BACKGROUND

Bile acid diarrhoea is a common cause of chronic diarrhoea, occurring as a primary condition or secondary to ileal disease or resection. Many patients have reduced levels of the ileal hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), an inhibitory regulator of hepatic bile acid synthesis, secreted in response to farnesoid X receptor (FXR) activation.

OBJECTIVE

To investigate whether obeticholic acid, a potent FXR agonist, could increase FGF<em>19</em> in patients with bile acid diarrhoea, and produce clinical benefits.

METHODS

After a 2 week run-in when bile acid sequestrants were discontinued, patients with previously diagnosed primary bile acid diarrhoea (n = 10), secondary bile acid diarrhoea (n = 10) or idiopathic chronic diarrhoea (n = 8), received oral obeticholic acid 25 mg daily for 2 weeks. Serum FGF<em>19</em>, total bile acids and 7α-OH-4-cholesten-3-one (C4) were measured, symptoms recorded and a diarrhoea index calculated.

RESULTS

In primary bile acid diarrhoea, obeticholic acid increased median fasting FGF<em>19</em> (133-237 pg/mL, P = 0.007) and significantly reduced fasting C4 and bile acid responses. Improvements occurred in median stool frequency (-24% after 2 weeks treatment, P = 0.03), stool form (-14%, P = 0.05) and diarrhoea index (-34%, P = 0.005). In the secondary bile acid diarrhoea group, significant clinical improvements were found predominantly in patients with shorter ileal resections. Symptoms of abdominal pain and urgency improved. FGF<em>19</em> and bile acids changed in the control group, without significant clinical improvement. Total and LDL-cholesterol increased and triglycerides decreased. Obeticholic acid treatment was well tolerated.

CONCLUSIONS

This proof-of-concept study indicates that obeticholic acid stimulates FGF<em>19</em>, reduces bile acid synthesis and produces clinical benefits in bile acid diarrhoea. FXR agonists have therapeutic potential in chronic diarrhoea. EudraCT 2011-003777-28; Clinical Trials: NCT01585025.

Connective tissue <em>growth</em> <em>factor</em> (CTGF), which is regulated by transforming <em>growth</em> <em>factor</em>-ss (TGFss), has recently been implicated in the pathogenesis of fibrotic diseases and tumor stroma. Inasmuch as generation of desmoplastic tissue is characteristic for pancreatic cancer, it is not known whether it gives pancreatic cancer cells a <em>growth</em> advantage or is a reaction of the body to inhibit cancer cell progression. In the present study we analyzed the expression and localization of CTGF and evaluated whether it influences the prognosis of pancreas cancer. Tissue samples were obtained from 25 individuals (6 women, <em>19</em> men) undergoing pancreatic resection for pancreatic cancer. Tissue samples from 13 previously healthy organ donors (5 women, 8 men) served as controls. Expression of CTGF was studied by Northern blot analysis. In situ hybridization and immunohistochemistry localized the respective mRNA moieties and proteins in the tissue samples. Northern blot analysis revealed that pancreatic cancer tissue samples exhibited a 46-fold increase in CTGF mRNA expression ( p < 0.001) over that of normal controls. In vitro studies confirmed that pancreatic stellate cells are the major source of CTGF mRNA expression and revealed a large variance in basal and TGFss-induced CTGF expression in cultured pancreatic cancer cells. This could also be confirmed by in situ hybridization, indicating that CTGF mRNA signals were located principally in <em>fibroblasts</em>, with only weak signals in the cancer cells. High CTGF mRNA levels in the tissue samples correlated with better tumor differentiation ( p < 0.03). In addition, patients whose tumors exhibited high CTGF mRNA levels >> onefold increase above normal controls) lived significantly longer than those whose tumors expressed low CTGF mRNA levels (none to onefold) ( p < 0.04 multivariate analysis). Our present data indicate that CTGF, as a downstream mediator of TGFss, is overexpressed in connective tissue cells and to a lesser extent in pancreatic cancer cells. Because patients with high CTGF mRNA expression levels have a better prognosis, our findings indicate that the desmoplastic reaction provides a <em>growth</em> disadvantage for pancreatic cancer cells.

Bile acids are increasingly recognized as key regulators of systemic metabolism. While bile acids have long been known to play important and direct roles in nutrient absorption, bile acids also serve as signalling molecules. Bile acid interactions with the nuclear hormone receptor farnesoid X receptor (FXR) and the membrane receptor G-protein-coupled bile acid receptor 5 (TGR5) can regulate incretin hormone and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) secretion, cholesterol metabolism, and systemic energy expenditure. Bile acid levels and distribution are altered in type 2 diabetes and increased following bariatric procedures, in parallel with reduced body weight and improved insulin sensitivity and glycaemic control. Thus, modulation of bile acid levels and signalling, using bile acid binding resins, TGR5 agonists, and FXR agonists, may serve as a potent therapeutic approach for the treatment of obesity, type 2 diabetes, and other components of the metabolic syndrome in humans.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) pathway promotes tumor <em>growth</em> and angiogenesis in many solid tumors. Although there has long been interest in FGF pathway inhibitors, development has been complicated: An effective FGF inhibitor must block the activity of multiple mitogenic FGF ligands but must spare the metabolic hormone FGFs (FGF-<em>19</em>, FGF-21, and FGF-23) to avoid unacceptable toxicity. To achieve these design requirements, we engineered a soluble FGF receptor 1 Fc fusion protein, FP-1039. FP-1039 binds tightly to all of the mitogenic FGF ligands, inhibits FGF-stimulated cell proliferation in vitro, blocks FGF- and vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-induced angiogenesis in vivo, and inhibits in vivo <em>growth</em> of a broad range of tumor types. FP-1039 antitumor response is positively correlated with RNA levels of FGF2, FGF18, FGFR1c, FGFR3c, and ETV4; models with genetic aberrations in the FGF pathway, including FGFR1-amplified lung cancer and FGFR2-mutated endometrial cancer, are particularly sensitive to FP-1039-mediated tumor inhibition. FP-1039 does not appreciably bind the hormonal FGFs, because these ligands require a cell surface co-receptor, klotho or β-klotho, for high-affinity binding and signaling. Serum calcium and phosphate levels, which are regulated by FGF-23, are not altered by administration of FP-1039. By selectively blocking nonhormonal FGFs, FP-1039 treatment confers antitumor efficacy without the toxicities associated with other FGF pathway inhibitors.

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 <em>fibroblasts</em>. Estimated [Ca++]i of serum-deprived, subconfluent <em>fibroblasts</em> was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells <em>growing</em> in serum, whose [Ca++]i was 50 (+/-<em>19</em>) nM. Serum, partially purified platelet-derived <em>growth</em> <em>factor</em> (PDGF), and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal <em>growth</em> <em>factor</em> (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit <em>growth</em> <em>factor</em>-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent <em>fibroblasts</em>, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate <em>growth</em> <em>factor</em>-induced increases in [Ca++]i.

Alteration in the expression of <em>growth</em> <em>factors</em> is widely accepted as being one of several critical defects in the generation of the malignant cell. In the present study, <em>19</em> human metastatic melanoma cell lines were compared to 14 normal human foreskin melanocyte cell lines for the production of RNA transcripts specific for 11 different <em>growth</em> <em>factors</em>. Using the extremely sensitive technique of polymerase chain reaction to amplify <em>growth</em> <em>factor</em>-specific complementary DNAs, we analyzed the following: transforming <em>growth</em> <em>factor</em> (TGF) types alpha, beta 1, beta 2, and beta 3, acidic (a) <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), basic (b) FGF, FGF-5, keratinocyte <em>growth</em> <em>factor</em> (KGF), HST, and platelet-derived <em>growth</em> <em>factor</em> (PDGF) types A and B. There were clear distinctions among the patterns of <em>growth</em> <em>factor</em> RNA expression by normal melanocytes and malignant melanoma cells. The prototypic melanocyte pattern of expression included TGF beta 1, TGF beta 3, and KGF. A subset of melanocyte cell lines also expressed PDGFA transcripts. In contrast, melanoma cells characteristically expressed RNA transcripts of TGF beta 1, TGF beta 2, TGF beta 3, TGF alpha, bFGF, KGF, and PDGFA. Subsets of melanoma cell lines also expressed aFGF, FGF-5, and PDGFB. The results presented indicated that TGF beta 2, TGF alpha, and bFGF may be particularly important in melanomagenesis and that these, as well as FGF-5, aFGF, and PDGFB, can be used as markers of transformation in this tumor type.

Although radiation-induced fibrosis has long been characterized by excess <em>fibroblast</em> proliferation and extracellular matrix deposition, the origin of cell activation in these complications of radiotherapy or radiation accidents is still controversial. The present work was designed to test the hypothesis that the abnormal production of TGF-beta 1 in irradiated tissues results in continuous signals for tissue repair and long-term cell activation. We examined gene expression of this <em>growth</em> <em>factor</em> in a well-characterized pig model of radiation-induced fibrosis, using Northern-blot and slot-blot hybridizations and indirect immunofluorescence. We found that the TGF-beta 1 mRNA level was increased <em>19</em>-fold in the irradiated skin during the early erythematous phase, which started 3 weeks after irradiation. During the later phases of fibrosis, from 6 to 12 months after irradiation, the TGF-beta 1 gene was highly expressed in the repaired skin and the underlying muscular fibrotic tissue, with 10- and 8-fold maximal increases, respectively. In addition, we found that the beta-actin mRNA level was increased in the fibrotic tissue. Immunostaining for TGF-beta 1 revealed the presence of the protein in endothelial cells of capillaries, in myo<em>fibroblast</em>s, and in the collagenous matrix of the fibrotic tissue. These results suggest that TGF-beta 1 may be one of the key cytokines involved in the cascade of events that leads to radiation-induced fibrosis, at both early and late stages.

Cancer patients treated for venous thromboembolism with low molecular weight heparin (LMWH) have a better survival rate than patients treated with unfractionated heparin (UFH). Because fibrin-associated angiogenesis is an important determinant in the progression and metastasis of many solid tumors, the effects of heparins on in vitro angiogenesis were investigated. Both UFH and LMWH inhibited bFGF-induced proliferation of human microvascular endothelial cells (hMVECs) to the same the extent (36-60%). VEGF165-induced proliferation was inhibited to a to a lesser extent (<em>19</em>-33%). Turbidity measurements and electron microscopy showed that the presence of LMWH during polymerization of the fibrin matrix led to a more transparent rigid network with thin fibrin bundles, whereas the presence of UFH resulted in a more opaque more porous network with thick fibrin fibers. We used a human in vitro angiogenesis model, which consisted of hMVECs seeded on top of a fibrin matrix, and stimulated the cells with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> plus tumor necrosis <em>factor</em> a to induce capillary-like tubular structures. The formation of capillary-like tubular structures was retarded with matrices polymerized in the presence of LMWH (46% inhibition compared with a control matrix for both 1.5 and 10 units/ml LMWH), whereas matrices polymerized in the presence of UFH facilitated tubular structure formation (72 and 36% stimulation compared with a control matrix for 1.5 and 10 units/ml UFH, respectively). Similar results were obtained for cells stimulated with vascular endothelial <em>growth</em> <em>factor</em> plus tumor necrosis <em>factor</em> alpha. These data demonstrate the inhibitory effect of heparins on proliferation of hMVECs and provide a novel mechanism by which LMWH may affect tumor progression, namely reduced in<em>growth</em> of microvascular structures in a fibrinous stroma matrix by rendering it less permissive for invasion.

OBJECTIVE

Human thoracic aortic aneurysms (TAAs) are characterized by extracellular matrix breakdown associated with progressive smooth muscle cell (SMC) rarefaction. These features are present in all types of TAA: monogenic forms [mainly Marfan syndrome (MFS)], forms associated with bicuspid aortic valve (BAV), and degenerative forms. Initially described in a mouse model of MFS, the transforming growth factor-β1 (TGF-β1)/Smad2 signalling pathway is now assumed to play a role in TAA of various aetiologies. However, the relation between the aetiological diversity and the common cell phenotype with respect to TGF-β signalling remains unexplained.

RESULTS

This study was performed on human aortic samples, including TAA [MFS, n = 14; BAV, n = 15; and degenerative, n = 19] and normal aortas (n = 10) from which tissue extracts and human SMCs and fibroblasts were obtained. We show that all types of TAA share a complex dysregulation of Smad2 signalling, independent of TGF-β1 in TAA-derived SMCs (pharmacological study, qPCR). The Smad2 dysregulation is characterized by an SMC-specific, heritable activation and overexpression of Smad2, compared with normal aortas. The cell specificity and heritability of this overexpression strongly suggest the implication of epigenetic control of Smad2 expression. By chromatin immunoprecipitation, we demonstrate that the increases in H3K9/14 acetylation and H3K4 methylation are involved in Smad2 overexpression in TAA, in a cell-specific and transcription start site-specific manner.

CONCLUSIONS

Our results demonstrate the heritability, the cell specificity, and the independence with regard to TGF-β1 and genetic backgrounds of the Smad2 dysregulation in human thoracic aneurysms and the involvement of epigenetic mechanisms regulating histone marks in this process.

FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Their potent effects on normalizing glucose, lipid, and energy homeostasis in disease models have made them an interesting focus of research for combating the growing epidemics of diabetes and obesity. Despite overlapping functions, FGF19 and FGF21 have many discrete effects, the most important being that FGF19 has both metabolic and proliferative effects, whereas FGF21 has only metabolic effects. Here we identify the structural determinants dictating differential receptor interactions that explain and distinguish these two physiological functions. We also have generated FGF19 variants that have lost the ability to induce hepatocyte proliferation but that still are effective in lowering plasma glucose levels and improving insulin sensitivity in mice. Our results add valuable insight into the structure-function relationship of FGF19/FGF21 and identify the structural basis underpinning the distinct proliferative feature of FGF19 compared with FGF21. In addition, these studies provide a road map for engineering FGF19 as a potential therapeutic candidate for treating diabetes and obesity.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of cancer, autoimmune disease, and various pathologic conditions characterized by excessive fibrosis. In this study, we investigated the expression of MMP-9 and its clinical significance in systemic sclerosis (SSc). The patients (n = 42) with SSc had higher concentrations of MMP-9 and of tissue inhibitor of metalloproteinase-1 (TIMP-1) and a higher ratio of MMP-9 to TIMP-1 in sera than healthy controls (n = 32). Serum MMP-9 concentrations were significantly higher in the diffuse type (n = 23) than the limited type of SSc (n = <em>19</em>). Serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score and with serum concentrations of transforming <em>growth</em> <em>factor</em> beta. Moreover, dermal <em>fibroblasts</em> from patients with SSc produced more MMP-9 than those from healthy controls when they were stimulated with IL-1beta, tumor necrosis <em>factor</em> alpha, or transforming <em>growth</em> <em>factor</em> beta. Such an increase in MMP-9 production was partially blocked by treatment with cyclosporin A. In summary, the serum MMP-9 concentrations were elevated in SSc patients and correlated well with skin scores. The increased MMP-9 concentrations may be attributable to overproduction by dermal <em>fibroblasts</em> in SSc. These findings suggest that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) 15/<em>19</em> and 21 belong to the FGF endocrine subfamily. They present the intriguing characteristic to be transcribed and secreted in certain tissues and to act as hormones. The insulin-mimetic properties of FGF21 and the regulatory role of FGF15/<em>19</em> in bile acid and glucose homeostasis endorse these hormones as druggable targets in metabolic disorders. Here, we present details on discoveries, identification, transcriptional regulation, and mechanism of actions of FGF15/<em>19</em> and FGF21 with a critical perspective view on their putative role as metabolic integrators in the liver.

BACKGROUND

Fibroblast growth factor-23 (FGF-23) is a phosphate regulatory hormone that directly stimulates left ventricular hypertrophy in experimental models. The role of FGF-23 in cardiovascular disease development in the general population is unclear. We tested associations of FGF-23 with major subclinical and clinical cardiovascular disease outcomes in a large prospective cohort.

RESULTS

We evaluated 6547 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) who were initially free of cardiovascular disease. We measured serum FGF-23 using the Kainos immunoassay. The MESA measured left ventricular mass by MRI, coronary calcium by computed tomography, and carotid intima-media thickness by ultrasound. The MESA adjudicated incident heart failure, coronary heart disease, and stroke by medical record review. After adjustment, the highest FGF-23 quartile was associated with an estimated 2.4-g greater left ventricular mass (95% confidence interval, 0.4-4.5 greater) and a 26% greater odds of higher coronary calcium scores (95% confidence interval, 9%-46% greater) compared with the lowest quartile. During 7.5-year follow-up, each 20-pg/mL higher FGF-23 concentration was associated with a 19% greater risk of heart failure (95% confidence interval, 3%-37% greater) and a 14% greater risk of coronary heart disease (95% confidence interval, 1%-28% greater). FGF-23 was not associated with carotid intima-media thickness or stroke.

CONCLUSIONS

Higher serum FGF-23 concentrations are associated with subclinical cardiac disease and with new heart failure and coronary disease events, but not with carotid intima-media thickness or stroke. FGF-23 may be a novel cardiovascular risk factor in the general population.

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5'-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein gamma-parvin and the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.

miRNAs (microRNAs) have been shown to play a role in myocardial fibrosis. The present study was designed to analyse whether alterations in miRNA expression contribute to the progression of myocardial fibrosis in AS (aortic valve stenosis) patients through up-regulation of the pro-fibrotic <em>factor</em> TGF-β1 (transforming <em>growth</em> <em>factor</em>-β type 1). Endomyocardial biopsies were obtained from 28 patients with severe AS, and from the necropsies of 10 control subjects. AS patients presented increased myocardial CVF (collagen volume fraction) and TGF-β1 compared with the controls, these parameters being correlated in all patients. Patients were divided into two groups by cluster analysis according to their CVF: SF (severe fibrosis; CVF >15%; n=15) and non-SF (CVF ≤15%; n=13). TGF-β1 was increased in patients with SF compared with those with non-SF. To analyse the involvement of miRNAs in SF, the miRNA expression profile of 10 patients (four with non-SF and six with SF) was analysed showing that 99 miRNAs were down-regulated and <em>19</em> up-regulated in the SF patients compared with the non-SF patients. Those miRNAs potentially targeting TGF-β1 were validated by real-time RT (reverse transcription)-PCR in the whole test population, corroborating that miR-122 and miR-18b were down-regulated in patients with SF compared with those with non-SF and the control subjects. Additionally, miR-122 was inversely correlated with the CVF, TGF-β1 and the TGF-β1-regulated PCPE-1 (procollagen C-terminal proteinase enhancer-1) in all patients. Experiments in human <em>fibroblasts</em> demonstrated that miR-122 targets and inhibits TGF-β1. In conclusion, for the first time we show that myocardial down-regulation of miR-122 might be involved in myocardial fibrosis in AS patients, probably through TGF-β1 up-regulation.

OBJECTIVE

PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study.

METHODS

This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2).

RESULTS

Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C(max) correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure. No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for>>or=6 months.

CONCLUSIONS

The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

A test of the mitogenic activity of greater than 40 purified and crude sources of hormones and <em>growth</em> <em>factors</em> revealed that epidermal <em>growth</em> <em>factor</em>, high density lipoprotein, an extract of bovine pituitary, hypothalamus, or whole brain, and the medium conditioned by differentiated human hepatoma cells were mitogenic for cultured endothelial cells derived from human umbilical vein. The four active agents combined with an improved nutrient medium and a collagen- or fibronectin-coated culture surface supported the <em>growth</em> of the endothelial cell population at a rate of 0.70-0.80 generations per day at both low and high cell densities in serum-free medium. The brain-derived activity exhibited properties reported by Maciag et al. [Maciag, T., Hoover, A. & Weinstein, R. (<em>19</em>82) J. Biol. Chem. 257, 5333-5336] and Gordon et al. [Gordon, P. B., Sussman, I. I. & Hatcher, V. B. (<em>19</em>83) In Vitro <em>19</em>, 661-671]. The liver cell-derived activity was a specific product of differentiated hepatoma cells. The medium from HeLa cells, relatively undifferentiated rat liver cell lines, and human <em>fibroblasts</em> was inactive. Purified plasma proteins of liver origin could not substitute for the hepatoma cell-conditioned medium. The hepatoma cell-derived activity was non-dialyzable, heat-labile, stable between pH 4 to 11, inactivated by trypsin and mercaptoethanol treatment, and stable after treatment with 6 M urea and phenylmethylsulfonyl fluoride. The results provide a simplified model for elucidation of the endocrinology of human endothelial cell <em>growth</em>, function, and aging. We suggest an endocrine role of both the nervous system and liver in the regeneration of endothelial cells.

1. Stimulation of purinergic receptors on retinal pigment epithelial (RPE) cells can increase the rate of fluid transport or decrease phagocytosis. This study aims to: determine whether the purine ATP can be released from RPE cells, begin probing the mechanism of any release and test whether cells degrade ATP extracellularly. 2. ATP release was monitored from cultured human ARPE-<em>19</em> cells with the luciferin-luciferase assay. Biphasic release of ATP was triggered by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), by the pyrimidine uridine triphosphate (UTP) and by hypotonicity. 3. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited release of ATP, suggesting that release was associated with Cl(-) channels. 4. Elevating intracellular Ca(2+) directly with ionomycin was insufficient to trigger ATP release. 5. UTP induced a biphasic elevation in intracellular Ca(2+). NPPB inhibited the second phase, suggesting autostimulation by released ATP. 6. Cells grown on permeable supports showed apical release of ATP, analogous to release into subretinal space in vivo. 7. The presence of ecto-ATPases on ARPE-<em>19</em> cell membranes was suggested by the degradation of ATP added to intact cells. 8. Phagocytosis of fluorescent beads was inhibited by ATP, but the ecto-5'-nucleotidase inhibitor alpha, beta-methylene ADP prevented this, suggesting that inhibition was mediated by extracellular conversion of ATP to adenosine. 9. These results suggest that <em>growth</em> <em>factors</em>, pyrimidines and changes in tonicity could trigger ATP release into subretinal space. The levels of ATP released may be capable of autocrine stimulation of ATP receptors, while conversion to adenosine by ecto-enzymes could alter phagocytosis.

Tight control of glucose excursions has been a long-standing goal of treatment for patients with type 2 diabetes mellitus in order to ameliorate the morbidity and mortality associated with hyperglycemia. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> is a hormone-like enterokine released postprandially that emerged as a potential therapeutic agent for metabolic disorders, including diabetes and obesity. Remarkably, FGF<em>19</em> treatment has hypoglycemic actions that remain potent in models of genetic and acquired insulin resistance. Here, we provided evidence that the central nervous system responds to FGF<em>19</em> administered in the periphery. Then, in two mouse models of insulin resistance, leptin-deficiency and high-fat diet feeding, third intra-cerebro-ventricular infusions of FGF<em>19</em> improved glycemic status, reduced insulin resistance and potentiated insulin signaling in the periphery. In addition, our study highlights a new mechanism of central FGF<em>19</em> action, involving the suppression of AGRP/NPY neuronal activity. Overall, our work unveils novel regulatory pathways induced by FGF<em>19</em> that will be useful in the design of novel strategies to control diabetes in obesity.

OBJECTIVE

Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel.

METHODS

Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers.

RESULTS

A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified.

CONCLUSIONS

Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFβ (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a heparin-binding protein expressing potent mitogenic and angiogenic properties. Elevated levels of bFGF have recently been described in human glioma cell lines. The high degree of vascularity and invasiveness which characterize human gliomas suggest that activated expression of bFGF or similar proteins may be related to the aberrant <em>growth</em> patterns of these tumors. The influence of endogenous bFGF on glioma cell <em>growth</em> in vitro was evaluated in the present study by down-regulating bFGF expression using antisense oligonucleotide primers. The addition of 50 microM bFGF-specific antisense primer to the human glioma cell line SNB-<em>19</em> resulted in an 80% inhibition in glioma <em>growth</em>. This effect was saturable and specific. Antisense primers directed to two different sites of bFGF mRNA were effective in suppressing SNB-<em>19</em> <em>growth</em>, whereas sense strand primer was ineffective. Furthermore, only the antisense primer significantly reduced the specific activity of bFGF protein in SNB-<em>19</em> cell extracts. Neither antisense or sense primers inhibited the <em>growth</em> of non-transformed human glia. bFGF mRNA was detected in both transformed and non-transformed human glia by polymerase chain reaction analysis suggesting that alterations in bFGF isoform content or activity may be specifically related to abnormal <em>growth</em> control in human gliomas.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> is an atypical member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family of signaling molecules. FGF<em>19</em>, FGF21, and FGF23 comprise a phylogenetic subfamily with attributes that distinguish them from typical FGFs. The FGF<em>19</em> subfamily has reduced heparin binding resulting from a disrupted beta-trefoil domain. Reduced heparin binding allows these FGFs to diffuse beyond their site of origin and act as endocrine hormones. This family of FGFs is regulated, at least in part, by nuclear hormone receptors. FGF<em>19</em> expression is regulated by the farnesoid X receptor, a nuclear hormone receptor that is a key regulator of bile acid biosynthesis and transport. In line with its regulation by a bile acid receptor, FGF<em>19</em> is involved in the regulation of bile acid biosynthesis and gallbladder filling. FGF<em>19</em> originates from intestine and signals to liver via the portal circulation with a pronounced diurnal pattern. FGF<em>19</em> is the only FGF to not have a closely related mouse homologue. The mouse homologue of FGF<em>19</em>, called FGF15, is only 53% identical to the human FGF<em>19</em>. FGF<em>19</em> transgenic mice and mice administered exogenous FGF<em>19</em> are resistant to the effects of a high fat diet, suggesting FGF<em>19</em> may play a role in metabolic signaling pathways. Hepatocellular carcinoma is seen in mice, predominantly female mice, exposed to FGF<em>19</em>. Further investigation into the cellular mechanisms involved in these activities will allow better understanding of FGF<em>19</em> biology in the context of human physiology.

After skin wounding, the repair process is initiated by the release of <em>growth</em> <em>factors</em>, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as <em>fibroblast</em> invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. <em>19</em>:359-369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736-748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of alpha-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

OBJECTIVE

Fibrotic sequelae remain the most important dose-limiting toxicity of radiation therapy to soft tissue. Functionally, this is reflected in loss of range of motion and muscle strength and the development of limb edema and pain. Tumor necrosis factor alpha and fibroblast growth factor 2 (FGF2), which are abnormally elevated in irradiated tissues, may mediate radiation fibrovascular injury.

METHODS

In an open label drug trial, we studied the effects of pentoxifylline (400 mg orally tid for 8 weeks) on 30 patients who displayed late, radiation-induced fibrosis at 1 to 29 years posttreatment (40 to 84 Gy). The primary outcome measurement was change in physical impairments thought to be secondary to radiation, including active and passive range of motion (AROM and PROM), muscle strength, limb edema, and pain. Plasma levels of cytokines (tumor necrosis factor alpha and FGF2) also were measured. Twenty-seven patients completed baseline and 8-week assessments, and 24 patients completed baseline, 8-week, and 16-week assessments.

RESULTS

After 8 weeks of pentoxifylline intervention, 20 of 23 patients with impaired AROM and 19 of 22 with impaired PROM improved; 11 of 19 patients with muscle weakness showed improved motor strength; five of seven patients with edema had decreased limb girth; and nine of 20 patients had decreased pain. Pretreatment FGF2 levels dropped from an average of 44.9 pg/mL to 24.0 pg/mL after 8 weeks of treatment.

CONCLUSIONS

Patients receiving pentoxifylline demonstrated improved AROM, PROM, and muscle strength and decreased limb edema and pain. Reversal of these delayed radiation effects was associated with a decrease in circulating FGF2.