Pregnancy is associated with progressive hypercholanemia, hypercholesterolemia, and hypertriglyceridemia, which can result in metabolic disease in susceptible women. Gut signals modify hepatic homeostatic pathways, linking intestinal content to metabolic activity. We sought to identify whether enteric endocrine signals contribute to raised serum bile acids observed in human and murine pregnancies, by measuring <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em>/15 protein and mRNA levels, and 7α-hydroxy-4-cholesten-3-one. Terminal ileal farnesoid X receptor (FXR)-mediated gene expression and apical sodium bile acid transporter (ASBT) protein concentration were measured by qPCR and western blotting. Shotgun whole-genome sequencing and ultra-performance liquid chromatography tandem mass spectrometry were used to determine the cecal microbiome and metabonome. Targeted and untargeted pathway analyses were performed to predict the systemic effects of the altered metagenome and metabolite profiles. Dietary CA supplementation was used to determine whether the observed alterations could be overcome by intestinal bile acids functioning as FXR agonists. Human and murine pregnancy were associated with reduced intestinal FXR signaling, with lower FGF<em>19</em>/15 and resultant increased hepatic bile acid synthesis. Terminal ileal ASBT protein was reduced in murine pregnancy. Cecal bile acid conjugation was reduced in pregnancy because of elevated bile salt hydrolase-producing Bacteroidetes. CA supplementation induced intestinal FXR signaling, which was not abrogated by pregnancy, with strikingly similar changes to the microbiota and metabonome as identified in pregnancy. Conclusion: The altered intestinal microbiota of pregnancy enhance bile acid deconjugation, reducing ileal bile acid uptake and lowering FXR induction in enterocytes. This exacerbates the effects mediated by reduced bile acid uptake transporters in pregnancy. Thus, in pregnant women and mice, there is reduced FGF<em>19</em>/15-mediated hepatic repression of hepatic bile acid synthesis, resulting in hypercholanemia.

Considerable effort is currently being devoted to understanding the physiological and pharmacological action of the endocrine <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). These three proteins (FGF15/<em>19</em>, FGF21 and FGF23) act in a tissue-specific manner through a membrane-complex consisting of an FGF-receptor and α/βKlotho. FGF15/<em>19</em> is produced in the intestine and regulates postprandial liver metabolism and gallbladder filling. FGF21 is largely liver-derived and co-ordinates adaptive changes in response to nutritional and physiological stresses. FGF23 signals from the bone to the kidney to maintain phosphate homeostasis. In pharmacological settings, FGF15/<em>19</em>, FGF21, and the prototypical FGF1, potentially represent novel treatments for obesity and diabetes. This review summarises the recent advances in our understanding of the biology of these important metabolic regulators.

BACKGROUND

Roux-en-Y gastric bypass (RYGB) surgery is an effective long-term intervention for weight loss maintenance, reducing appetite, and also food reward, via unclear mechanisms.

OBJECTIVE

To investigate the role of elevated satiety gut hormones after RYGB, we examined food hedonic-reward responses after their acute post-prandial suppression.

METHODS

These were randomized, placebo-controlled, double-blind, crossover experimental medicine studies.

METHODS

Two groups, more than 5 months after RYGB for obesity (n = 7-11), compared with nonobese controls (n = 10), or patients after gastric banding (BAND) surgery (n = 9) participated in the studies.

METHODS

Studies were performed after acute administration of the somatostatin analog octreotide or saline. In one study, patients after RYGB, and nonobese controls, performed a behavioral progressive ratio task for chocolate sweets. In another study, patients after RYGB, and controls after BAND surgery, performed a functional magnetic resonance imaging food picture evaluation task.

METHODS

Octreotide increased both appetitive food reward (breakpoint) in the progressive ratio task (n = 9), and food appeal (n = 9) and reward system blood oxygen level-dependent signal (n = 7) in the functional magnetic resonance imaging task, in the RYGB group, but not in the control groups.

RESULTS

Octreotide suppressed postprandial plasma peptide YY, glucagon-like peptide-1, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> after RYGB. The reduction in plasma peptide YY with octreotide positively correlated with the increase in brain reward system blood oxygen level-dependent signal in RYGB/BAND subjects, with a similar trend for glucagon-like peptide-1.

CONCLUSIONS

Enhanced satiety gut hormone responses after RYGB may be a causative mechanism by which anatomical alterations of the gut in obesity surgery modify behavioral and brain reward responses to food.

Hepatocellular carcinoma (HCC) is the sixth most common type of cancer, with an increasing mortality rate. Aberrant expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>-<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (<i>FGF<em>19</em>-FGFR4</i>) is reported to be an oncogenic-driver pathway for HCC patients. Thus, the <i>FGF<em>19</em>-FGFR4</i> signaling pathway is a promising target for the treatment of HCC. Several pan-<i>FGFR</i> (1-4) and <i>FGFR4</i>-specific inhibitors are in different phases of clinical trials. In this review, we summarize the information, recent developments, binding modes, selectivity, and clinical trial phases of different available <i>FGFR4</i>/pan-<i>FGF</i> inhibitors. We also discuss future perspectives and highlight the points that should be addressed to improve the efficacy of these inhibitors.

<b>Introduction</b>: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs). <b>Methods</b>: Human iPSCs were generated from normal dermal <em>fibroblasts</em> by transfecting plasmids encoding for six <em>factors</em>: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined <em>growth</em> media. The cells were also tested <i>in vivo</i> in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology. <b>Results</b>: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks <i>in vivo</i>. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the <i>in vitro</i> study, namely they still expressed the notochordal markers Keratin 18, Keratin <em>19</em>, Noto and Brachyury. <b>Conclusion</b>: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype <i>in vitro</i> and <i>in vivo</i>, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the <i>in vitro</i> studies with MSCs.

BACKGROUND

The fibroblast growth factor receptor 4 (FGFR4) pathway is an essential regulatory component of bile acid synthesis, and its relationship with hepatocellular carcinoma (HCC) has been reported. We investigated the gene expression and clinical significance of FGFR4 and related pathways in intrahepatic cholangiocarcinoma (iCCA).

RESULTS

The median age was 56 years (range 30-78) and 34 patients (74%) were male. Six patients (13%) had hepatitis B virus infection, with or without liver cirrhosis. Overall survival was significantly associated with FGFR4 (p = 0.004), FGF19 (p = 0.047), FGF21 (p = 0.04), and KLB (p = 0.03) expression. In the multivariate analysis with potential prognostic factors, high expression of FGF19, FGF21, and FGFR4 was significantly associated with better survival. In the analysis using the TCGA iCCA dataset, mRNA overexpression of at least 1 of the FGFR4-related genes was significantly associated with better disease-free survival (p = 0.02).

METHODS

We assessed the expression of 98 genes in formalin-fixed paraffin embedded tumor tissue specimens from 46 patients with surgically resected iCCA using a NanoString platform. This included 10 FGF pathway genes (e.g. FGFR1-4, KLB, FGF3, 4, 19, 21, and 23), 19 distal marker genes (e.g. CYP7A1 and CYP17A1), 31 genes relevant to HCC and iCCA (e.g. AFP, TS), 18 copy number variation matched genes, and 20 control genes. Log-transformation of gene expression was performed for normalization and statistical analysis. Overall survival was correlated with gene expression (< median vs. ≥ median) using a log-rank test. The prognostic impact of FGFR4-related genes was validated using the public TCGA dataset for iCCA.

CONCLUSIONS

Our results indicate that mRNA expression of FGFR4-related genes may be a biomarker to define the distinctive molecular phenotype of iCCA. Future preclinical and clinical validation is required to define the role of the FGFR4 pathway in iCCA.

BACKGROUND

Vascular calcification and arterial stiffening are cardiovascular risk factors among chronic kidney disease patients. Elevated aortic pulse wave velocity (AoPWV) is an independent predictor of increased cardiovascular morbidity and mortality.

OBJECTIVE

The aim of the study was to analyze the relationships between inflammatory and vascular calcification parameters and arterial wall stiffness in chronic kidney disease (CKD) patients treated by peritoneal dialysis.

METHODS

The study included 57 patients (27 women and 30 men) aged from 19 to 75 years (mean age 53 ± 13), treated by peritoneal dialysis during 4-100 months (mean 30.4 months). The concentrations of albumin, lipids, interleukin-6 (IL-6), IL-18, high-sensitive C-reactive protein, transforming growth factor-β1 (TGF-β1), osteocalcin, osteoprotegerin (OPG), fibroblast growth factor 23, fetuin A, parathyroid hormone (iPTH), total calcium (Ca), and phosphates (Pi) were measured. AoPWV was performed using a tonometric method, common carotid artery intima-media thickness (CCA-IMT) by ultrasonography evaluation, and calcium scoring (CaSc) with multirow spiral computed tomography (MSCT).

RESULTS

In univariate analysis, AoPWV correlated negatively with osteocalcin (R = -0.37; P = 0.005) and positively with OPG (R = 0.41; P = 0.002). Additionally, AoPWV was significantly positively associated with inflammatory parameters: IL-6 (R = 0.35; P = 0.009), TGF-β1 (R = 0.27; P = 0.047), and white blood cell (WBC) count (R = 0.33; P = 0.01). There were also positive correlations between AoPWV and imaging data: CCA-IMT (R = 0.32; P = 0.02) and CaSc (R = 0.38; P = 0.004). AoPWV did not correlate with calcium, phosphate, Ca × Pi index, or iPTH concentration. After multiple adjustments, osteocalcin was the only significant predictor of AoPWV. In logistic regression adjusted for age, hypertension, and mean arterial pressure at AoPWV evaluation, only osteocalcin was significantly associated with high (above median) AoPWV values [odds ratio 0.96 (0.92-0.99) per unit increase in osteocalcin].

CONCLUSIONS

OPG concentration and some inflammatory markers (WBC count, IL-6, TGF-β1) influenced the severity of arterial wall stiffness in CKD patients. Measurement of osteocalcin seems to be the best predictor of AoPWV.

Modulating bile acid synthesis has long been considered a good strategy by which to improve cholesterol homeostasis in humans. The farnesoid X receptor (FXR), the key regulator of bile acid synthesis, was, therefore, identified as an interesting target for drug discovery. We compared the effect of four, structurally unrelated, synthetic FXR agonists in two fat-fed rodent species and observed that the three most potent and selective agonists decreased plasma cholesterol in LDL receptor-deficient (Ldlr (-/-)) mice, but none did so in hamsters. Detailed investigation revealed increases in the expression of small heterodimer partner (Shp) in their livers and of intestinal <em>fibroblast</em> <em>growth</em> <em>factor</em> 15 or <em>19</em> (Fgf15/<em>19</em>) in mice only. Cyp7a1 expression and fecal bile acid (BA) excretion were strongly reduced in mice and hamsters by all four FXR agonists, whereas bile acid pool sizes were reduced in both species by all but the X-Ceptor compound in hamsters. In Ldlr (-/-) mice, the predominant bile acid changed from cholate to the more hydrophilic β-muricholate due to a strong repression of Cyp8b1 and increase in Cyp3a11 expression. However, FXR agonists caused only minor changes in the expression of Cyp8b1 and in bile acid profiles in hamsters. In summary, FXR agonist-induced decreases in bile acid pool size and lipophilicity and in cholesterol absorption and synthesis could explain the decreased plasma cholesterol in Ldlr (-/-) mice. In hamsters, FXR agonists reduced bile acid pool size to a smaller extent with minor changes in bile acid profile and reductions in sterol absorption, and consequently, plasma cholesterol was unchanged.

OBJECTIVE

To investigate the effects of the vascular endothelial <em>growth</em> <em>factor</em>-neutralizing agent aflibercept on primary cultures of human trabecular meshwork cells (hTMC), human scleral <em>fibroblasts</em> (hFibro), and a retinal pigment epithelial cell line (ARPE-<em>19</em>).

METHODS

Various concentrations of aflibercept were incubated with confluent cell cultures for 24 hours. Ranibizumab was used as an active control for comparison. Assays of cellular metabolism (MTT assay) and cell viability (calcein dye uptake) were performed.

RESULTS

Compared with untreated controls (100% live), a 24-hour exposure to 1 mg/mL aflibercept had no significant effect on cell viability in hTMC (100.1 ± 1.7%), hFibro (102.4 ± 2.4%), or ARPE-<em>19</em> (99.3 ± 3.9%) cells. Aflibercept vehicle controls also had no detrimental effect. Aflibercept (1 mg/mL) had no statistically significant effect on metabolic activity in hTMC (84.3 ± 10.2%), hFibro (102.7 ± 4.3%), and ARPE-<em>19</em> (104.6 ± 12.6%) cells. When compared side-by-side in ARPE-<em>19</em> cells, aflibercept and the anti-vascular endothelial <em>growth</em> <em>factor</em> agent ranibizumab had no toxicity at the highest concentration tested (1 mg/mL).

CONCLUSIONS

The authors' data reveal that concentrations of aflibercept in the range expected to occur in the human vitreous after intraocular injection are not harmful in an in vitro cell assay.

Bile acid nuclear receptor farnesoid X receptor (FXR) is a key molecular mediator of many metabolic processes, including the regulation of bile acid, lipid and glucose homeostasis. A significant component of FXR-mediated events essential to its biological activity is attributed to induction of the enteric endocrine hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>19</em> or its rodent ortholog, FGF15. In this report, we compared the properties of human FGF<em>19</em> and murine FGF15 in the regulation of hepatocarcinogenesis and metabolism in various mouse models of disease.

Tumorigenicity was assessed in three mouse models (db/db, diet-induced obese, and multi-drug resistance 2 [Mdr2]-deficient) following continuous exposure to FGF<em>19</em> or FGF15 via adeno-associated viral-mediated gene delivery. Glucose, hemoglobin A1c and β-cell mass were characterized in db/db mice. Oxygen consumption, energy expenditure, and body composition were evaluated in diet-induced obese mice. Serum levels of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase were assessed in Mdr2-deficient mice. Expression profiles of genes encoding key proteins involved in bile acid synthesis and hepatocarcinogenesis were also determined.

Both FGF15 and FGF<em>19</em> hormones repressed bile acid synthesis (p<0.001 for both). However, murine FGF15 lacked the protective effects characteristic of human FGF<em>19</em> in db/db mice with overt diabetes, such as weight-independent HbA1c-lowering and β-cell-protection. Unlike FGF<em>19</em>, FGF15 did not induce hepatocellular carcinomas (HCC) in three mouse models of metabolic diseases (db/db, diet-induced obese, and multi-drug resistance 2 [Mdr2]-deficient mice), even at supra-pharmacological exposure levels.

Fundamental species-associated differences between FGF<em>19</em> and FGF15 may restrict the relevance of mouse models for the study of the FXR/FGF<em>19</em> pathway, and underscore the importance of clinical assessment of this pathway, with respect to both safety and efficacy in humans.

Activation of the nuclear receptor, FXR, leads to the production of a hormone called <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) and subsequently regulation of multiple metabolic processes. Synthetic activators of FXR have been recently approved or are currently in clinical development for treatment of chronic liver diseases, including primary biliary cholangitis (PBC) and non-alcoholic steatohepatitis (NASH). The safety of these activators was partly assessed in mice exposed for prolonged periods of time. However, the results of this study show that mouse FGF15 and human FGF<em>19</em> exhibit fundamentally different biological activities in mice. This could raise the concern of relying on rodent models for safety assessment of FXR activators. The potential risk of HCC development in patients treated with FXR agonists may need to be monitored.

<AbstractText>No antiangiogenic treatment is yet approved for extrapancreatic neuroendocrine tumors (NET). Surufatinib (HMPL-012, previously named sulfatinib) is a small-molecule inhibitor targeting vascular endothelial <em>growth</em> <em>factor</em> receptors, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 and colony-stimulating <em>factor</em> 1 receptor. We conducted a single-arm phase Ib/II study of surufatinib in advanced NETs.</AbstractText><AbstractText>Patients with histologically well-differentiated, low or intermittent grade, inoperable or metastatic NETs were enrolled into a pancreatic or extrapancreatic NET cohort. Patients were treated with surufatinib 300 mg orally, once daily. The primary endpoints were safety and objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors (version 1.1).</AbstractText><AbstractText>Of the 81 patients enrolled, 42 had pancreatic NETs and 39 had extrapancreatic NETs. Most patients had radiologic progression within 1 year prior to enrollment (32 patients in each cohort). In the pancreatic and extrapancreatic NET cohorts, ORRs were <em>19</em>% [95% confidence intervals (CI), 9-34] and 15% (95% CI, 6-31), disease control rates were 91% (95% CI, 77-97) and 92% (95% CI, 79-98), and median progression-free survival was 21.2 months (95% CI, 15.9-24.8) and 13.4 months (95% CI, 7.6-<em>19</em>.3), respectively. The most common grade ≥3 treatment-related adverse events were hypertension (33%), proteinuria (12%), hyperuricemia (10%), hypertriglyceridemia, and diarrhea (6% for each), and increased alanine aminotransferase (5%).</AbstractText><AbstractText>Surufatinib showed encouraging antitumor activity and manageable toxicities in patients with advanced NETs. Two ongoing phase III studies, validating the efficacy of surufatinib in patients with NETs, will contribute to the clinical evidence.</AbstractText>

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) is a member of the hormone-like FGF family and has activity as an ileum-derived postprandial hormone. It shares high binding affinity with β-Klotho and together with the FGF receptor (FGFR) 4, is predominantly targeted to the liver. The main function of FGF<em>19</em> in metabolism is the negative control of bile acid synthesis, promotion of glycogen synthesis, lipid metabolism and protein synthesis.

METHODS

Drawing on in vitro and in vivo studies, this review discusses FGF<em>19</em> and some underlying mechanisms of action of FGF<em>19</em> as an endocrine hormone in several liver diseases. The molecular pathway of the FGF<em>19</em>-FGFR4 axis in non-alcoholic liver disease and hepatocellular carcinoma are discussed. Furthermore, definition of function and pharmacological effects of FGF<em>19</em> for liver disease are also presented.

CONCLUSIONS

A series of studies have highlighted a crucial role of FGF<em>19</em> in liver disease. However, the conclusions of these studies are partly paradoxical and controversial. An understanding of the underlying biological mechanisms which may explain inconsistent findings is especially important for consideration of potential biomarker strategies and an exploration of the putative therapeutic efficacy of FGF<em>19</em> for human liver disease.

Treatment with the farnesoid X receptor (FXR) agonist obeticholic acid is ineffective in some patients with non-alcoholic steatohepatitis (NASH) but the explanation is uncertain. We investigated hepatic FXR expression, and measurements of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) and bile acids (BAs) in children with NAFLD to investigate relationships with NASH.

33 children with NAFLD who underwent diagnostic liver biopsy were studied. Hepatic FXR protein levels and circulating FGF<em>19</em> concentrations were compared with those analysed in five control subjects with proven normal liver histology. NASH was defined by the Paediatric NAFLD Histological Score (PNHS). Binary logistic regression with adjustment for covariates and potential confounders was undertaken to test <em>factor</em>s independently associated with: a) NASH and b) hepatic FXR protein levels.

Mean ± SD age was 13.7 ± 1.9 years. Nineteen patients had NASH (PNHS ≥ 85) and 14 did not have NASH (PNHS < 85). Hepatic FXR level and plasma FGF<em>19</em> concentration varied ~10-fold and 5-fold, respectively, between groups, and was highest in control subjects, intermediate in NAFLD without NASH, and lowest in NASH (between group differences P < .001 and P < .01 respectively). NASH was independently associated with both FXR protein levels (OR = 0.18, 95% CI 0.09, 0.38) and FGF<em>19</em> concentration (OR = 0.55, 95% CI 0.20, 0.89).

FXR protein levels vary markedly between normal liver, NAFLD without NASH, and NASH. Low levels of FXR are independently associated with NASH.

BACKGROUND

Circumscribed cartilage defects are considered as prearthritic lesions and lead to differential intra-articular cytokine expression. Mechanisms of associated pain development and influence of smoking behavior are not yet fully understood in humans.

OBJECTIVE

This study aimed to reveal relations between synovial cytokine levels in knees with circumscribed cartilage defects and pain sensation.

METHODS

Descriptive laboratory study.

METHODS

In a clinical trial, knee lavage fluids of 42 patients with circumscribed cartilage lesions treated by either microfracturing (n = <em>19</em>) or by autologous chondrocyte implantation (n = 23) and fluids of 5 healthy control individuals were prospectively collected. Preoperative knee pain was evaluated according to frequency and strength; subjective knee function was assessed using a visual analog scale and the International Knee Documentation Committee (IKDC) score. Synovial concentrations of aggrecan, insulin-like <em>growth</em> <em>factor</em> (IGF)-I, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), interleukin (IL)-1β, bone morphogenetic protein (BMP)-2, and BMP-7 were determined by enzyme-linked immunosorbent assay.

RESULTS

Pain strength showed a highly significant association with intra-articular IGF-1 levels (ρ = .48, P < .01), but no correlation with synovial concentrations of aggrecan, bFGF, IL-1β, BMP-2, and BMP-7. Although pain strength and frequency demonstrated a statistically significant relationship, no substantial association between pain frequency and any of the examined cytokine levels was found. Intra-articular IGF-1 concentrations significantly correlated with the area of cartilage damage (ρ = .35, P < .02); the other investigated cytokines failed to show this association. Neither of the determined intra-articular mediators demonstrated statistically significant correlations with subjective knee function or IKDC score. Only intra-articular concentrations of IGF-1 and BMP-2 statistically significantly correlated with age; total protein content was negatively associated with body mass index (P < .05). In smokers, synovial expression of total protein content, IGF-1, and bFGF was significantly diminished compared to nonsmokers (P < .05).

CONCLUSIONS

Insulin-like growth factor-I is present in knees with circumscribed cartilage lesions in a size-dependent manner. IGF-1 levels correlated with indicators of pain perception; smoking negatively influenced synovial cytokine expression related to cartilage metabolism, but pain perception was not altered.

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of <em>19</em> experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.

We investigated the effects of environmental enrichment (EE) on the function of transplanted adipose stem cells (ASCs) and the combined effect of EE and ASC transplantation on neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in 7-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At 6 weeks of age, the mice were randomly injected with either ASCs or PBS into the striatum and were randomly assigned to either EE or standard cages (SC), comprising ASC-EE (n=18), ASC-SC (n=<em>19</em>), PBS-EE (n=12), PBS-SC (n=17), and untreated controls (n=23). Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. The fate of transplanted cells and the levels of endogenous neurogenesis, astrocyte activation, and paracrine <em>factors</em> were also measured. As a result, EE and ASC transplantation synergistically improved rotarod latency, forelimb-use asymmetry, and grip strength compared to those of the other groups. The number of engrafted ASCs and βIII-tubulin(+) neurons derived from the transplanted ASCs was significantly higher in mice in EE than those in SC. EE and ASC transplantation also synergistically increased BrdU(+)βIII-tubulin(+) neurons, GFAP(+) astrocytic density, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) level but not the level of CS-56(+) glial scarring in the striatum. In conclusion, EE and ASC transplantation synergistically improved neurobehavioral functions. The underlying mechanisms of this synergism included enhanced repair processes such as higher engraftment of the transplanted ASCs, increased endogenous neurogenesis and astrocytic activation coupled with upregulation of FGF2.

Levels of fibroblast growth factor 23 (FGF23) are elevated in chronic kidney disease (CKD) and strongly associated with left ventricular hypertrophy, heart failure, and death. Whether FGF23 is an independent risk factor for atrial fibrillation in CKD is unknown.

To investigate the association of FGF23 with atrial fibrillation in CKD.

Prospective cohort study of 3876 individuals with mild to severe CKD who enrolled in the Chronic Renal Insufficiency Cohort Study between June 19, 2003, and September 3, 2008, and were followed up through March 31, 2013.

Baseline plasma FGF23 levels.

Prevalent and incident atrial fibrillation.

The study cohort comprised 3876 participants. Their mean (SD) age was 57.7 (11.0) years, and 44.8% (1736 of 3876) were female. Elevated FGF23 levels were independently associated with increased odds of prevalent atrial fibrillation (n = 660) after adjustment for cardiovascular and CKD-specific factors (odds ratio of highest vs lowest FGF23 quartile, 2.30; 95% CI, 1.69-3.13; P < .001 for linear trend across quartiles). During a median follow-up of 7.6 years (interquartile range, 6.3-8.6 years), 247 of the 3216 participants who were at risk developed incident atrial fibrillation (11.9 events per 1000 person-years). In fully adjusted models, elevated FGF23 was independently associated with increased risk of incident atrial fibrillation after adjustment for demographic, cardiovascular, and CKD-specific factors, and other markers of mineral metabolism (hazard ratio of highest vs lowest FGF23 quartile, 1.59; 95% CI, 1.00-2.53; P = .02 for linear trend across quartiles). The results were unchanged when further adjusted for ejection fraction, but individual adjustments for left ventricular mass index, left atrial area, and interim heart failure events partially attenuated the association of elevated FGF23 with incident atrial fibrillation.

Elevated FGF23 is independently associated with prevalent and incident atrial fibrillation in patients with mild to severe CKD. The effect may be partially mediated through a diastolic dysfunction pathway that includes left ventricular hypertrophy, atrial enlargement, and heart failure events.

BACKGROUND

Interleukin (IL)-<em>19</em>, a member of the IL-10 cytokine family, is involved in keratinocyte proliferation in psoriasis.

OBJECTIVE

We investigated the role of IL-<em>19</em> in the wound-healing process in vivo and in vitro.

METHODS

Two full-thickness circular wounds (4mm in diameter) were punched into the skin of BALB/C mice. IL-<em>19</em> and keratinocyte growth factor (KGF) mRNA in wounded skin were determined using real-time PCR. The wounds were treated with PBS, vehicle, IL-<em>19</em> (400ng/mL), or IL-20 (400ng/mL) (n=6 in each group) twice daily and the percentage of wound healing was measured daily for 7days. In vitro, human skin fibroblast CCD966-SK cells and keratinocyte HaCaT cells were treated with IL-<em>19</em> or KGF. Cell proliferation and migration were determined using bromodeoxyuridine (BrdU) and transwell assays, respectively. The expression of IL-<em>19</em> and KGF mRNA was also analyzed.

RESULTS

In wounded mouse skin, IL-<em>19</em> mRNA was upregulated at 12h, and KGF at 24h after the injury. Both increases in gene expression declined 72h after the skin had been wounded. The percentage of wound healing in IL-<em>19</em>-treated mice was higher than in control mice. In vitro, IL-<em>19</em> upregulated KGF expression in the CCD966-SK cells; IL-<em>19</em> was upregulated in KGF-treated HaCaT cells. KGF but not IL-<em>19</em> promoted HaCaT cell proliferation. However, IL-<em>19</em> significantly increased the migration of HaCaT cells. HaCaT cells treated with the cultured supernatants of IL-<em>19</em>-stimulated CCD966-SK cells showed significantly more proliferation than in controls.

CONCLUSIONS

IL-<em>19</em> is important for cutaneous wound healing because it upregulates KGF expression.

Although genetic amplification and overexpression of the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) gene are found in human breast cancer, mechanisms that contribute to such functional alterations remain elusive. We report here that high expression of FGF<em>19</em> is associated with the aggressive malignant behavior and poor survival outcome of breast cancer patients. FGF<em>19</em> is particularly highly expressed in luminal molecular subtype of breast tumors and its expression levels are positively associated with its secretion levels from breast cancer cells. Genetic knockout of FGF<em>19</em> significantly induces repression of breast tumor progression and metastasis in either an orthotopic mouse model of breast cancer or an experimental metastasis model. The FGF<em>19</em> specific receptor, FGFR4, can be activated and subsequently upregulate AKT signaling in breast cancer cell upon FGF<em>19</em>, which is critical for oncogenic role of FGF<em>19</em>. Inactivation of FGFR4 by its inhibitor BLU9931 significantly attenuates FGF<em>19</em>-induced tumor-promoting activity, suggesting interruption of FGFR4 function is sufficient to affect FGF<em>19</em>-driven breast cancer. Overall, these insights support the idea that targeting FGFR4 in breast cancer cells overexpressing FGF<em>19</em> may represent an effective strategy to suppress cancer development, progression, and metastasis.

mRNA for basic <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-<em>19</em> kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.

BACKGROUND

It is now clear that the progression of renal disease is closely correlated to the degree of renal interstitial fibrosis. We have previously demonstrated that the renal proximal tubular epithelial cell may contribute to the fibrotic response by the generation of profibrotic cytokines. Transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) are two of a group of profibrotic cytokines that have been associated with the development of renal interstitial fibrosis. In this study, we have examined the influence of TGF-beta1 on the generation of bFGF by renal tubular epithelial cells.

METHODS

HK2 cells were grown to confluence and were serum deprived and stimulated with recombinant TGF-beta1 under serum-free conditions. Subsequently, supernatant, cell-associated, intracellular, and matrix-associated bFGF concentrations were determined by enzyme-linked immunosorbent assay (ELISA). bFGF mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

The exposure of confluent serum-deprived HK2 cells to TGF-beta1 led to a significant increase in bFGF concentration in the cell culture supernatant. Twenty-four hours following the addition of 10 ng/ml TGF-beta1, this represented a twofold increase in bFGF concentration (control, 102 pg/ml, N = 24, vs. 202 pg/ml, N = 19, P = 0.0001). Despite the increase in bFGF concentration in the supernatant, there was no change in the expression of bFGF mRNA following the addition of TGF-beta1. The addition of 10 ng/ml of TGF-beta1 led to a 30% decrease in the total cell-associated bFGF concentration (control, 8.51 ng/ml, N = 16, TGF-beta1, 6.01 ng/ml, N = 13, P = 0.0042). This decrease in intracellular bFGF was associated with a 15% reduction in anti-bFGF antibody binding to fixed permeabilized cells, following the addition of 10 ng/ml of recombinant TGF-beta1 (N = 9, P = 0.0007), suggesting that the mechanism of stimulation of bFGF by TGF-beta1 involved the release of preformed bFGF from within the cells. In addition, following the addition of TGF-beta1, there was a significant dose-dependent decrease in the amount of bFGF sequestered in the extracellular matrix. At a dose of 10 ng/ml TGF-beta, this represented a greater than sevenfold decrease (N = 9, P = 0.0007) in matrix-bound bFGF, although this represented less than 3% of the total bFGF released into the supernatant.

CONCLUSIONS

The data presented suggest that the main mechanism by which TGF-beta1 stimulates bFGF generation by proximal tubular epithelial cells is by stimulation of the secretion of preformed cytokine from within the cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually lethal interstitial lung disease of unknown etiology characterized by aberrant activation of epithelial cells that induce the migration, proliferation and activation of <em>fibroblasts</em>. The resulting distinctive <em>fibroblast</em>ic/myo<em>fibroblast</em>ic foci are responsible for the excessive extracellular matrix (ECM) production and abnormal lung remodeling. We have recently found that matrix metalloproteinase <em>19</em> (MMP-<em>19</em>)-deficient (Mmp<em>19</em>-/-) mice develop an exaggerated bleomycin-induced lung fibrosis, but the mechanisms are unclear. In this study, we explored the effect of MMP-<em>19</em> deficiency on <em>fibroblast</em> gene expression and cell behavior. Microarray analysis of Mmp<em>19</em>-/- lung <em>fibroblasts</em> revealed the dysregulation of several profibrotic pathways, including ECM formation, migration, proliferation, and autophagy. Functional studies confirmed these findings. Compared with wild-type mice, Mmp<em>19</em>-/- lung <em>fibroblasts</em> showed increased α1 (I) collagen gene and collagen protein production at baseline and after transforming <em>growth</em> <em>factor</em>-β treatment and increased smooth muscle-α actin expression (P < 0.05). Likewise, Mmp<em>19</em>-deficient lung <em>fibroblasts</em> showed a significant increase in proliferation (P < 0.01) and in transmigration and locomotion over Boyden chambers coated with type I collagen or with Matrigel (P < 0.05). These findings suggest that, in lung <em>fibroblasts</em>, MMP-<em>19</em> has strong regulatory effects on the synthesis of key ECM components, on <em>fibroblast</em> to myo<em>fibroblast</em> differentiation, and in migration and proliferation.

BACKGROUND

Acute kidney injury (AKI) carries a large burden of morbidity and mortality. Early diagnosis may lead to better strategies of clinical care. Cardiac surgery involving cardiopulmonary bypass is associated with a significant incidence of AKI. The study objective was to determine whether or not preoperative fibroblast growth factor-23 (FGF23) levels differed among pediatric patients who did or did not develop AKI following cardiac surgery.

METHODS

A nested case-control study was performed. FGF23 levels were measured pre- and post-operatively in 19 children without chronic kidney disease (CKD) who underwent cardiopulmonary bypass. Five patients developed AKI and 14 patients served as controls.

RESULTS

FGF23 levels in patients who developed AKI following cardiac surgery were elevated above normal levels, both pre-operatively and post-operatively compared with those patients who did not develop AKI. Relative risk of developing AKI when the pre-operative FGF23 level was >86 RU/mL was 2.0 (p = 0.033). Preoperative FGF23 levels correlated with post-operative fluid gain (correlation coefficient 0.607, p = 0.0059).

CONCLUSIONS

FGF23 may serve as a pre-operative prognostic indicator of the development of AKI following cardiopulmonary bypass surgery in pediatric patients without CKD. Identifying patients more likely to have AKI following surgery provides a means of achieving closer clinical management of AKI and fluid balance.

The lesioned CA3 region of the young adult hippocampus is very conducive for robust survival and integration of fetal hippocampal CA3 cell grafts when transplanted at an early postlesion delay of 4 days. However, similar CA3 cell grafts placed at 45 days postlesion display significantly diminished cell survival, implying that the receptivity of the lesioned young adult host hippocampus to grafts decreases considerably with a prolonged postlesion transplantation delay. We hypothesize that decreased cell survival in grafts placed into the chronically lesioned hippocampus is due to a reduced level of host neurotrophic <em>factors</em> that support fetal hippocampal cells; hence, pretreatment and grafting of donor fetal CA3 cells with <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) considerably enhances graft neuronal integration into the chronically lesioned young adult hippocampus. We employed the optical fractionator cell counting method and rigorously quantified the number of surviving cells and neurons derived from 5'-bromodeoxyuridine-labeled Embryonic Day <em>19</em> CA3 cell grafts pretreated and transplanted with FGF-2 into the lesioned CA3 region of the young adult rat hippocampus, at a delay of 60 days after a unilateral intracerebroventricular administration of the kainic acid. For comparison, we also analyzed the survival of standard fetal CA3 cell grafts (i.e., without FGF-2 treatment) after similar transplantation. Pre treatment and transplantation of CA3 cell grafts with FGF-2 resulted in a robust yield of surviving cells (115% of injected cells) and neurons (100% of injected cells) from grafts. In contrast, standard CA3 cell grafts exhibited a reduced yield of surviving cells (29%) and neurons (25%). Thus, the yield of neurons from fetal hippocampal CA3 cell grafts placed into the chronically lesioned young adult hippocampus can be greatly enhanced by a simple pretreatment and grafting of donor fetal CA3 cells with FGF-2. These results have significance toward advancement of clinically feasible cell grafting strategies for repair of the damaged young adult hippocampus, particularly at extended periods after the injury or the onset of neurodegenerative diseases.