OBJECTIVE

The diagnosis of mitochondrial disorders (MDs) is occasionally difficult because patients often present with solitary, or a combination of, symptoms caused by each organ insufficiency, which may be the result of respiratory chain enzyme deficiency. Growth differentiation factor 15 (GDF-15) has been reported to be elevated in serum of patients with MDs. In this study, we investigated whether GDF-15 is a more useful biomarker for MDs than several conventional biomarkers.

METHODS

We measured the serum levels of GDF-15 and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>), as well as other biomarkers, in 48 MD patients and in 146 healthy controls in Japan. GDF-15 and FGF-<em>21</em> concentrations were measured by enzyme-linked immunosorbant assay and compared with lactate, pyruvate, creatine kinase, and the lactate-to-pyruvate ratio. We calculated sensitivity and specificity and also evaluated the correlation based on two rating scales, including the Newcastle Mitochondrial Disease Rating Scale (NMDAS).

RESULTS

Mean GDF-15 concentration was 6-fold higher in MD patients compared to healthy controls (2,711 ± 2,459 pg/ml vs 462.5 ± 141.0 pg/mL; p < 0.001). Using a receiver operating characteristic curve, the area under the curve was significantly higher for GDF-15 than FGF-<em>21</em> and other conventional biomarkers. Our date suggest that GDF-15 is the most useful biomarker for MDs of the biomarkers examined, and it is associated with MD severity.

CONCLUSIONS

Our results suggest that measurement of GDF-15 is the most useful first-line test to indicate the patients who have the mitochondrial respiratory chain deficiency.

UNASSIGNED

Coronary artery calcification (CAC) is highly prevalent in dialysis-naive patients with chronic kidney disease (CKD). However, there are sparse data on the association of CAC with subsequent risk of cardiovascular disease and all-cause mortality in this population.

UNASSIGNED

To study the prospective association of CAC with risk of cardiovascular disease and all-cause mortality among dialysis-naive patients with CKD.

UNASSIGNED

The prospective Chronic Renal Insufficiency Cohort study recruited adults with an estimated glomerular filtration rate of 20 to 70 mL/min/1.73 m2 from 7 clinical centers in the United States. There were 1541 participants without cardiovascular disease at baseline who had CAC scores.

UNASSIGNED

Coronary artery calcification was assessed using electron-beam or multidetector computed tomography.

UNASSIGNED

Incidence of cardiovascular disease (including myocardial infarction, heart failure, and stroke) and all-cause mortality were reported every 6 months and confirmed by medical record adjudication.

UNASSIGNED

During an average follow-up of 5.9 years in 1541 participants aged <em>21</em> to 74 years, there were 188 cardiovascular disease events (60 cases of myocardial infarction, 120 heart failures, and 27 strokes; patients may have had >1 event) and 137 all-cause deaths. In Cox proportional hazards models adjusted for age, sex, race, clinical site, education level, physical activity, total cholesterol level, high-density lipoprotein cholesterol level, systolic blood pressure, use of antihypertensive treatment, current cigarette smoking, diabetes status, body mass index, C-reactive protein level, hemoglobin A1c level, phosphorus level, troponin T level, log N-terminal pro-B-type natriuretic peptide level, <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 level, estimated glomerular filtration rate, and proteinuria, the hazard ratios associated with per 1 SD log of CAC were 1.40 (95% CI, 1.16-1.69; P < .001) for cardiovascular disease, 1.44 (95% CI, 1.02-2.02; P = .04) for myocardial infarction, 1.39 (95% CI, 1.10-1.76; P = .006) for heart failure, and 1.19 (95% CI, 0.94-1.51; P = .15) for all-cause mortality. In addition, inclusion of CAC score led to an increase in the C statistic of 0.02 (95% CI, 0-0.09; P < .001) for predicting cardiovascular disease over use of all the above-mentioned established and novel cardiovascular disease risk <em>factors</em>.

UNASSIGNED

Coronary artery calcification is independently and significantly related to the risks of cardiovascular disease, myocardial infarction, and heart failure in patients with CKD. In addition, CAC improves risk prediction for cardiovascular disease, myocardial infarction, and heart failure over use of established and novel cardiovascular disease risk factors among patients with CKD; however, the changes in the C statistic are small.

OBJECTIVE

Fibroblast growth factor-2 (FGF-2) administration and bone marrow stromal cell (MSC) transplantation could improve neurological deficits after occlusive cerebrovascular disease. In the present study, we examined the effects of neurological improvement after transient middle cerebral artery occlusion (MCAO) in rats by a novel therapeutic strategy with FGF-2 gene-transferred MSCs by the herpes simplex virus type 1 (HSV-1) vector.

METHODS

Adult Wistar rats were anesthetized. Nonmodified MSCs, FGF-2-modified MSCs with HSV-1 1764/-4/pR19/ssIL2-FGF-2, or PBS was administered intracerebrally 24 hours after transient right MCAO. All animals underwent behavioral tests for 21 days, and the infarction volume with 2-3-5-triphenylterazolium was detected 3 days and 14 days after the MCAO. Three days and 7 days after the MCAO, the FGF-2 production in the ipsilateral hemisphere of the MCAO was measured with ELISA. Seven and 14 days after the MCAO, immunohistochemical staining for FGF-2 was applied.

RESULTS

The stroke animals receiving FGF-2-modified MSCs demonstrated significant functional recovery compared with the other groups. Fourteen days after the MCAO, there was a significant reduction in infarction volume only in FGF-2-modified MSC-treated group. FGF-2 production in the FGF-2-modified MSC-treated brain was significantly higher compared with the other groups at 3 and 7 days after MCAO. Administrated FGF-2-modified MSCs strongly expressed the FGF-2 protein, which was proven by ELISA.

CONCLUSIONS

Our data suggest that the FGF-2 gene-modified MSCs with the HSV-1 vector can contribute to remarkable functional recovery after stroke compared with MSCs transplantation alone.

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription <em>factors</em>. Several NRs are still characterized as orphan receptors because ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice, and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist, and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo.

The release of <em>growth</em> <em>factors</em> from ischaemic retina has been hypothesized as the central stimulus for retinal neovascularization in proliferative diabetic retinopathy. Two of the <em>growth</em> <em>factors</em> implicated are insulin-like <em>growth</em> <em>factor</em>-I and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. We examined the effect of insulin-like <em>growth</em> <em>factor</em>-I on in vivo neovascularization using the established angiogenic model of the rabbit cornea (n = 30), and also compared the effects of insulin-like <em>growth</em> <em>factor</em>-I and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> using two new in vivo systems. Either supraphysiologic concentrations of each <em>growth</em> <em>factor</em> (600 micrograms) were injected intravitreally into pigmented rabbits (n = <em>21</em>) or porous polyfluorotetraethylene chambers filled with an emulsion containing collagen and <em>growth</em> <em>factor</em> (500 ng) were placed on the retina surface (n = 8). Our results demonstrate that when insulin-like <em>growth</em> <em>factor</em>-I was implanted together with a slow release carrier into the pocket of the normally avascular cornea, insulin-like <em>growth</em> <em>factor</em>-I (10 micrograms/pellet) induced angiogenesis in all rabbits. This degree of angiogenesis was comparable to that previously shown for basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. For the intravitreal studies, the fibrotic component was greater in the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> injected eyes, whereas the vascular component was accentuated in the eyes injected with insulin-like <em>growth</em> <em>factor</em>-I. Light and electron microscopy demonstrated areas of vascular proliferation in both groups. Porous polyfluorotetraethylene chamber studies with insulin-like <em>growth</em> <em>factor</em>-I and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> demonstrated vascular proliferation in the vicinity of the chamber similar to the intravitreal injected eyes, but to a lesser degree than the injected eyes. Our experiments overall support the angiogenic potential of both insulin-like <em>growth</em> <em>factor</em>-I and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and support distinct but complimentary roles for each <em>growth</em> <em>factor</em> in the pathogenesis of proliferative diabetic retinopathy.

The insulin-like <em>growth</em> <em>factors</em> (IGF)-I and -II are bound to specific carrier proteins in the circulation. For investigation of their physiological role, the acid-stable subunit of the major binding protein (SmBP) was isolated from human plasma Cohn fraction IV. Its effect on the mitogenic activity of IGF-I was studied with baby hamster kidney <em>fibroblasts</em> (BHK-<em>21</em>) and human skin <em>fibroblasts</em>. While free IGF-I had no effect on thymidine incorporation into DNA with BHK-<em>21</em> cells and only a moderate effect with human <em>fibroblasts</em> under standard conditions, DNA synthesis was significantly enhanced with both cell lines if IGF-I was complexed with SmBP before the experiment. The enhancement was optimal at an approximately equimolar ratio of both peptides. In contrast to experiments in which large concentrations of IGF-I were added at the beginning, repeated addition of small quantities of free IGF-I at hourly intervals clearly stimulated DNA synthesis in BHK-<em>21</em> cells. Binding studies with radiolabeled SmBP revealed no evidence for direct interaction with either cell line. It is concluded that SmBP acts as a reservoir, releasing continuously low amounts of IGF-I and thereby creating a steady state situation of receptor occupancy, which appears to be a better mitogenic stimulus than temporary large concentrations of IGF-I.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF<em>21</em> analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF<em>21</em> for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF<em>21</em> ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF<em>21</em> analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF<em>21</em> at a specific and preferred site confers superior metabolic pharmacology.

Hepatic glucagon action increases in response to accelerated metabolic demands and is associated with increased whole body substrate availability, including circulating lipids. The hypothesis that increases in hepatic glucagon action stimulate AMP-activated protein kinase (AMPK) signaling and peroxisome proliferator-activated receptor-α (PPARα) and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) expression in a manner modulated by fatty acids was tested in vivo. Wild-type (gcgr(+/+)) and glucagon receptor-null (gcgr(-/-)) littermate mice were studied using an 18-h fast, exercise, and hyperglucagonemic-euglycemic clamps plus or minus increased circulating lipids. Fasting and exercise in gcgr(+/+), but not gcgr(-/-) mice, increased hepatic phosphorylated AMPKα at threonine 172 (p-AMPK(Thr(172))) and PPARα and FGF<em>21</em> mRNA. Clamp results in gcgr(+/+) mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, p-AMPK(Thr(172)), or PPARα and FGF<em>21</em> mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPARα and FGF<em>21</em> expression. All effects were absent in gcgr(-/-) mice. These findings demonstrate that glucagon exerts a critical regulatory role in liver to stimulate pathways linked to lipid metabolism in vivo and shows for the first time that effects of glucagon on PPARα and FGF<em>21</em> expression are amplified by a physiological increase in circulating lipids.

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-19, FGF-<em>21</em>, 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.

Increased hepatic expression is held responsible for elevated serum levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) in non-alcoholic fatty liver disease (NAFLD) but the underlying molecular mechanism is unclear. In the present study we tested the postulate that the metabolic hormone FGF<em>21</em> is regulated by endoplasmic reticulum (ER) stress, a condition that is observed in a number of diseases including NAFLD and results in activation of an adaptive response known as the unfolded protein response (UPR). ER stress stimuli were found to induce expression of Fgf<em>21</em> mRNA in H4IIE hepatoma cells and in isolated rat hepatocytes. Moreover, intraperitoneal injection of the ER stressor tunicamycin induced hepatic Fgf<em>21</em> expression in mice and resulted in marked elevation of serum FGF<em>21</em> levels. The effect of ER stress on FGF<em>21</em> expression could be mimicked by overexpression of ATF4, a transcriptional effector of the PERK-branch of the UPR. In silico analysis revealed the presence of two binding sites for ATF4 in the FGF<em>21</em> promoter region. Combined disruption of these elements, abrogated FGF<em>21</em> promoter activity induced by ER stress or ATF4 overexpression. These findings implicate the PERK/eIF2alpha/ATF4 cascade in ER stress regulation of FGF<em>21</em>. A consequence of this notion is that other intracellular stress signaling pathways that converge at eIF2alpha, can regulate FGF<em>21</em> expression. Indeed, both nutrient (amino acid deprivation) and oxidative stress (arsenite) were found to induce Fgf<em>21</em> expression in hepatoma cells and isolated rat hepatocytes. In conclusion, FGF<em>21</em> expression is regulated by ER stress and additional intracellular stress signaling pathways. Our findings suggest that increased cellular stress in fatty livers may underlie the elevated FGF<em>21</em> levels observed in patients with NAFLD.

Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis <em>factor</em>-alpha mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day <em>21</em> were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung <em>fibroblast</em> cell line WI-38. IL-10 significantly reduced constitutive and transforming <em>growth</em> <em>factor</em>-beta-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived <em>growth</em> <em>factor</em>. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.

Primary varicella-zoster virus (VZV) infection in humans produces varicella (chickenpox), after which the virus becomes latent in ganglionic neurons. Analysis of the physical state of viral nucleic acid and virus gene expression during latency requires postmortem acquisition of fresh human ganglia. To provide an additional way to study the VZV-host relationship in neurons, we developed an in vitro model of infected differentiated human neural stem cells (NSCs). NSCs were induced to differentiate in culture dishes coated with poly-l-lysine and mouse laminin in the presence of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), nerve <em>growth</em> <em>factor</em> (NGF), brain-derived neurotropic <em>factor</em> (BDNF), dibutyryl cyclic AMP, and retinoic acid. Immunostaining with neuronal (MAP2a and β-tubulin), astrocyte (GFAP), and oligodendrocyte (CNPase) markers revealed that differentiated neurons constituted approximately 90% of the cell population. These neurons were maintained in culture for up to 8 weeks. No cytopathic effect (CPE) developed in neurons infected with cell-free VZV (Zostavax vaccine) compared to human fetal lung <em>fibroblasts</em> infected with VZV. Weeks later, VZV DNA virus-specific transcripts (open reading frames [ORFs] <em>21</em>, 29, 62, and 63) were detected in infected neurons, and dual immunofluorescence staining revealed the presence of VZV IE63 and gE exclusively in healthy-appearing neurons, but not in astrocytes. Neither the tissue culture medium nor a homogenate prepared from VZV-infected neurons produced a CPE in <em>fibroblasts</em>. VZV induced apoptosis in <em>fibroblasts</em>, as shown by activation of caspase 3 and by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, but not in neurons. This model provides a unique in vitro system to study the VZV-neuronal relationship.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hepatic hormone involved in the regulation of lipid and carbohydrate metabolism. This study aims to test the hypothesis that elevated FGF<em>21</em> concentrations are associated with the change of renal function and the presence of left ventricular hypertrophy (LVH) in the different stages of chronic kidney disease (CKD) progression.

RESULTS

240 subjects including 200 CKD patients (146 outpatients and 54 long-term hemodialytic patients) and 40 healthy control subjects were recruited. All CKD subjects underwent echocardiograms to assess left ventricular mass index. Plasma FGF<em>21</em> levels and other clinical and biochemical parameters in all subjects were obtained based on standard clinical examination methods. Plasma FGF<em>21</em> levels were significantly increased with the development of CKD from early- and end-stage (P<0.001 for trend), and significantly higher in CKD subjects than those in healthy subjects (P<0.001). Plasma FGF<em>21</em> levels in CKD patients with LVH were higher than those in patients without LVH (P = 0.001). Furthermore, plasma FGF<em>21</em> level correlated positively with creatinine, blood urea nitrogen (BUN), β2 microglobulin, systolic pressure, adiponectin, phosphate, proteinuria, CRP and triglyceride, but negatively with creatinine clearance rate (CCR), estimated glomerular filtrate rate (eGFR), HDL-c, LDL-c, albumin and LVH after adjusting for BMI, gender, age and the presence of diabetes mellitus. Multiple stepwise regression analyses indicated that FGF<em>21</em> was independently associated with BUN, Phosphate, LVMI and β2 microglobulin (all P<0.05).

CONCLUSIONS

Plasma FGF<em>21</em> levels are significantly increased with the development of early- to end-stage CKD and are independently associated with renal function and adverse lipid profiles in Chinese population. Understanding whether increased FGF<em>21</em> is associated with myocardial hypertrophy in CKD requires further study.

An immunohistochemical study combined with morphometry was carried out to examine the time-dependent expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) using 53 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-4 days, III: 7-14 days and IV: 17-<em>21</em> days). In the human wound specimens aged 4-12 h, neutrophils recruited at the wound showed no positive signals for VEGF. With an increase in wound ages of>> or =7 days, granulation tissue and angiogenesis were observed, with the migration of macrophages and <em>fibroblasts</em> of which the cytoplasm expressed VEGF-positive reactions. Morphometrically, the average VEGF-positive ratio was highest in group III, followed by that of group IV. In groups III and IV, 13 out of 26 wound samples had VEGF-positive ratios of more than 50%. However, all of the wound samples in groups I and II showed VEGF-positive ratios of less than 50%. With regard to the practical applicability and forensic validity, these observations suggest that a VEGF-positive ratio of more than 50% possibly indicates a wound age of 7 days or more.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is emerging as a key regulator of energy homeostasis and presents a novel target for the development of therapies for the treatment of diabetes, cardiovascular disease and obesity. Recent publications have demonstrated that FGF<em>21</em> resides downstream of a complex network of transcriptional regulators which modulate its expression in response to a wide array of physiological stimuli or pharmacologic agents. The manner in which these mechanisms are integrated to regulate FGF<em>21</em> transcription, production and subsequent secretion is poorly understood. While FGF<em>21</em> is detected in many metabolically active tissues and is regulated by several of the known transcription <em>factors</em> involved in metabolic control little is known about how these pathways are integrated. In this review, we discuss the data presented to date on regulation of FGF<em>21</em> by a wide array of transcription <em>factors</em> and explore how it relates to metabolic regulation in vivo.

Continuous ambulatory peritoneal dialysis (CAPD) has emerged as an important dialysis treatment modality worldwide. One of the major complications is bacterial peritonitis, which may result in subsequent technique failure because of loss of peritoneal clearance or peritoneal fibrosis. Bacterial peritonitis leads to the release of proinflammatory cytokines from resident and infiltrating cells in the peritoneal cavity. We studied 35 patients undergoing CAPD with acute bacterial peritonitis. All patients treated with antibiotics for 2 weeks after the clinical diagnosis of peritonitis had a good recovery. Peritoneal dialysate effluent (PDE) was collected on days 1, 3, 5, 10, <em>21</em>, and 42 after the start of treatment. Cell populations were monitored by flow cytometry. PDE levels of interleukin-1beta (IL-1), IL-6, transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) were measured by enzyme-linked immunosorbent assay. Gene transcription of TGF-beta in macrophages from PDE was measured by quantitative polymerase chain reaction. Bacterial peritonitis was associated with a sharp increase in total cell and neutrophil counts (400-fold) in PDE up to 3 weeks after peritonitis despite clinical remission (P < 0.0001). There was an increased absolute number of macrophages during the first 3 weeks despite the reduced percentage of macrophages among total cells in PDE compared with noninfective PDE. There was a progressive increase in the percentage of mesothelial cells or dead cells in the total cell population in PDE over the entire 6-week period. PDE levels of IL-1, IL-6, TGF-beta, and FGF increased markedly on day 1 before their levels decreased gradually. PDE levels of these cytokines or <em>growth</em> <em>factors</em> were significantly greater than those in noninfective PDE (n = 76) throughout the study period (P < 0.01). Similarly, TGF-beta complementary DNA (cDNA) molecules per macrophage were significantly greater than those of macrophages in noninfective PDE throughout this period (P < 0.01). There was no significant correlation between PDE levels of TGF-beta and TGF-beta cDNA molecules per macrophage, suggesting that peritoneal macrophages are not the only source of TGF-beta in PDE. We conclude there is an active release of proinflammatory cytokines and sclerogenic <em>growth</em> <em>factors</em> through at least 6 weeks despite apparent clinical remission of peritonitis. The peritoneal cytokine networks after peritonitis may potentially affect the physiological properties of the peritoneal membrane.

The adult healing response of the rotator cuff tendon-to-bone insertion site differs from the ordered process of insertion site development. Healing is characterized by disorganized scar and a lack of fibrocartilage formation, in contrast to the well organized fibrocartilaginous transition which forms during the normal development of the tendon-to-bone insertion. The purpose of this study was to localize the expression of a number of extracellular matrix and <em>growth</em> <em>factor</em> genes during insertion site development in order to guide future strategies for augmenting adult rotator cuff healing. The rotator cuff was morphologically distinct at 13.5 dpc (days postconception). Neo-tendon was evident as a condensation of cells adjacent to bone. The interface between tendon and bone did not form into a mature fibrocartilaginous insertion until <em>21</em>-days postnatally, based upon the appearance of four distinct zones with a mineralized humeral head. <em>Fibroblasts</em> of the supraspinatus tendon expressed type I collagen at all timepoints. Type II collagen was first expressed by chondrocytes in the fibrocartilage and mineralized fibrocartilage at 7 days and persisted in the mineralized fibrocartilage at 56 days. Type X collagen was first expressed by the chondrocytes in the mineralized fibrocartilage at 14 days and persisted in the mineralized fibrocartilage at 56 days. A shift from TGF-beta3 to TGF-beta1 expression occurred at 15.5 dpc.

Lipid metabolism is tightly regulated by nuclear receptors, transcription <em>factors</em>, and cellular enzymes. In this study, we demonstrated that the liver-enriched transcription <em>factor</em> CREBH (cAMP-responsive element binding protein, hepatocyte specific) and peroxisome proliferator-activated receptor α (PPARα) function as binary transcriptional activators to regulate lipid metabolism by activating <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a hepatic hormone that regulates whole-body energy homeostasis. Gain- and loss-of-function studies indicated that CREBH regulates triglyceride and fatty acid metabolism in animals under fasting or on an atherogenic high-fat (AHF) diet. CREBH and PPARα act as interactive trans-activators that regulate each other for their expression. Activated CREBH protein interacts with PPARα to form a functional complex upon fasting or the AHF diet, and both <em>factors</em> are required for induction of the metabolic hormone FGF<em>21</em>. The CREBH-PPARα complex was found to bind to integrated CRE-PPAR-responsive element-binding motifs in the FGF<em>21</em> gene promoter. Whereas CREBH and PPARα function in synergy to activate FGF<em>21</em> gene expression, PPARα relies on CREBH to exert its trans-activation effect on FGF<em>21</em>. Supporting the key role of CREBH in regulating FGF<em>21</em>, infusion of recombinant FGF<em>21</em> protein can reverse hypertriglyceridemia and hypoketonemia and partially rescue nonalcoholic steatohepatitis developed in the CREBH-null mice after the AHF diet. Our study demonstrated a transcriptional regulatory axis of CREBH-PPARα-FGF<em>21</em> in maintaining lipid homeostasis under metabolic stress. The functional relationship between CREBH and PPARα in regulating FGF<em>21</em> may represent an important transcriptional coactivation mechanism that orchestrates the processes of energy supply upon metabolic alteration.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling is essential for endochondral bone formation. Mutations cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. Most previous work in this area has focused on embryonic chondrogenesis. To explore the role of FGF signaling in the postnatal <em>growth</em> plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Toward this aim, rat proximal tibial <em>growth</em> plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs <em>21</em> and 22. <em>Growth</em> plate expressed FGFs 2, 7, 18, and 22. Perichondrial expression was generally greater than <em>growth</em> plate expression, supporting the concept that perichondrial FGFs regulate <em>growth</em> plate chondrogenesis. Nevertheless, FGFs synthesized by <em>growth</em> plate chondrocytes may be physiologically important because of their proximity to target receptors. In <em>growth</em> plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL1, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing <em>growth</em> velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF1, FGF7, FGF18, and FGF22 were upregulated. In summary, we have analyzed the expression of all known FGFs and FGFRs in the postnatal <em>growth</em> plate using a method that is quantitative and highly sensitive. This approach identified ligands and receptors not previously known to be expressed in <em>growth</em> plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the <em>growth</em> plate. We also found temporal changes in FGF and FGFR expression which may contribute to <em>growth</em> plate senescence and thus help determine the size of the adult skeleton.

HMGCS2 (hydroxymethylglutaryl CoA synthase 2), the gene that regulates ketone body production, is barely expressed in cultured cell lines. In this study, we restored HMGCS2 expression and activity in HepG2 cells, thus showing that the wild type enzyme can induce fatty acid β-oxidation (FAO) and ketogenesis, whereas a catalytically inactive mutant C166A did not generate either process. Peroxisome proliferator-activated receptor (PPAR) α expression also induces fatty acid β-oxidation and endogenous HMGCS2 expression. Interestingly, PPARα-mediated induction was abolished when HMGCS2 expression was down-regulated by RNAi. These results indicate that HMGCS2 expression is both sufficient and necessary to the control of fatty acid oxidation in these cells. Next, we examined the expression pattern of several PPARα target genes in this now "ketogenic" HepG2 cell line. FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>) expression was specifically induced by HMGCS2 activity or by the inclusion of the oxidized form of ketone bodies (acetoacetate) in the culture medium. This effect was blunted by SirT1 (sirtuin 1) RNAi, so we propose a SirT1-dependent mechanism for FGF<em>21</em> induction by acetoacetate. These data suggest a novel feed-forward mechanism by which HMGCS2 could regulate adaptive metabolic responses during fasting. This mechanism could be physiologically relevant, because fasting-mediated induction of liver FGF<em>21</em> was dependent on SirT1 activity in vivo.

BACKGROUND

Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling.

RESULTS

Analysis was performed on mice subjected to 1, 7 and <em>21</em> days of hypoxia (FiO2 = 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and <em>21</em>, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the <em>growth</em> mediators PDGF-B, TGF-beta, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and <em>21</em> differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the <em>21</em> genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription <em>factors</em>. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media.

CONCLUSIONS

Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.

Skeletal muscle and adipose tissue are the two largest organs in the body. Skeletal muscle is an effector organ, and adipose tissue is an organ that stores energy; in addition, they are endocrine organs that secrete cytokines, namely myokines and adipokines, respectively. Myokines consist of myostatin, interleukin (IL)-8, IL-15, irisin, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, and myonectin; adipokines include leptin, adiponectin, resistin, chemerin, and visfatin. Furthermore, certain cytokines, such as IL-6 and tumor necrosis <em>factor</em>-α, are released by both skeletal muscle and adipose tissue and exhibit a bioactive effect; thus, they are called adipo-myokines. Recently, novel myokines or adipokines were identified through the secretomic technique, which has expanded our knowledge on the previously unknown functions of skeletal muscle and adipose tissue and provide a new avenue of investigation for obesity treatment or animal production. This review focuses on the roles of and crosstalk between myokines and adipokines in skeletal muscle and adipose tissue that modulate the molecular events in the metabolic homeostasis of the whole body.

Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived <em>growth</em> <em>factor</em>-BB and transforming <em>growth</em> <em>factor</em>-β1, in the presence of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal <em>fibroblasts</em>, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-<em>21</em>). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF<em>21</em> during chronic food restriction is associated with reduced bone <em>growth</em> and <em>growth</em> hormone (GH) insensitivity. In light of the inhibitory effects on <em>growth</em> plate chondrogenesis mediated by other FGFs, we hypothesized that FGF<em>21</em> causes <em>growth</em> inhibition by acting directly at the long bones' <em>growth</em> plate. We first demonstrated the expression of FGF<em>21</em>, FGFR1 and FGFR3 (two receptors known to be activated by FGF<em>21</em>) and β-klotho (a co-receptor required for the FGF<em>21</em>-mediated receptor binding and activation) in fetal and 3-week-old mouse <em>growth</em> plate chondrocytes. We then cultured mouse <em>growth</em> plate chondrocytes in the presence of graded concentrations of rhFGF<em>21</em> (0.01-10 μg/ml). Higher concentrations of FGF<em>21</em> (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF<em>21</em> in a concentration-dependent manner. In addition, FGF<em>21</em> reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF<em>21</em> on GH action were all prevented, supporting a specific effect of this <em>growth</em> <em>factor</em> in chondrocytes. Our findings suggest that increased expression of FGF<em>21</em> during food restriction causes <em>growth</em> attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the <em>growth</em> plate. In addition, high concentrations of FGF<em>21</em> may directly suppress <em>growth</em> plate chondrocyte proliferation and differentiation.