There has been obtained and is being maintained in vitro for over 12 years a stable cellular line DAPT, derived from a dedifferentiated human astrocytoma. In monolayer cultures the cells have passed 270 times. During <em>22</em> month (63 passages) the cells had a <em>fibroblast</em>-like shape, their <em>growth</em> having thereafter assumed an epithelioid character. On being impregnated with chlorous gold after Ramon-and-Kachal the cellular cytoplasma is stained purple-violet. In the periods of the <em>fibroblast</em>-like and epithelioid <em>growth</em> the time of the population doubling was 145 and 74 hours, respectively. The mitotic index amounted to 18%. When investigated during the 3d (106th passage) and 11th (250th passage) years of its cultivation the population consisted largely of cells containing 60-65 chromosomes. The carotype of the DAPT cells retained the main traits common to the structure of the human caryotype. After 7-9 months of cultivation (16-25th passages) the DAPT cells demonstrated the presence therein of an active glutamineketoacidic aminotransferase. Further on the activity of the enzyme declined. <em>Factors</em> influencing the changeability of the stable cellular DAPT line are discussed.

OBJECTIVE

The purpose of this study was to evaluate the oral health-related quality of life of patients treated with implant-supported mandibular overdentures and to compare the attachment systems used.

METHODS

The presence of myofibroblasts as well as transforming <em>growth</em> <em>factor</em>-beta1 was examined in twenty cases of fibrous epulis and <em>22</em> ossifying fibrous epulis, using immunohistochemistry.

RESULTS

Myofibroblasts positive for alpha smooth muscle actin and vimentin but negative to desmin were found in 20% and 45% in fibrous epulis and ossifying fibrous epulis, respectively. Myofibroblasts were distributed in areas with and without inflammatory infiltration and their presence in inflammatory areas was not related with the degree of inflammatory infiltration. A percentage of 21 - 60% of fibroblasts and chronic inflammatory cells expressed transforming growth factor-beta1 in all cases.

CONCLUSIONS

These data suggest that transforming growth factor-beta1 and myofibroblasts contribute to the formation of collagenous connective tissue in fibrous epulis and ossifying fibrous epulis. Myofibroblasts are mainly presented in ossifying fibrous epulis than in fibrous epulis. It seems to be no relationship between the presence of myofibroblasts and the degree of inflammatory infiltration of the lesions.

Cellular Infiltrate as well as class I and II HLA molecule expression, on <em>22</em> nasal polyps and on 12 samples of corresponding hypsilateral mucous membrane were studied by means of immuno-histological methods. These nasal polyps were classified according to their histopathological structure. Five polyps, with a fibrous connective core infiltrated by cells of the monocyte-macrophage lineage, were classified mixed. The remaining seventeen polyps were characterized by the presence of central oedematous connective tissue infiltrated almost exclusively by eosinophils and either contained (glandular type) or did not contain (oedematous type) glands. A comparative study of different types of nasal polyps and corresponding hypsilateral nasal mucous membranes was carried out on atopic and non-atopic patients. No correlation between atopic status and polyp presence or polyp typology was found. On the other hand, different polyp types appear to have a structural correlation with the corresponding hypsilateral mucous membrane regarding infiltrate cell type, oedematous or fibrous connective tissue presence and expression of on HLA antigen positivity pattern. The characteristic histological structure of hypsilateral mucous membranes in patients with different types of polyps appeared to be brought about by a multifactorial etiology involving mucosal hyperreactivity. Lastly, both polyps and parapolypal nasal mucous membranes were found to be infiltrated mainly in the peripheral subepithelial connective tissue by lymphocytes (55%) as well as by other leukocyte types. The presence of <em>growth</em> <em>factors</em> capable of enhancing an increase of <em>fibroblasts</em>, endothelial cells, together with focal distrupture on the basal membrane, might well be a general mechanism responsible for polyp sprouting.

Background: Angiogenesis disturbances are common in women with polycystic ovary syndrome (PCOS). Vitamin E has antiangiogenic properties. Data on the effects of vitamin E on angiogenesis in PCOS is limited, so the current study was conducted to evaluate its effects on angiogenic indices in PCOS patients.

<strong class="sub-title"> Materials and methods: </strong> This randomized, double-blind, placebo-controlled trial was performed on 43 women aged 20-40 years, diagnosed with PCOS (Rotterdam criteria). It was performed at the referral clinic affiliated to Tabriz University of Medical Sciences, Tabriz, Iran, from April 2017 to September 2017. Patients were randomly assigned into two groups to receive either 400 IU/day vitamin E -as alpha tocopheryl acetate- (n=<em>22</em>) or placebo (n=21), for 8 weeks. Anthropometric, and angiogenic parameters including body weight, fat mass and fat free mass, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), angiopoietin-1 (Ang-1), and angiopoietin- 2 (Ang-2) were measured by standard methods at the beginning and at the end of study. Statistical Package for Social Science version 25 was used for statistical analysis and P<0.05 were considered significant.

Results: After adjusting for potential confounders, we observed that vitamin E supplementation significantly reduced body weight, fat mass, Ang-1, Ang-1/Ang-2 ratio and VEGF (P<0.01). We did not observe any considerable effect for vitamin E on Ang-2 level or bFGF.

Conclusion: Vitamin E supplementation for 8 weeks in the PCOS women had beneficial effects on body weight, Ang- 1, Ang-1/Ang-2 ratio, and VEGF level (Registration number: IRCT201610193140N18).

Keywords: Angiopoietins; Basic Fibroblast Growth Factor; Polycystic Ovary Syndrome; Vascular Endothelial Growth Factor; Vitamin E.

OBJECTIVE

To study the expression changes of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and bone morphogenetic protein 2 (BMP-2) in femoral neck fracture, traumatic, and non-traumatic avascular necrosis of femoral head (ANFH), and to study the relationship between the expressions of VEGF, bFGF, BMP-2 mRNA and bone mass so as to explore the pathogenesis of ANFH and provide the experimental basis for individual treatment of ANFH.

METHODS

Femoral head specimens were obtained from 59 donors undergoing total hip replacement, including 22 cases of traumatic ANFH (group A, 13 cases of Ficat stage III and 9 cases of Ficat stage IV), 19 cases of non-traumatic ANFH (group B, 11 cases of Ficat stage III and 8 cases of Ficat stage IV; 10 cases of steroid-induced ANFH, 7 cases of alcoholic ANFH, and 2 cases of unexplained ANFH), and 18 cases of fresh femoral neck fracture (group C). There was no significant difference in the general data among 3 groups (P>> 0.05). The bone mineral density (BMD) at weight-bearing area of the femoral head was measured with dual energy X-ray absorptiometry. The pathological changes were observed by using optical microscope and scanning electron microscope. The percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The expressions of VEGF, bFGF, and BMP-2 mRNA in femoral head were detected by use of in-situ hybridization technique.

RESULTS

The BMD in groups A and B were significantly lower than that in group C (P < 0.05), and there was significant difference between group A and group B (P < 0.05). In the necrosis area of groups A and B, the bone trabecula was rarefactive and not of integrity, with a great number of empty bone lacuna. In healthy area, more fiber hyperplasia was observed in group A, the proliferated and hypertrophic fat cells in the medullary cavity in group B. Scanning electron microscope showed that many osteocytes underwent fatty degeneration and necrosis, and that the proliferation of fat cells in bone matrix was observed in groups A and B. While in group C, the femoral head had intact articular cartilage and intact bone trabeculae, and osteocytes were clearly seen. The percentage of empty bone lacuna was significantly higher (P < 0.05) and the percentage of trabecular bone area was significantly lower (P < 0.05) in groups A and B than group C; and there was significant difference in the percentage of empty bone lacuna between groups A and B (P < 0.05). The expressions of VEGF, bFGF, and BMP-2 mRNA were significantly lower in groups A and B than group C (P < 0.05), and the expressions of BMP-2 and bFGF mRNA in group A were significantly higher than those in group B (P < 0.05). There were positive linear correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and the BMD and percentage of trabecular bone area, respectively. While there were significantly negative correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and percentage of empty bone lacuna.

CONCLUSIONS

The repair capacity of local femoral head in traumatic ANFH is stronger than that in non-traumatic ANFH. The expressions of VEGF mRNA, bFGF mRNA, and BMP-2 mRNA decline in traumatic and nontraumatic ANFH.

Background: Sestrin2 (Sesn2) is involved in the maintenance of metabolic homeostasis and aging via modulation of the 5' AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) pathway.

<strong class="sub-title"> Methods: </strong> Wild-type and Sesn2 knockout (KO) mice of the 129/SvJ background were maintained in a pathogen-free authorized facility under a 12-hour dark/light cycle at 20°C-<em>22</em>°C and 50%-60% humidity. Mouse embryonic <em>fibroblasts</em> (MEFs) were prepared from 13.5-day-old embryos derived from Sesn2-KO mice mated with each other.

Results: The MEFs from Sesn2-KO mice showed enlarged and flattened morphologies and senescence-associated β-galactosidase activity, accompanied by an elevated level of reactive oxygen species. These senescence phenotypes recovered following treatment with N-acetyl-cysteine. Notably, the mRNA levels of NADPH oxidase 4 (NOX4) and transforming growth factor (TGF)-β were markedly increased in Sesn2-KO MEFs. Treatment of Sesn2-KO MEFs with the NOX inhibitor diphenyleneiodonium and the TGF-β inhibitor SB431542 restored cell growth inhibited by Sesn2-KO.

Conclusion: Sesn2 attenuates cellular senescence via suppression of TGF-β- and NOX4-induced reactive oxygen species generation and subsequent inhibition of AMPK.

Keywords: NOX4; Reactive oxygen species; Senescence; Sestrin2.

Apert syndrome is a rare genetic disorder that manifests as craniosynostosis, craniofacial and limb dysmorphic features. Mutations in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) gene account for almost all cases. Given the impact it can have throughout life, prenatal management becomes a challenge. A healthy 33-year-old woman, gravida 4, para 0, was referred to routine ultrasound at <em>22</em> weeks of gestation. Atypical cranial morphology with prominent forehead, ocular proptosis, hypertelorism and mitten hands were detected. Genetic investigation revealed an FGFR2 gene mutation (c.755C>G(p.Ser252Trp)), confirming the diagnosis. Magnetic resonance showed brachycephaly, turricephaly and cortical malformation. Following counselling, parents requested medical termination of pregnancy. Macroscopic features were consistent with ultrasound findings. This case emphasises the importance of early diagnosis to provide the best family counselling and prenatal management. A multidisciplinary team, consisting of an obstetrician with ultrasonography experience, a medical geneticist and a fetal pathologist, should conduct these cases.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), <em>fibroblast</em> <em>growth</em> <em>factor</em> 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100ng/ml), FGF16 (10ng/ml) or BMP15 combined with FGF16; and assessed at 0 and <em>22</em> hours of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Keywords: FGF16; Glucose Consumption; IVM; IVM medium; Lactate Production; oocyte DNA fragmentation.

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non-pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO2 , 95% humidity for <em>22</em>-24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO2 incubator for 6-7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real-time polymerase chain reaction using sequence-specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2-cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre-implantation stages of embryo. It can be concluded that FGF-2 plays a significant role in pre-implantation and early development of embryos in sheep.

To determine the mechanism by which <em>fibroblast</em> <em>growth</em> <em>factor</em> 9 (FGF9) alters granulosa (GC) and theca (TC) cell proliferation, cell cycle proteins that regulate progression through G1 phase of the cell cycle, cyclin D1 (CCND1) and cyclin-dependent kinase-4 (CDK4; CCND1's catalytic partner), were evaluated. Ovaries were obtained from a local abattoir, GC were harvested from small (1-5 mm) and large (8-<em>22</em> mm) follicles, and TC were harvested from large follicles. GC and TC were plated in medium containing 10% fetal calf serum followed by various treatments in serum-free medium. Treatment with 30 ng/mL of either FGF9 or IGF1 significantly increased GC numbers and when combined, synergized to further increase GC numbers by threefold. Abundance of CCND1 and CDK4 mRNA in TC and GC were quantified via real-time PCR. Alone and in combination with IGF1, FGF9 significantly increased CCND1 mRNA expression in both GC and TC. Western blotting revealed that CCND1 protein levels were increased by FGF9 in TC after 6 h and 12 h of treatment, but CDK4 protein was not affected. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway inhibitor, U0126, significantly reduced FGF9-induced CCND1 mRNA expression to basal levels. For the first time we show that CCND1 mRNA expression is increased by FGF9 in bovine TC and GC, and that FGF9 likely uses the MAPK pathway to induce CCND1 mRNA production in bovine TC.

The objectives of the present study were (experiment 1) to characterized development and dynamics of the dominant follicles (DF) and the corpus luteum (CL) to determine patterns of two (W2) and three (W3) follicular waves in beef heifers, and (experiment 2) to determine gene expression of <em>growth</em> <em>factors</em> gene expression in follicular cells of W2 and W3 heifer. Twenty-eight Braford heifers were used. Dominant follicular and CL were monitored daily by ultrasonography to identify the development W2 and W3 in heifers. Pre-ovulatory DF were aspirated on day 19 in W2 and on day <em>22</em> in W3 heifers. In W2 and W3, follicular cells (FC) of gene expression of <em>growth</em> differentiation <em>factor</em> 9, bone morphogenetic protein 15 (BMP15), <em>fibroblast</em> <em>growth</em> <em>factor</em> basic, transforming <em>growth</em> <em>factor</em> beta receptor 1, bone morphogenetic protein receptor type IB and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 were evaluated. The regression of the DF of the first follicular wave and the emergency of the DF of the second follicular wave began later in the heifers W2 than in W3 (p = .02 and p < .01). The regression of the CL began earlier in the W2 than in W3 group (p < .01). Gene expression of <em>growth</em> <em>factors</em> and receptors was similar between groups. However, higher relative levels of BMP15 was observed in W2 group (p = .07). Results propose that wave patterns were regulated by the development time of the DF in the first wave and the life of the CL. Furthermore, higher levels of BMP15 could produce shorter life of CL. The present work suggest that ultrasonography associated with molecular assays could be used as an easy and effective tool to characterize follicular wave patterns.

Keywords: beef heifer; follicular cells; follicular waves; growth factors.

Diabetes is the major risk <em>factor</em> for nontraumatic lower extremity amputation (LEA). The role of genetic polymorphisms in predisposing diabetics to impaired wound healing leading to LEA has not been sufficiently explored. We investigated the association between a set of genes belonging to the angiogenesis/wound repair pathway with LEA in the Chronic Renal Insufficiency Cohort, a study of adults with chronic kidney disease (CKD) that includes a subgroup with diabetes. This study was performed on 3,772 Chronic Renal Insufficiency Cohort participants who were genotyped on the ITMAT-Broad-CARe array chip. A total of 1,017 single-nucleotide polymorphisms (SNPs) in <em>22</em> genes belonging to the angiogenesis/would repair pathway were investigated. LEA was determined from patient self-report. The association between genetic variants and LEA status was examined using logistic regression and additive genetic models after stratifying the cohort by race/ethnicity and diabetic status. Unadjusted analyses as well as analyses adjusted for age, sex, estimated glomerular filtration rate, body mass index, peripheral vascular disease, hemoglobin A1c, and population stratification were performed. In non-Hispanic white participants with diabetes, rs11938826 and rs1960669, both intronic SNPs in the gene basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2), were significantly associated with LEA in covariate-adjusted analysis (OR: 2.83 (95% CI: 1.73, 4.62); p-value: 0.000034; Bonferroni adjusted p-value: 0.0006) and (OR: 2.61 (95% CI: 1.48, 4.61); p-value: 0.00095; Bonferroni adjusted p-value: 0.02). In the same subgroup, rs10883688, an FGF8 SNP of unknown functional effect, was also associated with LEA (OR: 1.72 (95% Confidence Interval: 1.14, 2.6); p-value: 0.00999; Bonferroni adjusted p-value: 0.04). No statistically significant associations were identified in the other ethnic groups. In conclusion, variant/s in FGF2 and FGF8 may predispose diabetics with CKD to LEA. Dysregulation of the FGF2 gene represents an opportunity to understand further, and possibly intervene upon, mechanisms of wound healing in diabetics with CKD.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) belong to a large family comprising <em>22</em> FGF polypeptides that are widely expressed in tissues. Most of the FGFs can be secreted and involved in the regulation of skeletal muscle function and structure. However, the role of fasting on FGF expression pattern in skeletal muscles remains unknown. In this study, we combined bioinformatics analysis and in vivo studies to explore the effect of 24-h fasting on the expression of Fgfs in slow-twitch soleus and fast-twitch tibialis anterior (TA) muscle from male and female C57BL/6 mice. We found that fasting significantly affected the expression of many Fgfs in mouse skeletal muscle. Furthermore, skeletal muscle fibre type and sex also influenced Fgf expression and response to fasting. We observed that in both male and female mice fasting reduced Fgf6 and Fgf11 in the TA muscle rather than the soleus. Moreover, fasting reduced Fgf8 expression in the soleus and TA muscles in female mice rather than in male mice. Fasting also increased Fgf21 expression in female soleus muscle and female and male plasma. Fasting reduced Fgf2 and Fgf18 expression levels without fibre-type and sex-dependent effects in mice. We further found that fasting decreased the expression of an FGF activation marker gene-Flrt2 in the TA muscle but not in the soleus muscle in both male and female mice. This study revealed the expression profile of Fgfs in different skeletal muscle fibre types and different sexes and provides clues to the interaction between the skeletal muscle and other organs, which deserves future investigations.

Recombinant human erythropoietin (rhEpo) can improve human performance, but misuse remains difficult to detect. C-terminal <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (cFGF23) was recently demonstrated to increase following injection of a single high dose rhEpo, but the effect of more frequent low doses is unknown. Using a randomized double-blind placebo-controlled design, we investigated whether two weeks with three weekly subcutaneous injections of 50 IU/kg Eprex (low-dose) or 20 IU/kg Eprex (micro-dose) increase cFGF23 levels compared with saline (placebo) injections in 24 healthy males. Venous blood was sampled at day -3, 0, 1, 3, 11, 14, 18 and 25 of the treatment and analyzed for cFGF23 and erythropoietin concentration ([EPO]). The level of cFGF23 was similar at days -3, 0, 1, 3, 11, 14, 18 and 25 with the low-dose (23±4, 26±5, 23±7, 27±6, 25±8, 24±10, <em>22</em>±5 and 24±7 RU/ml, respectively), micro-dose (23±6, 25±5, 23±8, 28±9, 27±7, 25±9, 25±5 and 23±6 RU/ml, respectively) and placebo (23±6, 24±6, 26±7, 26±6, 31±6, 31±7, 24±4, and 27±8 RU/ml, respectively) treatment. The correlation coefficient between plasma [EPO] and plasma cFGF23 levels was R² =0.01 and insignificant. The results demonstrate that cFGF23 is not sensitive to low doses of subcutaneous rhEpo injections in healthy males.

The 8p11 myeloproliferative syndrome (EMS), a rare myeloproliferative disease, generally progresses rapidly and is characterized by chromosomal translocations of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) gene. The FGFR1 gene is located at chromosome 8p11 and may fuse with distinct partner genes. The breakpoint cluster region gene located at chromosome <em>22</em> is one of these partner genes. The patients' clinical phenotype is primarily dependant on the partner gene that translocates with FGFR1. Of all the available examinations, determination of the chromosome karyotype is most essential for the diagnosis of EMS. In addition, regarding treatment, allogeneic hematopoietic stem cell transplantation is currently the optimal method. The present study presented a case of 8p11 myeloproliferative syndrome with t(8;<em>22</em>)(p11;q11). This represents a total of 8 and 11 chromosomal translocations, which form a BCR/FGFR1 fusion gene in the patient to produce the abnormal karyotype: 46,XY,t(8;<em>22</em>)(p11;q11). The difference between the current case and other EMS incidences is that the patient progressed slowly and the clinical manifestation was similar to chronic myeloid leukemia (CML).

Severe bleeding after cardiothoracic surgery with cardiopulmonary bypass (CPB) is associated with increased morbidity and mortality in adults and children. <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>-2 (FGF-2) and Vascular Endothelial <em>Growth</em> <em>Factor</em>-A (VEGF-A) induce hemorrhage in murine models with heparin exposure. We aim to determine if plasma and urine levels of FGF-2 and VEGF-A in the immediate perioperative period can identify children with severe bleeding after CPB. We performed a prospective, observational biomarker study in 64 children undergoing CPB for congenital heart disease repair from June 2015 - January 2017 in a tertiary pediatric referral center. Primary outcome was severe bleeding defined as ≥ 20% estimated blood volume loss within 24-hours. Independent variables included perioperative plasma and urinary FGF-2 and VEGF-A levels. Analyses included comparative (Wilcoxon rank sum, Fisher's exact, and Student's <i>t</i> tests) and discriminative (receiver operator characteristic [ROC] curve) analyses. Forty-eight (75%) children developed severe bleeding. Median plasma and urinary FGF-2 and VEGF-A levels were elevated in children with severe bleeding compared to without bleeding (preoperative: plasma FGF-2 = 16[10-35] vs. 9[2-13] pg/ml; urine FGF-2= 28[15-76] vs. 14.5[1.5-<em>22</em>] pg/mg; postoperative: plasma VEGF-A = 146[34-379] vs. 53 [0-134] pg/ml; urine VEGF-A = 132 [52-257] vs. 45[0.1-144] pg/mg; all <i>p</i> < 0.05). ROC curve analyses of combined plasma and urinary FGF-2 and VEGF-A levels discriminated severe postoperative bleeding (AUC: 0.73-0.77) with mean sensitivity and specificity above 80%. We conclude that the perioperative plasma and urinary levels of FGF-2 and VEGF-A discriminate risk of severe bleeding after pediatric CPB.

Keywords: Cardiopulmonary bypass surgery; Fibroblast Growth Factor 2; bleeding; heparin.

<AbstractText>Insulin has anabolic effects on skeletal muscle. However, there is limited understanding of the molecular mechanisms underlying this effect in humans. We evaluated whether the skeletal muscle expression of satellite cell activator <em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and muscle <em>growth</em> and differentiation <em>factors</em> are modulated acutely by insulin during euglycemic hyperinsulinemic clamp (EHC).</AbstractText><AbstractText>This is a secondary investigation and analysis of samples obtained from a previously completed trial investigating the effect of testosterone replacement in males with hypogonadotropic hypogonadism and type 2 diabetes. Twenty men with type 2 diabetes underwent quadriceps muscle biopsies before and after 4 hours of EHC.</AbstractText><AbstractText>The infusion of insulin during EHC raised the expression of myogenic <em>growth</em> <em>factors</em>, myogenin (by 72±20%) and myogenin differentiation protein (MyoD; by 81±<em>22</em>%). Insulin reduced the expression of muscle hypertrophy suppressor, myogenic regulatory <em>factor</em> 4 (Mrf4) by 34±14%. In addition, there was an increase in expression of FGF receptor 2, but not FGF2, following EHC. The expression of myostatin did not change.</AbstractText><AbstractText>Insulin has an acute potent effect on expression of genes that can stimulate muscle differentiation and <em>growth</em>.</AbstractText>

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a mitogen that is thought to play a important role in myocyte <em>growth</em>. In previous work, we found the increase of immunoreactivity of bFGF in cardiac myocyte of young SHR and pressure over loaded rat heart, and it suggested that endogenous bFGF which contained high molecular form of bFGF contributes to myocardial hypertrophy. To examine this hypothesis, rhabdomyosarcoma (A 204 cells) derived bFGF was used as wild type bFGF. Extracted bFGF from A 204 cells and nonserum conditioned medium of A 204 cells included high molecular form (<em>22</em>, 24 kd), as well as low molecular (18 kd) bFGF by western blotting, and showed hypertrophic effect in cultured myocardial cells. The hypertrophic effects were indicated in remarkable enlargement of cell size and significant increase in phenylalanine incorporation (x 2.2). The maximum expression of c-myc mRNA by A 204 conditioned medium was observed 30 min after stimulation, and it was also accompanied by an increase of accumulation of alpha skeletal actin mRNA by primer extension. After absorption of heparin binding <em>growth</em> <em>factor</em> from A 204 conditioned medium, it indicated no hypertrophic effects. In conclusion, it is likely that wild type bFGF including high molecular weight form plays a important role in cardiac myocyte <em>growth</em> and hypertrophy.

<AbstractText>We sought to determine whether TRASCET could impact congenital diaphragmatic hernia (CDH).</AbstractText><p><div><b>METHODS</b></div>Twelve pregnant dams received Nitrofen on gestational day 9.5 (E9; term = <em>22</em> days) to induce fetal CDH. Fetuses were divided into three groups: untreated (n = 31) and two groups receiving volume-matched intraamniotic injections of either saline (n = 37) or a suspension of 2 × 10⁶ cells/mL of amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 65) on E17. Animals were euthanized at term. Expression of <em>fibroblast</em> <em>growth</em> <em>factor</em>-10 (FGF-10), vascular endothelial <em>growth</em> <em>factor</em>-A (VEGF-A), and surfactant protein-C (SPC) was quantified by qRT-PCR. Statistical analysis was by the Mann-Whitney U test with Bonferroni adjusted criterion (p ≤ 0.01).</p><AbstractText>Among survivors with CDH (n = 27/133), the TRASCET group showed significant downregulation of FGF-10 and VEGF-A gene expressions compared to the untreated (p < 0.001 for both) and saline groups (p = 0.005 and p = 0.004, respectively). SPC expression was higher in the TRASCET group compared to the untreated group (p = 0.01), but not the saline group (p = 0.043). Lung laterality had minimal impact on these comparisons.</AbstractText><AbstractText>Transamniotic stem cell therapy affects select processes of lung development in experimental congenital diaphragmatic hernia. Further scrutiny into this novel therapy as a potential component of the prenatal management of this disease is warranted.</AbstractText><AbstractText>N/A (animal and laboratory study).</AbstractText>

Previously reported serum-free defined media for muscle cell culture require supplementation with hormones, purified <em>growth</em> <em>factors</em> or attachment <em>factors</em>. This report describes a culture system that enhances embryonic chick, skeletal muscle cell <em>growth</em> and differentiation in a serum-free defined medium, without added specialized trophic <em>factors</em>. Myoblasts adhered more to and proliferated more rapidly on a reconstituted basement membrane substrate, Matrigel, than on rat-tail collagen. Matrigel contains several basement membrane attachment molecules which apparently obviate the need for added purified attachment <em>factors</em>. Matrigel also appeared to play a trophic role in subsequent development by enabling the serum-free <em>growth</em> of myotubes which suggests that Matrigel mediates the cellular interaction of <em>growth</em> or attachment <em>factors</em>. Collagen, on the other hand, did not support serum-free myotube <em>growth</em>. Supplementation of defined medium with increasing levels of horse serum enhanced total protein in myotubes grown on both substrates; protein was higher in Matrigel cultures for each medium tested. The serum-free defined medium supported complete morphological differentiation of myotubes grown on Matrigel and maintained myotube cultures up to <em>22</em> days. <em>Fibroblast</em> proliferation was higher in cultures on collagen in defined medium with high serum levels, but was virtually eliminated in cultures on Matrigel in serum-free defined medium. The culture system described supports the differentiation of embryonic muscle cells in a simple, serum-free defined medium, thus providing an in vitro model of developing myotubes which should be particularly useful for studies of regulation mediated by extracellular <em>factors</em>.

Cochlear ribbon synapses play a pivotal role in the prompt and precise acoustic signal transmission from inner hair cells (IHCs) to the spiral ganglion neurons, while noise and aging can damage ribbon synapses, resulting in sensorineural hearing loss. Recently, we described reduced <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>22</em> (FGF<em>22</em>) and augmented myocyte enhancer <em>factor</em> 2D (MEF2D) in an ototoxicity mouse model with impaired ribbon synapses. Here, we investigated the mechanisms that underlie the FGF<em>22</em>/MEF2D- regulated impairment of ribbon synapses. We generated adeno-associated virus (AAV) carrying FGF<em>22</em>, shFGF<em>22</em>, MEF2D, shMEF2D, calcineurin (CalN), shCalN or corresponding scramble controls for transduction of cultured mouse hair cells. We found that FGF<em>22</em> was a suppressor for MEF2D, but not vice versa. Moreover, FGF<em>22</em> likely induced increases in the calcium influx into IHCs to activate CalN, which subsequently inhibited MEF2D. Cochlear infusion of AAV-shFGF<em>22</em> activated MEF2D, reduced ribbon synapse number and impaired hearing function, which were all abolished by co-infusion of AAV-shMEF2D. Hence, our data suggest that the ribbon synapses may be regulated by FGF<em>22</em>/calcium/CalN/MEF2D signaling, which implied novel therapeutic targets for hearing loss.

Oxidative stress has been proposed as a possible pathogenic <em>factor</em> for diabetic complications. It is relevant in determining cell replicative capacity and life span, and in vitro antioxidant treatment is able to reverse the impaired proliferative activity of different cell types. It was recently demonstrated that cultured skin <em>fibroblasts</em> from insulin-dependent diabetic patients with nephropathy age prematurely and have a shorter life cell cycle. To test whether the <em>growth</em> phenotype of cells from patients with diabetic nephropathy was related to a lack of protection from oxidative stress, the effect of reduced glutathione (GSH) on cultured skin <em>fibroblasts</em> from 13 insulin-dependent diabetes mellitus (IDDM) patients with nephropathy (DN), 10 IDDM patients without kidney disease (D), and 10 nondiabetic control subjects (C), in normal (5 mM) glucose (NG) and high (<em>22</em> mM) glucose (HG) medium was studied. After 6 to 8 passages, <em>fibroblasts</em> from DN showed impaired <em>growth</em> both in NG (mean +/- SD fold increase over baseline counts in DN 1.17 +/- 0.6 versus D 1.7 +/- 0.5 versus C 1.95 +/- 0.8; P = 0.04 by ANOVA) and in HG (mean +/- SD fold increase over baseline counts DN 1.16 +/- 0.41 versus D 1.89 +/- 0.66 versus C 2.24 +/- 0.9; P = 0.003 by ANOVA). GSH prevented the <em>growth</em> abnormalities of cells from DN restoring it to values similar to that of the other two groups (mean +/- SD fold increase over baseline counts NG +/- GSH: DN 1.68 +/- 0.9 versus D 1.78 +/- 0.49 versus C 1.99 +/- 0.7, P = 0.6; and in HG + GSH: DN 1.66 +/- 0.69 versus D 1.87 +/- 0.75 versus C 2.2 +/- 0.9, P = 0.3). <em>Growth</em> rates were not affected by the addition of GSH in <em>fibroblasts</em> from D and C. The treatment of <em>fibroblasts</em> from D and C with the inhibitor of the gamma-glutamylcysteine synthetase activity, L-buthionine-S,R-sulfoximine, resulted in <em>growth</em> impairment, and the addition to the culture medium of another antioxidant, superoxide dismutase, corrected the <em>growth</em> abnormalities in <em>fibroblasts</em> from DN. The impaired <em>growth</em> of cultured <em>fibroblasts</em> from IDDM patients with nephropathy is prevented by GSH and superoxide dismutase and is independent of prevailing glucose concentrations. This suggests that oxidative stress is an important mechanism of intrinsic cell dysfunction in these patients.

We previously reported that human <em>fibroblasts</em> secrete a protease into their conditioned medium that cleaves insulin-like <em>growth</em> <em>factor</em> binding protein-5 (IGFBP-5) into non-IGF-I binding fragments. Because the protease activity in the <em>fibroblast</em> medium has characteristics of both serine and metalloproteases, the activity was purified and analyzed to determine whether it retained serine or metalloprotease properties. The protease was purified by heparin Sepharose affinity chromatography followed by alpha1 antichymotrypsin affinity or gelatin agarose chromatography. The heparin Sepharose purified material degraded IGFBP-5 into <em>22</em>-, 17-, and 16-kDa fragments. Amino acid sequencing showed that the <em>22</em>-kDa fragment contained the amino-terminus of the protein. The protease activity in the <em>fibroblast</em> conditioned medium that was purified by heparin Sepharose was inhibited by both serine and metalloprotease inhibitors. To attempt to separate these activities, the heparin Sepharose purified activity was further purified by gelatin agarose chromatography. The IGFBP-5 protease activity that did not bind to gelatin agarose was inhibited by serine protease inhibitors, such as 3,4 dichloroisocoumain (3,4 DCI), whereas tissue inhibitor metalloprotease-1 (TIMP-1) had minimal activity. When this same pool of protease activity that had been eluted from heparin Sepharose was applied to an alpha1 antichymotrypsin peptide affinity column, the protease activity that bound to the column was inhibited by 3,4 dichloroisocoumain, but was not inhibited by TIMP-1. In contrast, the activity that did not adhere to this column was inhibited by TIMP-1. IGFBP-5 zymography showed that the Mr estimate of the protease that was inhibited by serine protease inhibitors was 92 kDa, whereas gelatin zymography showed that the metalloproteases had Mr estimates of 72, 69, and 55 kDa. When the protease activity in the crude conditioned medium was analyzed by zymography, almost all of the detectable protease had an Mr estimate of 92 kDa, suggesting that the metalloproteases that were detected in the partially purified fractions were inactive in the medium. In summary, <em>fibroblasts</em> secrete a 92-kDa protease that cleaves IGFBP-5 into <em>22</em>-, 17-, and 16-kDa fragments. The protease inhibitor specificity results, chromatographic characteristics, and zymographic analyses suggest that this is a serine protease. Although metalloproteases are secreted by these cells, the 92-kDa serine protease is the predominate form of activity in the conditioned medium that cleaves IGFBP-5.

Limited proteolysis of insulin-like <em>growth</em> <em>factor</em> binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like <em>growth</em> <em>factor</em> (IGF) activity, its major effect being to increase IGF bioavialability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases. Two major peaks were purified by RP-HPLC. One contained a 16 kDa fragment and the other comprised two fragments of <em>22</em> and 25 kDa. Competitive binding experiments showed that the 16 kDa material had no affinity for IGFs. The <em>22</em>-25 kDa fragments had considerably reduced affinity, particularly for IGF-I. In a chick embryo <em>fibroblast</em> assay where DNA synthesis was stimulated by IGF-I or insulin, the <em>22</em>-25 kDa fragments weakly inhibited IGF-I-induced cell proliferation and had no effect on stimulation by insulin. The 16 kDa fragment unexpectedly proved to be a potent inhibitor of both IGF- and insulin-induced cell <em>growth</em>. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.