Dysregulation of the glutamatergic system has been implicated not only in the treatment of major depressive disorder (MDD), but also in the excitotoxic effects of stress and anxiety on the prefrontal cortex, which may precede the onset of a depressive episode. Our previous studies demonstrate marked deficits in prominent postsynaptic proteins involved in glutamate neurotransmission in the prefrontal cortex (PFC), Brodmann's area 10 (BA 10) from subjects diagnosed with major depressive disorder (MDD). In the same group of subjects we have identified deficits in expression and phosphorylation level of key components of the mammalian target of rapamycin (mTOR) signaling pathway, known to regulate translation initiation. Based on our previous findings, we have postulated that glutamate-dependent dysregulation of mTOR-initiated protein synthesis in the PFC may underlie the pathology of MDD. The aim of this study was to use the NanoString nCounter System to perform analysis of genes coding for glutamate transporters, glutamate metabolizing enzymes, neurotrophic <em>factors</em> and other intracellular signaling markers involved in glutamate signaling that were not previously investigated by our group in the PFC BA 10 from subjects with MDD. We have analyzed a total of 200 genes from <em>16</em> subjects with MDD and <em>16</em> healthy controls. These are part of the same cohort used in our previous studies. Setting our cutoff p-value≤0.01, marked upregulation of genes coding for mitochondrial glutamate carrier (GC1; p=0.0015), neuropilin 1 (NRP-1; p=0.0019), glutamate receptor ionotropic N-methyl-d-aspartate-associated protein 1 (GRINA; p=0.0060), and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR-1; p=0.010) was identified. No significant differences in expression of the remaining 196 genes were observed between MDD subjects and controls. While upregulation of FGFR-1 has been previously shown in MDD; abnormalities in GC-1, GRINA, and NRP-1 have not been reported. Therefore, this postmortem study identifies GC1, GRINA, and NRP-1 as novel <em>factors</em> associated with MDD; however, future studies will be needed to address the significance of these genes in the pathophysiology of depression and antidepressant activity.

BACKGROUND

In this Phase 4 international study, efficacy and safety of paricalcitol-centred therapy were compared with that of cinacalcet-centred therapy for the treatment of chronic kidney disease (CKD)-associated secondary hyperparathyroidism (SHPT) in patients undergoing haemodialysis (ClinicalTrials.gov identifier NCT00977080).

METHODS

Patients ≥ 18 years of age with Stage 5 CKD and SHPT [intact parathyroid hormone (iPTH) level of 300-800 pg/mL, calcium level of 8.4-10.0 mg/dL and phosphate concentration of ≤ 6.5 mg/dL] who were undergoing haemodialysis were included. Patients were randomized by mode of paricalcitol administration [i.e. intravenous (IV) or oral strata] to receive paricalcitol- or cinacalcet-centred therapy for ≤ 28 weeks. Changes in metabolic markers [total alkaline phosphatase (AP), bone-specific AP and <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23)] and the proportion of patients in each treatment group who achieved an iPTH level of 150-300 pg/mL during Weeks 8, <em>16</em> and 21-28 as a composite value were evaluated.

RESULTS

Compared with cinacalcet-centred therapy, levels of both bone turnover markers were significantly reduced from baseline with IV and oral paricalcitol-centred treatment (P < 0.05 for both dosing strata) at Weeks 8, <em>16</em> and 28. Levels of FGF-23 were increased with paricalcitol versus cinacalcet-centred treatment. A greater proportion of patients receiving paricalcitol-centred therapy achieved target iPTH levels (i.e. 150-300 pg/mL) throughout the study in the IV and oral dosing strata compared with patients receiving cinacalcet-centred treatment.

CONCLUSIONS

In patients with CKD and SHPT undergoing haemodialysis, paricalcitol-centred therapy reduced circulating bone turnover markers and iPTH levels and increased FGF-23 levels compared with cinacalcet-centred treatment.

BACKGROUND

ClinicalTrials.gov identifier NCT00977080.

Potential biomarkers were identified for in vitro sensitivity to the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% <em>growth</em> inhibition [GI(50)] < 1 microM), three had intermediate sensitivity (GI(50) 1-7 microM), and six were resistant (GI(50)>> 7 microM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis-driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E-cadherin (CDH1); transforming <em>growth</em> <em>factor</em>, alpha (TGF-alpha); amphiregulin (AREG); FLJ22662; EGFR; p21-activated kinase 6 (PAK6); glutathione S-transferase Pi (GSTP1); and ATP-binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis-free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF-alpha, CDH1, EGFR, keratin <em>16</em> [KRT<em>16</em>], <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme-linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

Vitamin D deficiency, low levels of fetuin-A, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF-23) are related to vascular calcification, which is associated with cardiovascular disease. We hypothesized that omega-3 fatty acid (FA), which has cardioprotective properties, modifies vitamin D status, fetuin-A, and FGF-23 levels in dialysis patients. In a randomized, open-label, controlled study, a total of 47 patients treated with dialysis for at least 1 year were randomized to treatment for 6 months with omega-3 FAs (Omacor, 3 g/d; Pronova, Sandefjord, Norway) or a control group. Levels of fetuin-A and FGF-23 were measured by enzyme-linked immunoassay, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured by radioimmunoassay. The mean age of the enrolled patients was 57.4 ± 10.4 years, and mean dialysis duration was 46.5 ± 28.1 months. Twenty-seven hemodialysis patients and <em>16</em> peritoneal dialysis patients finished this trial. After 6 months, the levels of 1,25-dihydroxyvitamin D and fetuin-A were significantly increased in the group taking the omega-3 FA supplement compared with baseline. Levels of calcium, phosphorous, parathyroid hormone, 25-hydroxyvitamin D, FGF-23, and lipid profiles were not significantly changed in the omega-3 FA-supplemented group after 6 months compared with baseline. The erythrocyte membrane contents of eicosapentaenoic acid and docosahexaenoic acid were significantly increased, and oleic acid content was significantly decreased in the omega-3 FA-supplemented group after 6 months compared with baseline. Regarding vascular calcification and cardiovascular disease, omega-3 FA supplementation may have a clinical benefit caused by activating vitamin D, increasing fetuin-A levels, and modifying erythrocyte membrane FA contents in dialysis patients.

BACKGROUND

TNP-470 (AGM-1470) is a potent inhibitor of angiogenesis with potential therapeutic applications in neoplastic and angio-proliferative diseases. This study evaluated its effect on cutaneous wound healing in a murine dorsal excisional wound model.

METHODS

Full-thickness wounds (1.60 cm2) were created on the dorsum of homozygous/hairless mice (7 to 9 weeks). Wound areas were measured on alternate days for <em>16</em> days. Experimental groups consisted of (1) TNP-470 administered in doses of 0.05, 0.5, and 5.0 mg/kg on Days 0, 2, and 4 or Days 0 through 6; (2) TNP-470 (5.0 mg/kg) coadministered with minocycline (4.0 and 10 mg/kg) on Days 0, 2, and 4; and (3) TNP-470 (5.0 mg/kg on Days 0, 2, and 4) coadministered with topical basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) 1. 0 microg/wound on Days 0, 1, and 2. Hematoxylin and eosin staining was used to compare experimental and control wounds.

RESULTS

TNP-470 administration significantly decreased wound healing in a dose-dependent manner versus controls (P <.05). The 5.0 mg/kg concentration yielded the greatest effect by maintaining an average wound area 20.4% greater than controls and a marked delay in wound healing on H&E staining. Alternate-day dosing was as effective as consecutive day administration. Minocycline did not augment the wound healing inhibition of TNP-470. Coadministration of TNP-470 and bFGF eliminated any rate-altering effect of TNP-470 upon wound healing and resulted in wound areas similar to controls.

CONCLUSIONS

Therapy with TNP-470 induces a significant delay in murine cutaneous wound healing. This effect may be exploited for use in situations where wound healing is excessive and debilitating. Topical application of bFGF can overcome TNP-470-induced wound healing inhibition.

OBJECTIVE

Pulmonary remodeling is a well recognized consequence of heart failure (HF). However, the cellular and molecular mechanisms orchestrating the structural alterations of the lungs in HF are poorly understood. We have previously reported induction of the profibrotic peptide connective tissue growth factor (CTGF) in myocardial tissue of rats with HF, suggesting a role of CTGF during myocardial remodeling. The aim of the present study was to explore the potential role of CTGF in pulmonary remodeling in HF.

METHODS

Pulmonary tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Real-time quantitative RT-PCR was employed to investigate mRNA levels. The cellular distribution of CTGF was analysed by immunohistochemistry.

RESULTS

Seven days after induction of myocardial infarction (MI) and HF in rats we found 2.3-fold and 1.9-fold increase of pulmonary transforming growth factor-beta1 and procollagen alpha1(I) mRNA levels, respectively, and typical morphological characteristics of pulmonary remodeling including interstitial fibrosis and medial thickening of pulmonary arteries. Pulmonary CTGF mRNA levels were substantially elevated in HF rats compared to sham-operated rats (4-fold; P<0.05) and corresponded with similar increase (3-fold; P<0.05) of pulmonary CTGF protein contents. Immunohistochemical analysis revealed increased pulmonary anti-CTGF immunoreactivity in HF, with immunostaining predominantly localized to alveolar macrophages and interstitial fibroblasts. Isolated alveolar macrophages from HF rats demonstrated substantial induction of CTGF mRNA expression (16-fold; p<0.05). Interestingly, platelets caused robust induction of CTGF mRNA expression in alveolar macrophages upon co-culture in vitro.

CONCLUSIONS

Pulmonary CTGF was substantially increased in parallel with pulmonary remodeling in rats with HF. Our data indicate that alveolar macrophages are a major source of increased pulmonary CTGF in HF and that CTGF may be a player in the profibrotic mechanisms associated with HF.

Human tubal epithelial cells in primary culture were transfected with simian virus 40 (SV40) large T antigen plasmid, and an immortalized ciliated cell line, named as NT/T-S, was established without crisis. Transmission electron microscopy proved that NT/T-S cells had cilia, microvilli, junctional complexes, rough endoplasmic reticula, free ribosomes and microtubules. NT/T-S cells were evaluated preliminarily on the basis of co-culture study using surplus embryos at the 4- to 8-cell stage in our IVF and embryo transfer programme. All of the 133 embryos had>>/=10% fragments (based on the surface area) and were unworthy of cryopreservation. Up to 57% (<em>16</em>/28) of the embryos with 10-30% fragments reached the blastocyst stage by co-culture. In contrast, blastocyst formation was observed in <10% of the control embryos, some of which were co-cultured with NFL/T cells (the immortalized human fetal liver epithelial cells) (1/<em>16</em>), and the others were incubated with the co-culture medium alone (1/18). Various cytokines/<em>growth</em> <em>factors</em> such as leukaemia inhibitory <em>factor</em> (LIF), interleukin (IL)-6, IL-8 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were secreted by NT/T-S cells as well as by the tubal epithelial cells in primary culture. The establishment of a ciliated cell line will provide a valuable resource for the further studies of the Fallopian tube in the early events of pregnancy.

OBJECTIVE

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

METHODS

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

RESULTS

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

CONCLUSIONS

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

BACKGROUND

Sodium glucose cotransporter-2 (SGLT2) inhibitors are the most recently approved class of drugs for type 2 diabetes and provide both glycemic efficacy and cardiovascular risk reduction. A number of safety issues have been identified, including treatment-emergent bone fractures. To understand the overall clinical profile, these safety issues must be balanced against an attractive efficacy profile. Our study was designed to investigate pathophysiological mechanisms mediating treatment-emergent adverse effects on bone health.

METHODS

We conducted a single-blind randomized crossover study in hospitalized healthy adults (n = 25) receiving either canagliflozin (300 mg/d) or placebo for 5 days. The primary end-point was the drug-induced change in AUC for plasma intact fibroblast growth factor 23 (FGF23) immunoactivity between 24 and 72 hours.

RESULTS

Canagliflozin administration increased placebo-subtracted mean levels of serum phosphorus (+16%), plasma FGF23 (+20%), and plasma parathyroid hormone (PTH) (+25%), while decreasing the level of 1,25-dihydroxyvitamin D (-10%). There was substantial interindividual variation in the magnitude of each of these pharmacodynamic responses. The increase in plasma FGF23 was correlated with the increase in serum phosphorus, and the decrease in plasma 1,25-dihydroxyvitamin D was correlated with the increase in plasma FGF23.

CONCLUSIONS

Canagliflozin induced a prompt increase in serum phosphorus, which triggers downstream changes in FGF23, 1,25-dihydroxyvitamin D, and PTH, with potential to exert adverse effects on bone health. These pharmacodynamic data provide a foundation for future research to elucidate pathophysiological mechanisms of adverse effects on bone health, with the objective of devising therapeutic strategies to mitigate the drug-associated fracture risk.

BACKGROUND

ClinicalTrial.gov (NCT02404870).

BACKGROUND

Supported by the Intramural Program of NIDDK.

Chronic wounds, such as ulceration of the lower limb, represent a significant clinical challenge in today's ageing society. With the aim of identifying improved therapeutics, we have previously described a bioresponsive, dextrin-recombinant human epidermal <em>growth</em> <em>factor</em> conjugate (dextrin-rhEGF), that (i) protects rhEGF against proteolytic degradation by human chronic wound fluid; and (ii) mediates rhEGF release by α-amylase, capable of stimulating increased proliferation/migration in normal dermal and chronic wound <em>fibroblasts</em>; and keratinocytes, in vitro. The aim of this study was to extend these findings, by investigating the effects of dextrin-rhEGF on wound healing in the (db/db) diabetic mouse, a widely used in vivo model of delayed wound healing. Standardised, full-thickness excisional wounds, created in the dorsal flank skin, were treated topically with succinoylated dextrin (50 μg/mL), rhEGF (10 μg/mL) or dextrin-rhEGF (1 or 10 μg/mL). Treatments were applied immediately after injury and subsequently on post-wounding, days 3 and 8. Wound healing was assessed macroscopically, in terms of initiation of neo-dermal tissue deposition and wound closure (including wound contraction and re-epithelialisation), over a <em>16</em> day period. Wound healing was assessed histologically, in terms of granulation tissue formation/maturity; cranio-caudal wound contraction and wound angiogenesis (CD31 immuno-staining), using tissues harvested at day <em>16</em>. Blood samples were also analysed for α-amylase and rhEGF concentrations. In this established impaired wound healing model, the topically-applied dextrin-rhEGF significantly accelerated wound closure and neo-dermal tissue formation at the macroscopic level; and significantly increased granulation tissue deposition and angiogenesis at the histological level (p<0.05), relative to untreated, succinoylated dextrin and rhEGF alone controls. Overall, these findings support the further development of bioresponsive polymer conjugates, for tissue repair.

Insulin-like <em>growth</em> <em>factor</em>-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital <em>fibroblasts</em> in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these <em>fibroblasts</em> produce RANTES and IL-<em>16</em>, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD <em>fibroblasts</em> where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital <em>fibroblasts</em>. In GD <em>fibroblasts</em>, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD <em>fibroblasts</em>. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD <em>fibroblasts</em>. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived <em>fibroblasts</em>. Nuclear IGF-1R may play a role in disease pathogenesis.

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

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal <em>growth</em> <em>factor</em> (EGF), resulted in stimulation of <em>growth</em> from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The <em>growth</em>-inhibitory effect of exogenous transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (<em>16</em> kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the <em>16</em> kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their <em>growth</em> regulation at the autocrine level.

Transforming <em>growth</em> <em>factor</em> beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal <em>growth</em> <em>factor</em> (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other <em>growth</em> <em>factors</em>. It also modulates the expression of cellular products. TGF-beta causes <em>fibroblasts</em> to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a <em>16</em>-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

Diastolic dysfunction (DD), a hallmark of obesity and primary defect in heart failure with preserved ejection fraction, is a predictor of future cardiovascular events. We previously reported that linagliptin, a dipeptidyl peptidase-4 inhibitor, improved DD in Zucker Obese rats, a genetic model of obesity and hypertension. Here we investigated the cardioprotective effects of linagliptin on development of DD in western diet (WD)-fed mice, a clinically relevant model of overnutrition and activation of the renin-angiotensin-aldosterone system.

Female C56Bl/6 J mice were fed an obesogenic WD high in fat and simple sugars, and supplemented or not with linagliptin for <em>16</em> weeks.

WD induced oxidative stress, inflammation, upregulation of Angiotensin II type 1 receptor and mineralocorticoid receptor (MR) expression, interstitial fibrosis, ultrastructural abnormalities and DD. Linagliptin inhibited cardiac DPP-4 activity and prevented molecular impairments and associated functional and structural abnormalities. Further, WD upregulated the expression of TRAF3IP2, a cytoplasmic adapter molecule and a regulator of multiple inflammatory mediators. Linagliptin inhibited its expression, activation of its downstream signaling intermediates NF-κB, AP-1 and p38-MAPK, and induction of multiple inflammatory mediators and growth factors that are known to contribute to development and progression of hypertrophy, fibrosis and contractile dysfunction. Linagliptin also inhibited WD-induced collagens I and III expression. Supporting these in vivo observations, linagliptin inhibited aldosterone-mediated MR-dependent oxidative stress, upregulation of TRAF3IP2, proinflammatory cytokine, and growth factor expression, and collagen induction in cultured primary cardiac fibroblasts. More importantly, linagliptin inhibited aldosterone-induced fibroblast activation and migration.

Together, these in vivo and in vitro results suggest that inhibition of DPP-4 activity by linagliptin reverses WD-induced DD, possibly by targeting TRAF3IP2 expression and its downstream inflammatory signaling.

Obesity is a serious and costly issue to the medical welfare worldwide. Probiotics have been suggested as one of the candidates to resolve the obesity-associated problems, but how they combat obesity is not fully understood. Herein, we investigated the effects of Lactobacillus reuteri 263 (L. reuteri 263) on antiobesity using four groups of Sprague-Dawley rats (n=10/group), namely, C (normal diet with vehicle treatment), HE [high-energy diet (HED) with vehicle treatment], 1X (HED with 2.1×109 CFU/kg/day of L. reuteri 263) and 5X (HED with 1.05×1010 CFU/kg/day of L. reuteri 263), for 8 weeks. L. reuteri 263 improved the phenomenon of obesity, serum levels of proinflammatory <em>factors</em> and antioxidant enzymes. More importantly, L. reuteri 263 increased oxygen consumption in white adipose tissue (WAT). The mRNA expressions of thermogenesis genes uncoupling protein-1, uncoupling protein-3, carnitine palmitoyltransferase-1 and cell death-inducing DFFA-like effector-a were up-regulated in WAT of the 5X group. Moreover, L. reuteri 263 might induce browning of WAT due to the higher mRNA levels of browning-related genes peroxisome proliferator-activated receptor-γ, PR domain containing-<em>16</em>, Pparγ coactivator-1α, bone morphogenetic protein-7 and <em>fibroblast</em> <em>growth</em> <em>factor</em>-21 in the 1X and 5X groups compared to the HE group. Finally, L. reuteri 263 altered the expressions of genes involved in glucose and lipid metabolisms in WAT, including increasing the levels of glucose transporter type 4 and carbohydrate-responsive element-binding protein and decreasing the expression of Acetyl-CoA carboxylase-1. The results suggest that L. reuteri 263 may treat obesity through energy metabolism remodeling of WAT in the high-energy-diet-induced obese rats.

Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of <em>fibroblasts</em> in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the <em>16</em> U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, <em>growth</em> <em>factor</em> and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

BACKGROUND

Patients with chronic kidney disease (CKD) develop renal osteodystrophy with alterations in bone turnover, mineralization, and volume (TMV). A specific skeletal complication in children is growth impairment, which currently is treated by recombinant human growth hormone (rhGH). The effects on bone material properties are poorly understood. This study assesses the effects of rhGH treatment on bone matrix mineralization.

METHODS

Observational study.

METHODS

18 short children and adolescents (aged 3.6-16 years) with CKD on dialysis therapy.

METHODS

rhGH treatment for 1 year.

RESULTS

Tetracycline-labeled bone biopsy classified according to the TMV system.

METHODS

Bone mineralization density distribution (BMDD) was evaluated by quantitative backscattered electron imaging in trabecular and cortical compartments. Additional data for patients' height and biochemical bone serum parameters were obtained.

RESULTS

Prior to rhGH treatment, our cohort showed low bone turnover and high mineralization densities versus reference data: Ca(mean) (weighted mean calcium content) in cancellous bone, +3.3% (P = 0.04); Ca(mean) in cortical bone, +6.7% (P < 0.001); Ca(peak) (mode of the BMDD) in cancellous bone, +5.0% (P < 0.001); Ca(peak) in cortical bone, +8.2% (P < 0.001); Ca(width) (heterogeneity in mineralization), no significant difference for cancellous (P = 0.2) and cortical (P = 0.1) bone; Ca(high) (portion of fully mineralized bone) in cancellous bone, 5-fold greater (P < 0.001); Ca(high) in cortical bone, 14-fold greater (P < 0.001); Ca(low) (portion of low mineralized bone) in cancellous bone, +23.9% (P = 0.02); Ca(low) in cortical bone, -22.2% (P = 0.05). After rhGH treatment, height increased by 9.1 cm (P < 0.001) and bone turnover indices to normal values or beyond. Matrix mineralization was lesser and more heterogeneous compared to baseline: Ca(width) for cancellous bone, +15.3% (P < 0.001); Ca(width) for cortical bone, +34.1% (P < 0.001). Ca(mean), Ca(peak), and Ca(high) for cancellous bone and Ca(mean) and Ca(peak) for cortical bone were no longer significantly different from reference data. Ca(high) for cortical bone dramatically decreased after treatment but was still substantially greater than reference data.

CONCLUSIONS

Low case number per TMV subgroup, no measurements of fibroblast growth factor 23.

CONCLUSIONS

Children and adolescents with CKD and growth deficiency are at risk of having low bone turnover. rhGH treatment improves height and concomitantly bone modeling/remodeling, which appears beneficial for bone matrix mineralization.

BACKGROUND

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

RESULTS

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

CONCLUSIONS

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

BACKGROUND

Esophageal squamous cell carcinoma (ESCC) is an important cause of cancer-related death worldwide. To improve prognoses in patients with ESCC, we evaluated the potential of transforming growth factor-beta-induced protein (TGFBI), which is overexpressed in ESCC, as a therapeutic candidate.

METHODS

We examined the clinical significance of TBFBI in 102 ESCC samples using real-time RT-PCR. Immunohistochemical studies were conducted to examine the localization of TGFBI. Knockdown of TGFBI in cocultured fibroblasts was performed to determine the roles of TGFBI in migration and invasion.

RESULTS

The level of TGFBI in ESCC tissues was higher than that in normal tissues. The high TGFBI expression group (n = 16) had higher TGFB1 expression and more frequent hematogenous recurrence than the low-expression group (n = 86). High TGFBI expression was an independent prognostic factor in patients with ESCC. TGFBI was mainly localized in stromal cells of ESCC. Moreover, suppression of TGFBI in fibroblasts inhibited the migration and invasion capacity of TE8 ESCC cells.

CONCLUSIONS

High TGFBI expression in ESCC tissues could be a powerful biomarker of poor prognosis and hematogenous recurrence. TGFBI in stromal cells might be a promising molecular target for ESCC treatment.

BACKGROUND

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

METHODS

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

RESULTS

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

CONCLUSIONS

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

BACKGROUND

Lactate dehydrogenase A (LDHA) and Pyruvate Kinase M2 (PKM2) are important enzymes of glycolysis. Both of them can be phosphorylated and therefore regulated by Fibroblast growth factor receptor 1 (FGFR1). While phosphorylation of LDHA at tyrosine10 leads to tetramerization and activation, phosphorylation of PKM2 at tyrosine105 promotes dimerization and inactivation. Dimeric PKM2 is found in the nucleus and regulates gene transcription. Up-regulation and phosphorylation of LDHA and PKM2 contribute to faster proliferation under hypoxic conditions and promote the Warburg effect.

METHODS

Using western blot and SYBR Green Real time PCR we investigated 77 thyroid tissues including 19 goiter tissues, 11 follicular adenomas, 16 follicular carcinomas, 15 papillary thyroid carcinomas, and 16 undifferentiated thyroid carcinomas for total expression of PKM2, LDHA and FGFR1. Additionally, phosphorylation status of PKM2 and LDHA was analysed. Inhibition of FGFR was performed on FTC133 cells with SU-5402 and Dovitinib.

RESULTS

All examined thyroid cancer subtypes overexpressed PKM2 as compared to goiter. LDHA was overexpressed in follicular and papillary thyroid cancer as compared to goiter. Elevated phosphorylation of LDHA and PKM2 was detectable in all analysed cancer subtypes. The highest relative phosphorylation levels of PKM2 and LDHA compared to overall expression were found in undifferentiated thyroid cancer. Inhibition of FGFR led to significantly decreased phosphorylation levels of PKM2 and LDHA.

CONCLUSIONS

Our data shows that overexpression and increased phosphorylation of PKM2 and LHDA is a common finding in thyroid malignancies. Phospho-PKM2 and Phospho-LDHA could be valuable tumour markers for thyroglobulin negative thyroid cancer.

OBJECTIVE

Venous ulcer fibroblasts demonstrate decreased proliferative responses to growth factor stimulation, suggesting cellular senescence. However, the role of chronic venous insufficiency (CVI) disease progression and extracellular matrix (ECM) proteins in agonist-induced cellular proliferation is ill-defined. We hypothesize that CVI-induced fibroblast proliferative resistance to growth factors worsens with disease progression and is regulated by the composition of ECM.

METHODS

Fibroblast explants were isolated from biopsy specimens from two patients without CVI and 16 patients with CVI of the lower calf (LC) and lower thigh (LT) and stratified according to CEAP disease severity: non-CVI (NC; n = 2), class 2-3 (n = 5), class 4 (n = 5), class 5 (n = 3), and class 6 (n = 3). Proliferation experiments were standardized with a neonatal foreskin fibroblast cell line (HS68). A 10-day course and dose response experiment with 0, 0.5, 1.0, 2.5, 5, 10, and 20 ng/mL of transforming growth factor-beta(1) (TGF-beta(1)) demonstrated maximal cell proliferation at 5 ng/mL of TGF-beta(1) on day 4. Under these conditions, CVI dermal fibroblasts were challenged with and without TGF-beta(1) and evaluated for proliferative responses on plates coated with polystyrene, collagen, and fibronectin.

RESULTS

No differences in unstimulated proliferation were observed in LT and LC fibroblasts from patients with class 2-3 disease and LT fibroblasts from patients with class 4 and 5 disease, compared with NC and HS68 cells. LC fibroblasts from patients with class 4 disease (P <.05) and class 5 disease (P <.001), and LC (P <.001), and LT fibroblasts from patients with class 6 disease (P <.001) proliferated to a lesser degree than did NC and HS68 cells. The diminished proliferation observed in class 4 LC cells was reversible with TGF-beta(1) stimulation (P <.004); however, class 5 and class 6 LC and LT fibroblasts did not respond to stimulation with TGF-beta(1). Collagen increased proliferation of HS68 cells with (P <.05) and without (P <.01) TGF-beta(1), compared with cells grown on polystyrene, but did not increase proliferative responses in NC or CVI fibroblasts with and without TGF-beta(1). Similarly, fibronectin increased proliferation of HS68 cells (P <.05) compared with cells grown on polystyrene, but did not alter proliferation in CVI fibroblasts. Fibronectin did seem to inhibit TGF-beta(1)-induced proliferation observed in class 4 LC cells.

CONCLUSIONS

These data indicate that clinical disease progression correlates with cellular dysfunction. Fibroblasts from patients with class 2-3 disease retain their unstimulated and agonist- induced proliferative capacity, compared with NC and HS68 cells. The onset of inflammatory skin changes (class 4 and class 5 disease) diminishes agonist-induced proliferation, and ulcer formation (class 6 disease) severely inhibits it. In addition, the composition of ECM does not affect TGF-beta(1)-induced proliferation of fibroblasts in CVI.

Epithelio-mesenchymal interactions are active during the development of the root of the tooth and are regulated by a variety of <em>growth</em> <em>factors</em>, such as <em>fibroblast</em> <em>growth</em> <em>factors</em>. FGF-2, 3, 4, and 8 have all been shown to play a role in the development of the crown of the tooth, but less is known about the <em>factors</em> that govern root formation, particularly FGF-2. The aim of this study was thus to elucidate the spatial and temporal expression of FGF-2 in the root of the developing tooth, as this <em>growth</em> <em>factor</em> is believed to be a mediator of epithelio-mesenchymal interactions. Parasagittal sections of the maxillary and mandibular arches of post-natal mice were utilized and the roots of the molar teeth were studied. Immunocytochemistry utilizing an antibody to FGF-2 was performed on sections of teeth at various stages of development. Intense immunostaining for FGF-2 was observed in differentiating odontoblasts at the apical end of the tooth and in the furcation zone of the developing root at all the stages examined. FGF-2 localization was also observed in cementoblasts on post-natal days <em>16</em>, 20 and 24. The pattern of localization of FGF-2 in the developing root suggests that this <em>growth</em> <em>factor</em> may participate in the signaling network associated with root development.