em> <em>factors</em> by using three different MAPK assay procedures, an "in-gel MAP kinase assay," an immune-complex kinase assay, and western blotting with anti-active MAPK antibody which recognizes specifically activated forms of both ERK1 and ERK2. Results have demonstrated that in addition to ERK2, ERK1 is activated by FGF-2 and PDGF-BB in normal HOB and HBMS cells. The human ERK1 moved faster on SDS-polyacrylamide gel compared to rat and mouse, revealing differences in the apparent molecular weight of FRK1 in normal human osteoblastic and bone marrow osteoprogenitor cells, human (TE-85) and rat (ROS <em>17</em>/2.8 and UMR-106) osteosarcoma, and mouse (MC3T3E1) osteoblastic cells. ERK1 is less stable in the in-gel renaturation process compared to ERK2; thus, in-gel MAP kinase assay does not provide an accurate estimation of ERK1 activity. Results also showed that anti-active MAPK antibody can be used reliably and accurately to measure the activation of ERK1 and ERK2 in osteoblastic cells.

OBJECTIVE

To detect new predictive markers from the prostate cancer tissue, to study the expression by cultured cancer-associated fibroblasts (CAFs) of stromal factors implicated in prostate carcinogenesis, and to compare their expressions in localized, metastatic, castration-sensitive (CSCP), castration-resistant prostate tumors (CRCP) as well as in fibroblasts from benign prostatic hyperplasia (BPH).

METHODS

The genomic expression of 20 stroma-derived factors, including the androgen receptor (AR), growth factors (FGF2, FGF7, FGF10, HGF, TGFβ, PDGFB), protein implicated in invasion (MMP-2, MMP-9 and MMP-11), inflammation (IL-6, IL-17, STAT-3 and NFκB), stroma/epithelium interaction (CDH11, FAP, CXCL12 and CXCL14) and chaperones (HPA1A and HSF1), was evaluated in cultured fibroblasts both from BHP and prostate carcinomas (PCa). After isolation and culture of fibroblasts by biopsy specimens, RNA was isolated and genomic studies performed.

RESULTS

Finally, 5 BPH and 37 PCa specimens were selected: clinically localized (19), metastatic (5), CSCP (7) and CRPC (6). Interleukin-17 receptor (IL-17RB) was highly expressed in CAFs compared with fibroblasts from BPH. However, metalloproteinase-2 and chemokine ligand 14 (CXCL14) were expressed at higher levels by fibroblasts from BPH. The fibroblastic growth factor-7 was highly expressed by CAFs from localized tumors, but metalloproteinase-11 in metastatic tumors. MMP-11, androgen receptor (AR) and heat-shock-70kda-protein-1A (HSPA1A) expressions were significantly higher in CAFs from CRPC.

CONCLUSIONS

These results demonstrate a CAFs heterogeneity among prostate carcinomas with regard to some molecular profile expressions that may be relevant in tumor development (IL-17RB), progression (MMP-11) and castration resistance (AR, MMP-11 and HSPA1A).

Human pluripotent stem cell (hPSC) lines exhibit repeated patterns of genetic variation, which can alter in vitro properties as well as suitability for clinical use. We examined associations between copy-number variations (CNVs) on chromosome <em>17</em> and hPSC mesodiencephalic dopaminergic (mDA) differentiation. Among 24 hPSC lines, two karyotypically normal lines, BG03 and CT3, and BG01V2, with trisomy <em>17</em>, exhibited amplification of the WNT3/WNT9B region and rapid mDA differentiation. In hPSC lines with amplified WNT3/WNT9B, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) signaling through mitogen-activated protein kinase (MAPK)/ERK amplifies canonical WNT signaling by phosphorylating LRP6, resulting in enhanced undifferentiated proliferation. When bFGF is absent, noncanonical WNT signaling becomes dominant due to upregulation of SIAH2, enhancing JNK signaling and promoting loss of pluripotency. When bFGF is present during mDA differentiation, stabilization of canonical WNT signaling causes upregulation of LMX1A and mDA induction. Therefore, CNVs in <em>17</em>q21.31, a "hot spot" for genetic variation, have multiple and complex effects on hPSC cellular phenotype.

The recent findings of cancer-specific metabolic changes, including increased glucose and glutamine consumption, have provided new therapeutic targets for consideration. Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients exhibit several tumor cell-like characteristics; however, the role of glucose and glutamine metabolism in the aberrant proliferation of these cells is unclear. Here, we evaluated the role of these metabolic pathways in RA-FLS proliferation and in autoimmune arthritis in SKG mice.

The expression of glycolysis- or glutaminolysis-related enzymes was evaluated by real-time polymerase chain reaction (PCR) and Western blotting, and the intracellular metabolites were evaluated by metabolomic analyses. The effects of glucose or glutamine on RA-FLS cell growth were investigated using glucose- or glutamine-free medium. Glutaminase (GLS)1 small interfering RNA (siRNA) and the GLS1 inhibitor compound 968 were used to inhibit GLS1 in RA-FLS, and compound 968 was used to study the effect of GLS1 inhibition in zymosan A-injected SKG mice.

GLS1 expression was increased in RA-FLS, and metabolomic analyses revealed that glutamine metabolism was increased in RA-FLS. RA-FLS proliferation was reduced under glutamine-deprived, but not glucose-deprived, conditions. Cell growth of RA-FLS was inhibited by GLS1 siRNA transfection or GLS1 inhibitor treatment. Treating RA-FLS with either interleukin-17 or platelet-derived growth factor resulted in increased GLS1 levels. Compound 968 ameliorated the autoimmune arthritis and decreased the number of Ki-67-positive synovial cells in SKG mice.

Our results suggested that glutamine metabolism is involved in the pathogenesis of RA and that GLS1 plays an important role in regulating RA-FLS proliferation, and may be a novel therapeutic target for RA.

Tumor-induced osteomalacia (TIO) is a rare acquired form of hypophosphatemic osteomalacia, which is usually attributed to the overproduction of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF-23) by benign mesenchymal neoplasms. Localization and thereafter surgical resection of tumors lead to a cure. The present study aimed to investigate the clinical data, diagnostic methods, and follow-up after tumor resection at one medical center in Shanghai to characterize the profile of this rare disorder and to share our successful experience in diagnosis and treatment. Twenty-three patients with adult-onset hypophosphatemia osteomalacia seen in Shanghai Sixth People's Hospital from 2009 to 2014 and 95 normal individuals were enrolled. After taking a medical history and performing a physical examination, we analyzed the laboratory results (including the serum FGF-23 levels) and localized the tumors by 18F-fluorodeoxyglucose positron emission tomography and computed tomography (18F-FDG PET/CT), 99mTc-octreotide (99mTc-OCT) scintigraphy, and magnetic resonance imaging (MRI). On the basis of the results of laboratory tests and imaging findings, tumor resection was conducted in <em>17</em> patients with a certain diagnosis of TIO. The results demonstrated that the <em>17</em> patients (nine men and eight women, average age 46.6 ± 12.9 years) had TIO. FGF-23 level was elevated in 94.1 % of patients (16 of <em>17</em> patients) . Serum phosphorus level decreased in 100 % of patients. 18F-FDG PET/CT revealed five tumors, 99mTc-OCT scintigraphy revealed two tumors, physical examination revealed nine tumors, and MRI revealed one tumor, among which 58.8 % of the causative tumors (10 of <em>17</em> tumors) were located in the lower extremities. After tumor resection, serum phosphorus levels normalized in 100 % of patients (all <em>17</em> patients) in 4-21 days and FGF-23 levels decreased in 90 % of patients (nine of ten patients). We found 64.7 % of the tumors (11 of <em>17</em> tumors) were phosphaturic mesenchymal tumors or a phosphaturic mesenchymal tumor mixed connective tissue variant. Measurement of serum phosphorus and FGF-23 levels in patients with suspected TIO is of paramount importance for diagnosing of TIO. 18F-FDG PET/CT, 99mTc-OCT scintigraphy, and physical examination play a considerable role in revealing TIO-associated tumors. TIO-associated tumors were more frequently located in the lower extremities than in other places; thus, the lower extremities need to be carefully checked. Complete surgical resection results in normalization of parameters in laboratory tests and relief of symptoms of TIO patients.

Tumor necrosis <em>factor</em> (TNF)-like weak inducer of apoptosis (TWEAK) binds to its sole receptor <em>fibroblast</em> <em>growth</em> <em>factor</em>-inducible 14 (Fn14), participating in various inflammatory responses. Recently, TWEAK/Fn14 activation was found prominent in the lesions of cutaneous lupus erythematosus (CLE). This study was designed to further reveal the potential role of this pathway in Ro52-mediated photosensitization. TWEAK, Fn14, and Ro52 were determined in the skin lesions of patients with CLE. Murine keratinocytes received ultraviolet B (UVB) irradiation or plus TWEAK stimulation and underwent detection for Ro52 and proinflammatory cytokines. The chemotaxis of J774.2 macrophages was evaluated on TWEAK stimulation of cocultured keratinocytes. We found that TWEAK, Fn14, and downstream cytokines were highly expressed in CLE lesions that overexpressed Ro52. Moreover, TWEAK enhanced the UVB-induced Ro52 upregulation in murine keratinocytes. Meanwhile, TWEAK stimulation of keratinocytes favored the migration of macrophages through promoting the production of chemokine C-C motif ligands <em>17</em> and 22. Furthermore, Fn14 siRNA transfection or nuclear <em>factor</em>-kappa B (NF-κB) inhibitor abrogated the TWEAK enhancement of Ro52 expression in keratinocytes. Similarly, TNF receptor associated <em>factor</em> 2 (TRAF2) siRNA reduced the protein level of Ro52 in these cells upon TWEAK stimulation. Interestingly, UVB irradiation increased the expression of TNF receptor type 1 (TNFR1) but not affecting TNFR2 expression in keratinocytes. In conclusion, the TWEAK/Fn14 signaling participates in Ro52-mediated photosensitization and involves the activation of NF-κB pathway as well as the function of the TRAF2/TNFR partners.

Eicosanoids participate in the regulation of cellular proliferation. Thus, we observed that prostaglandin E(2) interaction with membrane receptors is involved in the control of 3T6 <em>fibroblast</em> <em>growth</em> induced by serum. However, our results suggested that another arachidonic acid pathway might be implicated in these events. Our results show that 3T6 <em>fibroblasts</em> synthesized hydroxyeicosatetraenoic acids (HETEs) such as 12-HETE through the cytochrome P-450 (CYP450) pathway. However, 3T6 <em>fibroblasts</em> did not produce leukotriene B(4) (LTB(4)), and lipoxygenase inhibitors and LT antagonists failed to inhibit 3T6 <em>fibroblast</em> <em>growth</em> induced by FBS. In contrast, we observed that CYP450 inhibitors such as SKF-525A, <em>17</em>-octadecynoic acid, 1-aminobenzotriazole, and 6-(2-propargyloxyphenyl)hexanoic acid reduced 12(S)-HETE levels, 3T6 <em>fibroblast</em> <em>growth</em>, and DNA synthesis induced by FBS. The impairment of DNA synthesis and 3T6 <em>fibroblast</em> <em>growth</em> induced by SKF-525A were reversed by exogenous addition of HETEs. Moreover, we report that 5-HETE, 12(S)-HETE, and 15(S)-HETE are mitogenic on 3T6 <em>fibroblast</em> in the absence of another <em>growth</em> <em>factor</em>, and this effect was dependent on the activation of the phosphatidylinositol-3-kinase pathway. In conclusion, our results show that HETEs, probably produced by CYP450, are involved in the control of 3T6 <em>fibroblast</em> <em>growth</em>.

We examined the effect of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-9 on primary cultures of rat basal forebrain cholinergic neurons (BFCN) obtained at embryonic day <em>17</em>. FGF-9 enhanced survival of AChE-positive neurons, increased their mean soma size, and up-regulated their choline acetyltransferase (ChAT) activity. The ChAT-promoting effect of FGF-9 was approximately as potent as that of nerve <em>growth</em> <em>factor</em> (NGF) and was greater than those of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), ciliary neurotrophic <em>factor</em> (CNTF), or glia-derived neurotrophic <em>factor</em> (GDNF). Simultaneous addition of FGF-9 and NGF induced extremely high ChAT levels, suggesting that FGF-9 and NGF may enhance cholinergic properties in BFCN via different pathways that can act synergistically. In immunocytochemical and in situ hybridization studies in cultured cells and also in sections of adult rat brain, BFCN showed cytoplasmic immunostaining for FGF-9 and expressed FGF-9 messenger RNA; thus, we concluded that FGF-9 acts on BFCN in an autocrine and/or paracrine manner. Although effective delivery of exogenous FGF-9 into the central nervous system remains a problem to be solved, FGF-9 may be a promising candidate for therapeutic trials in Alzheimer disease.

3-Dimensional conditions for the culture of Bone Marrow-derived Stromal/Stem Cells (BMSCs) can be generated with scaffolds of biological origin. Cardiogel, a cardiac <em>fibroblast</em>-derived Extracellular Matrix (ECM) has been previously shown to promote cardiomyogenic differentiation of BMSCs and provide protection against oxidative stress. To determine the matrix composition and identify significant proteins in cardiogel, we investigated the differences in the composition of this nanomatrix and a BMSC-derived ECM scaffold, termed as 'mesogel'. An optimized protocol was developed that resulted in efficient decellularization while providing the maximum yield of ECM. The proteins were sequentially solubilized using acetic acid, Sodium Dodecyl Sulfate (SDS) and Dithiothreitol (DTT). These proteins were then analyzed using surfactant-assisted in-solution digestion followed by nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). The results of these analyses revealed significant differences in their respective compositions and <em>17</em> significant ECM/matricellular proteins were differentially identified between cardiogel and mesogel. We observed that cardiogel also promoted cell proliferation, adhesion and migration while enhancing cardiomyogenic differentiation and angiogenesis. In conclusion, we developed a reproducible method for efficient extraction and solubilization of in vitro cultured cell-derived extracellular matrix. We report several important proteins differentially identified between cardiogel and mesogel, which can explain the biological properties of cardiogel. We also demonstrated the cardiomyogenic differentiation and angiogenic potential of cardiogel even in the absence of any external <em>growth</em> <em>factors</em>. The transplantation of Bone Marrow derived Stromal/Stem Cells (BMSCs) cultured on such a nanomatrix has potential applications in regenerative therapy for Myocardial Infarction (MI).

Heat shock proteins (Hsps) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA).

METHODS

Herein, Hsp70 cell surface and mRNA expression were studied in human <em>fibroblast</em>-like synovial cells, dermal <em>fibroblasts</em> and peripheral blood leukocytes derived from 24 RA patients, who underwent synovectomy by using flow-cytometric analysis and real-time quantitative reverse-transcriptase polymerase chain reaction. For comparison, peripheral blood leukocytes of <em>17</em> healthy controls were tested.

RESULTS

Significantly higher Hsp70 membrane positivity was found on fibroblast-like synovial cells in RA patients (average 18.3%, median 16.5%) than on autologous and healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%, p = 0.002; healthy controls: average 6.0%, median 4.5%, p = 0.002) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%, p < 0.001). Strong Hsp70 cell surface expression was also found on peripheral blood monocytes of RA patients (average 53.0%, median 58.1%) and healthy controls (average 49.4%, median 47.5%, p = 0.52). Peripheral blood granulocytes of healthy controls (average 41.8%, median 41.4%) showed significantly increased Hsp70 expression comparing with RA patients (average 10.7%, median 6.4%, p = 0.005). Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients (average 2.04, median 1.7) when compared with autologous peripheral blood leukocytes (average 0.75, median 0.68; p < 0.001). However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes (average 1.69, median 1.64) was not observed (p = 0.83). We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (p < 0.001). Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p < 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p < 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p < 0.001).

CONCLUSIONS

Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast-like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via Hsp receptors. As a physiological response to potentially harmful enviromental stress factors, skin dermis produces higher levels of Hsp70 comparing to the cells of internal organs and tissues.

BACKGROUND

Increased concentrations of circulating fibroblast growth factor 23 (FGF-23) have been associated with higher risk of cardiovascular disease. The association between FGF-23 and the risk of atrial fibrillation (AF), a common arrhythmia, is less defined. Thus, we explored whether FGF-23 concentration was associated with AF incidence in a large community-based cohort.

RESULTS

We studied 12 349 men and women enrolled in the Atherosclerosis Risk in Communities (ARIC) study, without prevalent AF at baseline in 1990-1992. Serum intact FGF-23 concentration was measured with the Kainos 2-site ELISA. Incident AF through 2010 was ascertained from study ECGs and hospital discharge codes. Cox proportional hazards models adjusted for potential confounding factors, including kidney function, were used to estimate the association between FGF-23 and AF risk. We identified 1572 AF events during a mean follow-up of 17 years. In multivariable analysis, a difference of 1 SD (16 pg/mL) in baseline FGF-23 was not associated with the risk of AF (hazard ratio [HR], 1.04; 95% confidence interval [CI], 0.99, 1.09). Results were similar when FGF-23 was modeled in quartiles (HR, 1.09; 95% CI, 0.94, 1.26, comparing extreme quartiles). Reduced kidney function was associated with increased AF risk across quartiles of FGF-23 levels.

CONCLUSIONS

In this large community-based cohort, baseline FGF-23 levels were not associated with AF risk independently of kidney function. Our results do not support a major role for FGF-23 as a risk factor for AF or as a mediator of the association between chronic kidney disease and AF.

BACKGROUND

Fibroblast growth factor (FGF)-23 is a key regulator of phosphate homeostasis. Higher FGF-23 levels are correlated with poor outcomes in cardiovascular diseases. FGF-23 can produce cardiac hypertrophy and increase intracellular calcium, which can change cardiac electrical activity. However, it is not clear whether FGF-23 possesses arrhythmogenic potential through calcium dysregulation. Therefore, the purposes of this study were to evaluate the electrophysiological effects of FGF-23 and identify the underlying mechanisms.

METHODS

Patch clamp, confocal microscope with Fluo-4 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis and calcium regulatory proteins in HL-1 atrial myocytes with and without FGF-23 (10 and 25 ng/mL) incubation for 24 h.

RESULTS

FGF-23 (25 ng/mL) increased L-type calcium currents, calcium transient and sarcoplasmic reticulum Ca(2+) contents in HL-1 cells. FGF-23 (25 ng/mL)-treated cells (n = 14) had greater incidences (57%, 17% and 15%, P < 0·05) of delayed afterdepolarizations than control (n = 12) and FGF-23 (10 ng/mL)-treated cells (n = 13). Compared with control cells, FGF-23 (25 ng/mL)-treated cells (n = 14) exhibited increased phosphorylation of calcium/calmodulin-dependent protein kinase IIδ and phospholamban (PLB) at threonine 17 but had similar phosphorylation extents of PLB at serine 16, total PLB and sarcoplasmic reticulum Ca(2+) -ATPase protein. Moreover, the FGF receptor inhibitor (PD173074, 10 nM), calmodulin inhibitor (W7, 5 μM) and phospholipase C inhibitor (U73122, 1 μM) attenuated the effects of FGF-23 on calcium/calmodulin-dependent protein kinase II phosphorylation.

CONCLUSIONS

FGF-23 increases HL-1 cells arrhythmogenesis with calcium dysregulation through modulating calcium-handling proteins.

Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressive <em>factor</em> in vitro that is neutralized by rabbit anti-transforming <em>growth</em> <em>factor</em> (TGF)-beta. Previous studies have suggested that the decidual suppressor <em>factor</em> (DSF) is smaller than TGF-beta 1, and in this paper, we show that DSF on HPLC-sieving columns also elutes later than TGF-beta 2. Nevertheless, DSF has the ability to promote anchorage-independent <em>growth</em> of NRK <em>fibroblasts</em> similar to TGF-beta s. Using turkey antibodies specific for TGF-beta 1 or beta 2, we show that DSF is related to TGF-beta 2 rather than TGF-beta 1, and this relationship was confirmed by using a panel of murine mAb to TGF-subtypes. PAGE and Western blotting showed that the TGF-beta 2-reactive molecules in HPLC-purified DSF was slightly smaller than TGF-beta 2 and approximately 20 to 23 kDa. The DSF molecule is therefore closely related to TGF-beta 2 but as released from decidua, differs in size. The TGF-beta 2-related decidual suppressor <em>factor</em> was also obtained from the decidua of synpregnant C.B.-<em>17</em> severe combined immune deficiency (SCID) and pregnant SCID-BG (C57BL/6 background) mice, confirming the lack of T or B cell dependence of DSF production and the generality of production of a TGF-beta-related suppressor <em>factor</em> by decidua associated with successful pregnancy in mice.