The secreted <em>growth</em> <em>factor</em> pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this <em>growth</em> <em>factor</em>, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival <em>factor</em> for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 <em>fibroblasts</em> PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell <em>growth</em> at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell <em>growth</em> (<em>21</em> +/- 1% relative to PTN), and was an antagonist of PTN-induced cell <em>growth</em> (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell <em>growth</em> required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 <em>fibroblasts</em> is via the MAP kinase pathway.

Scaffolds that better approximate the mechanical properties of cardiovascular and other soft tissues might provide a more appropriate mechanical environment for tissue development or healing in vivo. An ability to induce local angiogenesis by controlled release of an angiogenic <em>factor</em>, such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), from a biodegradable scaffold with mechanical properties more closely approximating soft tissue could find application in a variety of settings. Toward this end biodegradable poly(ester urethane)urea (PEUU) scaffolds loaded with bFGF were fabricated by thermally induced phase separation. Scaffold morphology, mechanical properties, release kinetics, hydrolytic degradation and bioactivity of the released bFGF were assessed. The scaffolds had inter-connected pores with porosities of 90% or greater and pore sizes ranging from 34-173 microm. Scaffolds had tensile strengths of 0.25-2.8 MPa and elongations at break of 81-443%. Incorporation of heparin into the scaffold increased the initial burst release of bFGF, while the initial bFGF loading content did not change release kinetics significantly. The released bFGF remained bioactive over <em>21</em> days as assessed by smooth muscle mitogenicity. Scaffolds loaded with bFGF showed slightly higher degradation rates than unloaded control scaffolds. Smooth muscle cells seeded into the scaffolds with bFGF showed higher cell densities than for control scaffolds after 7 days of culture. The bFGF-releasing PEUU scaffolds thus exhibited a combination of mechanical properties and bioactivity that might be attractive for use in cardiovascular and other soft tissue applications.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> is a unique member of the FGF family, with several molecular characteristics that differ from classical FGFs and exhibiting a pharmacologic profile that includes a variety of metabolic responses in vitro and when tested in vivo in animal models. FGF<em>21</em> represents a novel and attractive therapeutic agent for type 2 diabetes mellitus, because of its ability to modulate disease phenotype in preclinical settings without inducing any apparent adverse effects. Although FGF<em>21</em> was discovered relatively recently, the understanding of its biology and therapeutic utility is rapidly evolving. A number of key metabolically linked molecules and pathways have been suggested to be involved in the mechanism of action of FGF<em>21</em>, depending on the specific target tissue/organ. Further research into these mechanisms should lead to important advances in the understanding of FGF<em>21</em> biology and pave the way for novel therapeutic strategies. The specifics of FGF<em>21</em> activities both in cell culture and in vivo, its potential as a target for diabetes, and insights into the molecular mechanisms of FGF<em>21</em> metabolic actions will be discussed in this review.

It has been previously reported (M. Yashiro et al., Jpn. J. Cancer Res., 84: 883-886, 1994) that a <em>growth</em> <em>factor</em> secreted by human gastric <em>fibroblasts</em> stimulated proliferation of human scirrhous gastric carcinoma cells in vitro, suggesting a similar paracrine action in the gastric submucosa. The present study established the identity of the <em>growth</em> <em>factor</em> as keratinocyte <em>growth</em> <em>factor</em> (KGF). Increase in numbers and incorporation of [(3)H]thymidine in scirrhous gastric carcinoma cell lines (OCUM-2M and OCUM-11) in response to culture medium from a gastric <em>fibroblast</em> line (NF-8 and NF-<em>21</em>) were duplicated by substitution of KGF and inhibited by addition of anti-KGF antibody. Effects were specific for scirrhous carcinoma cells in distinction to well-differentiated gastric carcinoma cell lines. <em>Fibroblasts</em>, especially gastric <em>fibroblasts</em>, expressed KGF mRNA, whereas gastric cancer cells did not. Conversely, scirrhous gastric cancer cells expressed more KGF receptor mRNA than well-differentiated gastric adenocarcinoma cell, whereas gastric <em>fibroblasts</em> did not express this mRNA. ELISA detected high concentrations of KGF in medium from gastric <em>fibroblasts</em>, much lower concentration in medium from other <em>fibroblasts</em>, and no KGF in medium from gastric cancer cells. Western analysis indicated that KGF in gastric <em>fibroblasts</em> lysates had a molecular weight of M(r) 19,000, within the range suggested in our previous report. Thus, gastric <em>fibroblasts</em> secretion of KGF is likely to underline the remarkable proliferation of scirrhous gastric cancer cells in a paracrine manner.

Current studies suggest that short-term exposure of parathyroid glands to <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) reduces parathyroid hormone secretion. However, patients with chronic kidney disease (CKD) develop secondary hyperparathyroidism despite high levels of serum FGF23, indicating a parathyroid FGF23 'resistance'. Here we analyzed the expression of the FGF23 receptors Klotho and FGF receptor 1 (FGFR1) in 88 hyperplastic parathyroid glands from 31 patients with CKD (including <em>21</em> renal allograft recipients), and their regulation in isolated bovine and human hyperplastic parathyroid cells. Glandular expression was variable, yet the Klotho and FGFR1 mRNA levels declined in parallel with the decreasing glomerular filtration rate, significantly decreasing over CKD stages. We found no association between the expression of Klotho, FGFR1, and the proliferation marker Ki67. In vitro treatment of bovine cells with FGF23 or calcium reduced the Klotho level, whereas active vitamin D(3) compounds increased its expression. Phosphate and parathyroid hormone had no effect. Treatment had less impact on Klotho in cultured human cells than in the bovine healthy cell model, whereas FGFR1 expression was induced in the hyperplastic cells. Thus parathyroid Klotho and FGFR1 decrease with declining renal function, possibly because of alterations in mineral metabolism related to the failing kidney. This could explain the observed parathyroid resistance to FGF23 in late CKD.

The cJun NH2-terminal kinase (JNK) stress signaling pathway is implicated in the metabolic response to the consumption of a high-fat diet, including the development of obesity and insulin resistance. These metabolic adaptations involve altered liver function. Here, we demonstrate that hepatic JNK potently represses the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). Therefore, JNK causes decreased expression of PPARα target genes that increase fatty acid oxidation and ketogenesis and promote the development of insulin resistance. We show that the PPARα target gene <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (Fgf<em>21</em>) plays a key role in this response because disruption of the hepatic PPARα-FGF<em>21</em> hormone axis suppresses the metabolic effects of JNK deficiency. This analysis identifies the hepatokine FGF<em>21</em> as a critical mediator of JNK signaling in the liver.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) 15/19 and <em>21</em> 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 FGF<em>21</em> and the regulatory role of FGF15/19 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 FGF15/19 and FGF<em>21</em> with a critical perspective view on their putative role as metabolic integrators in the liver.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has recently emerged as a metabolic hormone involved in regulating glucose and lipid metabolism in mouse, but the regulatory mechanisms and actions of FGF<em>21</em> in humans remain unclear. Here we have investigated the regulatory mechanisms of the human FGF<em>21</em> gene at the transcriptional level. A deletion study of the human FGF<em>21</em> promoter (-1672 to +230 bp) revealed two fasting signals, including peroxisome proliferator-activated receptor α (PPARα) and glucagon signals, that independently induced human FGF<em>21</em> gene transcription in mouse primary hepatocytes. In addition, two feeding signals, glucose and xylitol, also dose-dependently induced human FGF<em>21</em> gene transcription and mRNA expression in both human HepG2 cells and mouse primary hepatocytes. FGF<em>21</em> protein expression and secretion were also induced by high glucose stimulation. The human FGF<em>21</em> promoter (-1672 to +230 bp) was found to have a carbohydrate-responsive element at -380 to -366 bp, which is distinct from the PPAR response element (PPRE). Knock-down of the carbohydrate response element binding protein by RNAi diminished glucose-induced human FGF<em>21</em> transcription. Moreover, we found that a region from -555 to -443 bp of the human FGF<em>21</em> promoter region exerts an important role in the activation of basic transcription. In conclusion, human FGF<em>21</em> gene expression is paradoxically and independently regulated by both fasting and feeding signals. These regulatory mechanisms suggest that human FGF<em>21</em> is increased with nutritional crisis, including starvation and overfeeding.

BACKGROUND

It has been suggested that the metabolic benefits of physical exercise could be mediated by myokines. We examined here the effect of exercise training on skeletal muscle expression of a panel of myokines in humans. Pathways regulating myokine expression were investigated in human myotubes.

METHODS

Eleven obese non-diabetic male subjects were enrolled in an 8-week endurance training program. Insulin sensitivity was assessed by an oral glucose tolerance test. Subcutaneous adipose tissue and Vastus lateralis muscle biopsy samples were collected before and after training. RNAs were prepared from adipose tissue and skeletal muscle. Primary culture of myoblasts was established.

RESULTS

As expected, exercise training improved aerobic capacity and decreased fat mass. No significant change in interleukin 6, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, myostatin (MSTN) or irisin mRNA level was found in muscle after training. A twofold increase in apelin mRNA level was found in muscle but not in adipose tissue. No change in circulating myokine and adipokine plasma levels was observed in the resting state in response to training. Interestingly, apelin was significantly expressed and secreted in primary human myotubes. Apelin gene expression was upregulated by cyclic AMP and calcium, unlike the other myokines investigated. Importantly, changes in muscle apelin mRNA levels were positively related to whole-body insulin sensitivity improvement.

CONCLUSIONS

Collectively, our data show that exercise training upregulates muscle apelin expression in obese subjects. Apelin expression is induced by exercise signaling pathways and secreted in vitro in human primary myotubes, and may behave as a novel exercise-regulated myokine with autocrine/paracrine action.

Postischemic cerebral blood flow and blood volume changes have been associated with angiogenesis; nevertheless, the spatiotemporal changes in vascular permeability, vascular density, and vessel size have not been investigated. Here we report a prolonged increase in vascular permeability from day 3 to day <em>21</em> after ischemia, in particular in the reperfused outer cortical layers and leptomeninges. Increased cerebral blood volume (CBV) was observed from day 3 to day 14, whereas increased blood volume in small vessels, primarily capillaries, was noticed from day 7 to day 14 in the reperfused cortex. An initial decrease in vascular density and a reciprocal increase in vessel size were observed within the reperfused cortex at days 1 and 3 after ischemia. Immunohistological analysis confirmed a similar decrease in microvessel density and an increase in vessel size in vessels with a diameter greater than 30 microm. These large-sized vessels exhibited intense basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and endothelial nitric oxide synthase immunoreactivity, suggesting the <em>growth</em> of collateral vessels. By contrast, a late increase in vascular density was noticed in the reperfused outer cortex at days 14 and <em>21</em> after ischemia. Together, these findings suggest that the early phase of CBV increase is likely because of the improvement in collateral circulation, whereas the late phase of CBV increase is attributed to the surge of angiogenesis.

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical <em>fibroblasts</em> (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) secretion (<em>21</em>1% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- <em>21</em>%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

BACKGROUND

In rheumatoid arthritis (RA), synovial fluid (SF)contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF),which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate the effects of GM-CSF on neutrophils.

METHODS

Neutrophils stimulated by GM-CSF were divided in to four subcellular fractions: cytosol, membrane/organelle, nuclei,and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time of-flight mass spectrometry (MALDI-TOF MS).

RESULTS

We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA.Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor(EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells.Conclusions Our results indicate that GM-CSF contributes to the pathogenesis of RA through upregulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the upregulated enzymes may therefore play an important role in RA.

BACKGROUND

Signalling of angiotensin II via angiotensin II type 1 receptor (AT1) promotes cardiac and renal fibrosis, but its role in lung fibrosis is little understood. Using a rat bleomycin (BLM) induced model of pulmonary fibrosis, we examined the expression of AT1 in the lung and the effect of an AT1 antagonist on pulmonary fibrosis.

METHODS

Adult male Sprague-Dawley rats were given 0.3 mg/kg BLM intratracheally. Two days earlier they had received 10 mg/kg/day of the AT1 antagonist candesartan cilexetil mixed in the drinking water. AT1 expression in the lungs was examined by immunohistochemistry and immunoblot methods. The effect of the AT1 antagonist on pulmonary fibrosis was studied by analysis of bronchoalveolar lavage (BAL) fluid, histopathology, and hydroxyproline assay.

RESULTS

Immunohistochemical studies showed overexpression of AT1 in inflammatory immune cells, alveolar type II cells, and <em>fibroblasts</em>. A quantitative assay for AT1 showed that AT1 expression was significantly upregulated in cells from BAL fluid after day 3 and in the lung homogenates after day <em>21</em>. Candesartan cilexetil significantly inhibited the increase in total protein and albumin, as well as the increase in total cells and neutrophils in BAL fluid. On day <em>21</em> candesartan cilexetil also ameliorated morphological changes and an increased amount of hydroxyproline in lung homogenates. In addition, BLM increased the expression of transforming <em>growth</em> <em>factor</em> (TGF)-beta1 in BAL fluid on day 7; this increase was significantly reduced by candesartan cilexetil.

CONCLUSIONS

AT1 expression is upregulated in fibrotic lungs. Angiotensin II promotes lung fibrosis via AT1 and, presumably, in part via TGF-beta1.

Nonwoven fiber mats of poly(epsilon-caprolactone) (PCL) and PCL blended with poly(ethylene oxide) (PEO) were generated by electrospinning. Differential scanning calorimetry, scanning electron microscopy, and gravimetric measurement confirm the removal of PEO after immersion in water, as well as an increase in the PCL crystallinity. The reorganization of PCL resulted in the macroscopic alteration of the electrospun mat, decreasing the peak pore diameter up to a <em>factor</em> of 3 while only minimally affecting the fiber diameter. This technique was used to create electrospun PCL scaffolds with similar fiber diameters but different pore diameters to examine the effect of pore diameter on cell <em>growth</em>. Human Dermal <em>Fibroblasts</em> (HDF) were seeded into multiple samples using a perfusion seeding technique to guarantee successful cell deposition. Fluorescence analysis at 7, 14, and <em>21</em> days found that cells proliferated at a faster rate on scaffolds with peak pore diameters greater than 6 microm, as determined by mercury porosimetry. Cell conformation was also found to change as the peak pore diameter grew from 12 to 23 microm; cells began aligning along single fibers instead of attaching to multiple fibers. Knowledge of the effect of void architecture on cell proliferation and conformation could lead to the development of more effective scaffolds for tissue engineering.

BACKGROUND

Tetraiodothyroacetic acid (tetrac) blocks angiogenic and tumor cell proliferation actions of thyroid hormone initiated at the cell surface hormone receptor on integrin alphavbeta3. Tetrac also inhibits angiogenesis initiated by vascular endothelial growth factor and basic fibroblast growth factor.

OBJECTIVE

We tested antiangiogenic and antiproliferative efficacy of tetrac and tetrac nanoparticles (tetrac NP) against human medullary thyroid carcinoma (h-MTC) implants in the chick chorioallantoic membrane (CAM) and h-MTC xenografts in the nude mouse.

METHODS

h-MTC cells were implanted in the CAM model (n = 8 per group); effects of tetrac and tetrac NP at 1 microg/CAM were determined on tumor angiogenesis and tumor growth after 8 d. h-MTC cells were also implanted sc in nude mice (n = 6 animals per group), and actions on established tumor growth of unmodified tetrac and tetrac NP ip were determined.

RESULTS

In the CAM, tetrac and tetrac NP inhibited tumor growth and tumor-associated angiogenesis. In the nude mouse xenograft model, established 450-500 mm(3) h-MTC tumors were reduced in size over 21 d by both tetrac formulations to less than the initial cell mass (100 mm(3)). Tumor tissue hemoglobin content of xenografts decreased by 66% over the course of administration of each drug. RNA microarray and quantitative real-time PCR of tumor cell mRNAs revealed that both tetrac formulations significantly induced antiangiogenic thrombospondin 1 and apoptosis activator gene expression.

CONCLUSIONS

Acting via a cell surface receptor, tetrac and tetrac NP inhibit growth of h-MTC cells and associated angiogenesis in CAM and mouse xenograft models.

Artificial nerve grafts are needed to reconstruct massive defects in the peripheral nervous system when autologous nerve grafts are not available in sufficient amounts. Nerve grafts containing Schwann cells display a suitable substrate for long-distance regeneration. We present here a comprehensive analysis of the in vivo effects of different isoforms of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) on peripheral nerve regeneration across long gaps. FGF-2 isoforms were provided by grafted, genetically modified Schwann cells over-expressing 18-kDa-FGF-2 and <em>21</em>-/23-kDa-FGF-2, respectively. Functional tests evaluated motor and sensory recovery. Additionally, morphometrical analyses of regenerated nerves were performed 3 and 6 months after grafting. Distinct regeneration promoting effects of the different FGF-2 isoforms were found. 18-kDa-FGF-2 mediated inhibitory effects on the grade of myelination of regenerating axons, whereas <em>21</em>-/23-kDa-FGF-2 mediated early recovery of sensory functions and stimulation of long-distance myelination of regenerating axons. The results contribute to the development of new therapeutic strategies in peripheral nerve repair.

Neuroblastoma is an embryonal tumor that typically arises in cells of the developing adrenal medulla. IGF-II mRNA is expressed at high levels in the adrenal cortex before birth but it is not detectable until after birth in the adrenal medulla. Neuroblastoma cell lines corresponding to early adrenal medullary precursors did not express IGF-II, although all three cell lines we tested were <em>growth</em> stimulated by IGF-II. Cell lines corresponding to more mature adrenal medullary cells expressed IGF-II, and one, SK-N-AS, grows by an IGF-II autocrine mechanism (J. Clin. Invest. 84:829-839) El-Badry, Romanus, Helman, Cooper, Rechler, and Israel. 1989. An examination of human neuroblastoma tumor tissues for IGF-II gene expression using in situ hybridization histochemistry revealed that IGF-II is expressed by tumor cells in only 5 of <em>21</em> neuroblastomas, but is detectable in cells of nonmalignant tissues including adrenal cortical cells, stromal <em>fibroblasts</em>, and eosinophils in all <em>21</em> tumors. These findings indicate that IGF-II may function as an autocrine <em>growth</em> <em>factor</em> for some neuroblastomas and as a paracrine <em>growth</em> <em>factor</em> for others. They suggest that the <em>growth</em> regulatory pathways utilized by neuroblastoma mimic those used in the precursor cell type from which individual tumors arise.

OBJECTIVE

To examine the correlation of plasma fibroblast growth factor (FGF)-23 and serum fetuin A levels with the coronary artery calcification score (CACS) in patients with normal kidney function.

BACKGROUND

Vascular calcification is an active process that may be aggravated by hyperphosphataemia and hypercalcaemia. FGF-23 and human fetuin-A have been associated with calcifying arteriosclerosis in renal failure. Plasma FGF-23 was identified as an independent factor negatively associated with peripheral vascular calcification. Fetuin-A acts as a systemic inhibitor of ectopic calcification in dialysis patients and can be correlated to the survival of these patients. Very few data exists on the role of FGF-23 and fetuin-A in coronary calcification of patients without impaired kidney function.

METHODS

Sixty-four patients, 21 females and 43 males, were subjected to 64-slice coronary computed tomography (CT) to evaluate coronary artery calcification (CAC). Plasma intact FGF-23 was determined by ELISA. Serum fetuin-A concentration were evaluated nephelometrically.

RESULTS

Mean plasma FGF-23 level was 20.4 +/- 9.1 pg/ml and serum fetuin-A was 0.46 +/- 0.09 g/l. There was no correlation between FGF-23 (P = 0.777) and fetuin-A (P = 0.767) levels and the CACS. No correlation was found between the presence of noncalcified plaques and coronary artery stenosis (CAS)>>or= 50%, and FGF-23 (P = 0.313 and P = 0.775) and fetuin-A levels (P = 0.601 and P = 0.659).

CONCLUSIONS

Plasma intact FGF-23 and serum fetuin-A concentration do not correlate with the CACS, the grade of stenosis or presence of noncalcified plaques of the coronary arteries in patients with normal kidney function.

OBJECTIVE

Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) play a critical role in neurogenesis. In the present study, we evaluated the additive effect of administering these two factors on post-ischemic progenitor cell proliferation, survival, and phenotypic maturation in the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) in the adult rat brain after transient middle cerebral artery occlusion.

METHODS

A combination of EGF+FGF-2 (each 1.44 ng/d) was continuously administered into the lateral ventricles for 3 days, 5-bromodeoxyuridine (BrdUrd) was injected (50 mg/Kg) twice daily for 3 days starting on Day 1 of reperfusion, and cohorts of rats were sacrificed on Day 5 and Day 21 of reperfusion.

RESULTS

Compared with sham controls, ischemic rats showed a significantly higher number of newly proliferated cells in both the DG (by 766 +/- 37%, P < 0.05) and the SVZ (by 650 +/- 43%, P < 0.05). Of the progenitor cells proliferated on Day 5 after ischemia, 41 +/- 6% in the DG and 28 +/- 5% in the SVZ survived to 3 weeks. Compared with vehicle control, the EGF + FGF-2 infusion significantly increased the post-ischemic progenitor cell proliferation (by 319 +/- 40%, P < 0.05 in the DG and by 366 +/- 32%, P < 0.05 in the SVZ) and survival (by 40 +/- 12%, P < 0.05 in the DG and by 522 +/- 47%, P < 0.05 in the SVZ) studied at 5 and 21 days, respectively. Furthermore, of the newly proliferated cells survived to 3 weeks after ischemia, EGF + FGF-2 infusion caused a significantly higher number of neuronal nuclear protein-BrdUrd double-positive mature neurons in the DG (46 +/- 9%, P < 0.05) compared with vehicle control. Neuronal nuclear protein and BrdUrd double-positive mature neurons were also found in the DG. Glial fibrillary acidic protein-positive astrocytes did not show double-positive staining in either region.

CONCLUSIONS

Specific growth factor infusion enhances post-ischemic progenitor cell proliferation by 5 days of reperfusion and neuronal maturation by 21 days of reperfusion in both the DG and SVZ in the adult rat brain.

Many studies have characterized the role of <em>growth</em> <em>factors</em> in multiple myeloma (MM) pathogenesis and have derived novel therapies to improve patient outcome based upon targeting cytokines and their signaling cascades both in the MM cell and in the bone-marrow (BM) microenvironment. These cytokines include interleukin 6 (IL-6), insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), tumor necrosis <em>factor</em> alpha (TNF-alpha), transforming <em>growth</em> <em>factor</em> beta (TGF-beta), stromal cell-derived <em>factor</em> 1alpha (SDF-1alpha), IL-<em>21</em>, B-cell stimulating <em>factor</em> 3 (BSF-3) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). These cytokines are secreted from stromal cells (SCs), endothelial cells and/or osteoclasts, and promote MM cell <em>growth</em>, survival and migration, as well as paracrine cytokine secretion and angiogenesis in the BM milieu. Thus inhibition of signaling cascades induced by these cytokine provides rationale for a therapeutic option for MM.

BACKGROUND

Several studies have shown antifibrotic effects of angiotensin converting enzyme (ACE) inhibitors as well as of angiotensin receptor 1 (AT1) antagonists, however, prospective trials with clinical end points comparing these effects do not exist. COL4A3-/- mice develop a non-hypertensive progressive renal fibrosis. We used this animal model to compare the potential of ACE inhibitor vs AT1 antagonist to prevent renal fibrosis irrespective of blood pressure-dependent involvement by the renin system.

METHODS

COL4A3-/- mice were treated with placebo, ramipril or candesartan. Blood pressure, proteinuria, serum urea and lifespan were monitored. Renal matrix was characterized by immuno-histochemistry, light and electron microscopy. Further biochemical analysis was provided using cDNA microarray and western blot techniques.

RESULTS

Untreated mice died of renal failure after 71+/-6 days. Ramipril and candesartan both delayed onset and reduced the extent of proteinuria. Both had minor effects on blood pressure and postponed onset of uraemia. Ramipril increased lifespan by 111% to 150+/-<em>21</em> days (P<0.01), whereas candesartan resulted in only a 38% prolongation to 98+/-16 days (P<0.01). Ramipril reduced glomerular and tubulo-interstitial fibrosis and numbers of activated <em>fibroblasts</em> to a greater extent than candesartan. Microarray and western blot analysis revealed a higher antifibrotic potential of ramipril in terms of downregulation of TGFbeta, connective tissue <em>growth</em> <em>factor</em>, metalloproteinases and extracellular matrix proteins.

CONCLUSIONS

The results indicate an antifibrotic, nephroprotective effect of ACE inhibitors and AT1 antagonists in an animal model of progressive renal fibrosis. The greater antifibrotic effect of ramipril at the maximal therapeutic doses employed may not be explained by different antiproteinuric or blood pressure lowering properties, but by-in contrast to candesartan-its ability to hinder the proinflammatory, profibrotic activation of the angiotensin receptor 2.

Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF<em>21</em> is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF<em>21</em>-deficient mice. Our results demonstrate the important role of FGF<em>21</em> in the regulation of lipid metabolism during amino acid starvation.

Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) in both lean and obese mice. FGF<em>21</em> expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response-driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF<em>21</em> induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF<em>21</em> axis.

OBJECTIVE

Methionine restriction (MR) is a dietary intervention that increases lifespan, reduces adiposity and improves insulin sensitivity. These effects are reversed by supplementation of the MR diet with cysteine (MRC). Genomic and metabolomic studies were conducted to identify potential mechanisms by which MR induces favorable metabolic effects, and that are reversed by cysteine supplementation.

METHODS

Gene expression was examined by microarray analysis and TaqMan quantitative PCR. Levels of selected proteins were measured by Western blot and metabolic intermediates were analyzed by mass spectrometry.

RESULTS

MR increased lipid metabolism in inguinal adipose tissue and quadriceps muscle while it decreased lipid synthesis in liver. In inguinal adipose tissue, MR not only caused the transcriptional upregulation of genes associated with fatty acid synthesis but also of Lpin1, Pc, Pck1 and Pdk1, genes that are associated with glyceroneogenesis. MR also upregulated lipolysis-associated genes in inguinal fat and led to increased oxidation in this tissue, as suggested by higher levels of methionine sulfoxide and 13-HODE + 9-HODE compared to control-fed (CF) rats. Moreover, MR caused a trend toward the downregulation of inflammation-associated genes in inguinal adipose tissue. MRC reversed most gene and metabolite changes induced by MR in inguinal adipose tissue, but drove the expression of Elovl6, Lpin1, Pc, and Pdk1 below CF levels. In liver, MR decreased levels of a number of long-chain fatty acids, glycerol and glycerol-3-phosphate corresponding with the gene expression data. Although MR increased the expression of genes associated with carbohydrate metabolism, levels of glycolytic intermediates were below CF levels. MR, however, stimulated gluconeogenesis and ketogenesis in liver tissue. As previously reported, sulfur amino acids derived from methionine were decreased in liver by MR, but homocysteine levels were elevated. Increased liver homocysteine levels by MR were associated with decreased cystathionine β-synthase (CBS) protein levels and lowered vitamin B6 and 5-methyltetrahydrofolate (5MeTHF) content. Finally, MR upregulated <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) gene and protein levels in both liver and adipose tissues. MRC reversed some of MR's effects in liver and upregulated the transcription of genes associated with inflammation and carcinogenesis such as Cxcl16, Cdh17, Mmp12, Mybl1, and Cav1 among others. In quadriceps muscle, MR upregulated lipid metabolism-associated genes and increased 3-hydroxybutyrate levels suggesting increased fatty acid oxidation as well as stimulation of gluconeogenesis and glycogenolysis in this tissue.

CONCLUSIONS

Increased lipid metabolism in inguinal adipose tissue and quadriceps muscle, decreased triglyceride synthesis in liver and the downregulation of inflammation-associated genes are among the factors that could favor the lean phenotype and increased insulin sensitivity observed in MR rats.