We isolated a population of proliferating cells from cultured human fetal hepatocytes of 16-<em>22</em> weeks gestational age. The cells shared a similar phenotype to that of mesenchymal stromal cells (MSCs) according to the International Society for Cellular Therapy (ISCT), including plastic adherence, antigen expression profile, and in vitro multilineage differentiation potential. Fetal liver (FL)-MSCs expressed the albumin gene, and harbored a subpopulation of CK18⁺ cells (20-40%), which defined their hepatic origin. However, when subjected to in vitro hepatic differentiation, FL-MSCs did not acquire significant liver functions. Quantitative analysis of conditioned medium (CM) collected from cultured cells revealed the presence of <em>growth</em> <em>factors</em> and chemokines with potential liver regenerative properties, the most relevant of which (concentration ≥3000 pg/ml) were SDF-1 alpha, IL-6, MCP-1, IL-8, MIP-1 beta, VEGF-A, Gro-alpha, and HGF. Culturing of FL-MSCs as spheroids significantly enhanced the secretion of HGF and bFGF (approximately 5-fold) compared with culture monolayers. Moreover, CM assessed in vitro induced capillary-like organization and migration of human umbilical vein endothelial cells (HUVECs) and <em>fibroblasts</em> as target cells. Interestingly, exosomes isolated from CM induced similar cellular responses in vitro with high efficiency and in a dose-dependent manner. FL-MSCs underwent several in vitro subcultivations, and did not stimulate allogenic T-cell proliferation thus suggesting a low immunogenicity. Furthermore, 5-year cryopreservation did not affect cell viability (approximately 90% of viable post-thawed FL-MSCs). These observations support the feasibility of a cell bank establishment for allogenic transplantation. We concluded that FL-MSCs or they secreted <em>factors</em> may be a valid alternative to hepatocyte transplantation in liver cell-based therapies.

Successful human spermatogonial stem cell (hSSC) culture could enable cell therapy for male infertility. Mammalian extracellular matrix (ECM) promotes mitogenesis, migration, and/or differentiation of various stem/progenitor cells and can plausibly facilitate hSSC survival in culture. Hydrogel forms of human testicular ECM (htECM), porcine testicular ECM (ptECM), porcine small intestinal submucosa ECM (SIS), and porcine urinary bladder ECM (UBM) were used to coat tissue culture plates for hSSC culture. In addition, hSSC were cultured on Sandos inbred mice (SIM) 6-thioguanine-resistance, ouabain-resistant (STO) mouse embryonic <em>fibroblast</em> feeder cells (control), murine laminin, or human laminin. Undifferentiated embryonic cell transcription <em>factor</em> 1-positive (UTF1+) human spermatogonia were quantified at days 0, 7, and 14 of culture. htECM was the only condition that retained a significantly higher number of UTF1+ cells than control STO feeder cell cultures (<em>22</em>% vs 3%). Overall, the number of hSSC declined during the 14 day culture period under all conditions. A multiparameter flow cytometry analysis of cells cultured on htECM and ptECM revealed that Stage-specific embryonic antigen 4 (SSEA4)+ undifferentiated spermatogonia may be lost to differentiation (cKIT+ spermatogonia) and apoptosis (annexin V+ spermatogonia). Proliferation of undifferentiated human spermatogonia (Ki67+) was limited suggesting that hSSCs may have different <em>growth</em> <em>factor</em> requirements than mouse SSCs. ECM from the homologous species (human) and homologous tissue (testis) was most effective substrate for hSSC and establishes a foundational feeder free, serum free condition for future iterative testing of culture conditions toward the long-term goal of stable hSSC cultures.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) regulatory axis is phylogenetically ancient, evolving into a large mammalian/human gene family of <em>22</em> ligands that bind to four receptor tyrosine kinases for a complex physiologic system controlling cell <em>growth</em>, differentiation, and metabolism. The tissue targets for the primary FGF function are mainly in cartilage and in bone for morphogenesis, mineralization, and metabolism. A multitude of complexities in the FGF ligand-receptor signaling pathways have made translation into therapies for FGF-related bone disorders such as osteomalacia, osteoarthritis, and osteoporosis difficult but not impossible.

The IGF system is thought to play a major role in adrenocortical tumorigenesis. In this study, we used the NCI H295R cell line as a model to investigate the effects of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), a potent mitogen for normal adrenal cells, on the proliferation and on the expression of the IGF system in cultured adrenocortical tumor cells. Three immunoreactive FGF-2 isoforms of molecular masses 18, <em>22</em>, and 24 kDa were detected in H295R cell extracts. Recombinant human FGF-2 stimulated the proliferation of adrenocortical tumor cells in a dose- and time-dependent manner, with a maximal effect at a concentration of about 1 ng/ml. Treatment of H295R cells with 10 ng/ml FGF-2 for 7 days had no significant effect on IGF-II messenger RNA levels. However, a marked increase in levels of intracellular IGF-II protein was detected by immunoblotting. In contrast, FGF-2 induced a marked decrease in the amount of IGF-II protein secreted, with the disappearance of mature IGF-II and secretion of higher molecular forms of the <em>growth</em> <em>factor</em>, suggesting modifications of IGF-II processing. Cell cultures in the presence of brefeldin A (1 microg/ml), a specific inhibitor of protein secretion, suggested that FGF-2 did not increase IGF-II synthesis but instead inhibited the secretion of pro-IGF-II from H295R cells, thereby impairing the final steps of IGF-II processing to the mature 7.5-kDa peptide. At the same concentrations, FGF-2 also decreased both IGFBP-2 messenger RNA and secreted protein, which might increase IGF-II bioavailability. No proteolysis of IGFBP-2 was detected in FGF-2-conditioned medium. Altogether, these results indicate that FGF-2 is mitogenic for NCI H295R tumor cells and regulates the expression of both IGF-II and IGFBP-2 in this tumor model. Moreover, this study shows a novel effect of FGF-2, by which this <em>growth</em> <em>factor</em> modulates the processing of pro-IGF-II.

Consumption of a low-protein, high-carbohydrate diet induces a striking increase in circulating <em>fibroblast</em> <em>growth</em> <em>factor</em>-21 (FGF21), which is associated with improved cardiometabolic health and increased longevity. Increased lifespan during this dietary protein "dilution" has been explained by resource-mediated trade-offs between reproduction and survival, such that fecundity is optimized at a greater relative intake of proteins/carbohydrates. The magnitude of this trade-off is thought to be sex-dependent. In this study, we tested the hypothesis that metabolic responses to dietary protein dilution are likewise dependent on sex. We maintained age-matched adult male and female C57BL/6J mice on isocaloric diets containing <em>22</em>% fat and differing in the ratio of protein/carbohydrate. The normal protein (NP) control diet contained 18% protein and 60% carbohydrate by kcal. The protein diluted (PD) diet contained 4% protein and 74% carbohydrate. Consistent with previous reports, PD males gained less weight and less fat than did normal protein controls and exhibited both improved glucose tolerance and decreased plasma lipids. In contrast, these metabolic benefits were absent among age-matched females maintained on the same diets. Likewise, whereas circulating FGF21 was increased up to 66-fold among PD male mice, this was substantially blunted among female counterparts. Sex differences in energy balance, glucose control, and plasma FGF21 were reversed upon ovariectomy. Collectively, our findings support that female mice are relatively less sensitive to the metabolic improvements observed following dietary protein dilution. This is accompanied by blunted circulating levels of FGF21 and requires an intact female reproductive system.

Low-grade osteosarcoma (LGOS) encompasses low-grade central osteosarcoma (LGCOS) and parosteal osteosarcoma (POS). LGOSs are characterized by a supernumerary ring and giant rod chromosomes containing the 12q13-15 amplicon. The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4. Recent studies identified consistent amplification of FRS2 gene in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma. The aim of this study was to evaluate the frequency of FRS2 amplification and its relationship with the clinicopathologic features of LGOSs. The amplification of FRS2 and MDM2 genes were analyzed by fluorescence in situ hybridization using <em>22</em> LGOSs (3 LGCOSs, 14 classic POSs, and 5 dedifferentiated POSs) and 85 control samples of bone and soft tissue. The clinicopathologic features of the <em>22</em> LGOSs were described. Amplification of FRS2 was detected in 21/<em>22</em> (95%) of the LGOSs, including 3 (100%) LGCOSs and 18 (95%) POSs. All <em>22</em> LGOSs showed MDM2 amplification (100%). The only MDM2/FRS2 LGOS was dedifferentiated POS (the dedifferentiated component was conventional osteosarcoma). In the control group, all of the atypical lipomatous tumor/well-differentiated liposarcoma/dedifferentiated liposarcomas (DDLs) (10/10, 100%) were FRS2-amplified, whereas the remaining 75 control cases were FRS2-nonamplified. These findings indicate that the FRS2 gene is consistently amplified in classic and dedifferentiated LGOSs but not in their histologic mimics. These results offer another avenue for investigating the biology of LGOSs. Whether FRS2-nonamplified tumors exhibit unusual clinicopathologic features needs further investigation. Some so-called "high-grade osteosarcomas harboring 12q13-15 amplification" may be unrecognized dedifferentiated LGOSs.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are a superfamily of <em>22</em> proteins related to cell proliferation and tissue repair after injury. A subgroup of three proteins, FGF19, FGF21, and FGF23, are major endocrine mediators. These three FGFs have low affinity to heparin sulfate during receptor binding; in contrast they have a strong interaction with the cofactor Klotho/β-Klotho. FGF21 has received particular attention because of its key role in carbohydrate, lipids, and energy balance regulation. FGF21 improves glucose and lipids metabolism as well as increasing energy expenditure in animal models and humans. Conditions that induce human physical stress such as exercise, lactation, obesity, insulin resistance, and type 2 diabetes influence FGF21 circulating levels. FGF21 also has an anti-oxidant function in human metabolic diseases which contribute to understanding the FGF21 compensatory increment in obesity, the metabolic syndrome, and type 2 diabetes. Interestingly, energy expenditure and weight loss is induced by FGF21. The mechanism involved is through "browning" of white adipose tissue, increasing brown adipose tissue activity and heat production. Therefore, clinical evaluation of therapeutic action of exogenous FGF21 administration is warranted, particularly to treat diabetes and obesity.

Interleukin (IL)-<em>22</em> regulates tissue inflammation and repair. Here we report participation of the liver in IL-<em>22</em>-mediated cardiac repair after acute myocardial infarction (MI). Methods: We induced experimental MI in mice by ligation of the left ascending artery and evaluated the effect of IL-<em>22</em> on post-MI cardiac function and ventricular remodeling. Results: Daily subcutaneous injection of 100 µg/kg mouse recombinant IL-<em>22</em> for seven days attenuated adverse ventricular remodeling and improved cardiac function in mice at 28 days after left anterior descending coronary artery ligation-induced MI. Pharmacological inhibition of signal transducer and activator of transcription (STAT3) muted these IL-<em>22</em> activities. While cardiomyocyte-selective depletion of STAT3 did not affect IL-<em>22</em> activities in protecting post-MI cardiac injury, hepatocyte-specific depletion of STAT3 fully muted these IL-<em>22</em> cardioprotective activities. Hepatocyte-derived <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF21) was markedly increased in a STAT3-dependent manner following IL-<em>22</em> administration and accounted for the cardioprotective benefit of IL-<em>22</em>. Microarray analyses revealed that FGF21 controlled the expression of cardiomyocyte genes that are involved in cholesterol homeostasis, DNA repair, peroxisome, oxidative phosphorylation, glycolysis, apoptosis, and steroid responses, all of which are responsible for cardiomyocyte survival. Conclusions: Supplementation of IL-<em>22</em> in the first week after acute MI effectively prevented left ventricular dysfunction and heart failure. This activity of IL-<em>22</em> involved crosstalk between the liver and heart after demonstrating a role of the hepatic STAT3-FGF21 axis in IL-<em>22</em>-induced post-MI cardiac protection.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a serious complication in SLE. Although the mechanism of NPSLE remains unclear, cytokines and chemokines are considered to be involved in their pathogenesis. Here we used Bio-Plex Pro assays to examine 27 types of cytokines and chemokines in the cerebrospinal fluid (CSF) of 32 NPSLE patients. We used the CSF of 20 patients with multiple sclerosis (MS) and <em>22</em> patients with neuromyelitis optica (NMO) as a disease control group. Fourteen of 27 cytokines/chemokines were significantly higher in the NPSLE patients compared to the MS/NMO patients. We could identify six "minimum predictive markers" by using a weighted-voting algorithm that could distinguish NPSLE from MS and NMO: interleukin (IL)-17, IL-2, interferon (IFN)-γ, IL-5, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-basic and IL-15. The determination of various types of CSF cytokine profiles may contribute to the diagnosis of NPSLE and may help elucidate the mechanisms underlying this disease.

<AbstractText>To identify potential biomarkers to distinguish adult-onset Still's disease (AOSD) from sepsis.</AbstractText><AbstractText>We recruited 70 patients diagnosed with AOSD according to the Yamaguchi criteria, <em>22</em> patients with sepsis, and 118 age-matched controls. Serum levels of 40 cytokines were analyzed using multi-suspension cytokine array. We performed a cluster analysis of each cytokine in the AOSD and sepsis groups in order to identify specific molecular networks. Further, multivariate classification (random forest analysis) and logistic regression analysis were used to rank the cytokines by their importance and determine specific biomarkers for distinguishing AOSD from sepsis.</AbstractText><AbstractText>Seventeen of the 40 cytokines were found to be suitable for further analyses. The serum levels of eleven were significantly higher in patients with AOSD than healthy controls. Levels of serum <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), granulocyte colony-stimulating <em>factor</em> (G-CSF), and interleukin (IL)-18 were significantly elevated in patients with AOSD compared with those with sepsis, and cytokine clustering patterns differed between these two groups. Multivariate classification followed by logistic regression analysis revealed that measurement of both FGF-2 and IL-18 could distinguish AOSD from sepsis with high accuracy (cutoff value for FGF-2 = 36 pg/mL; IL-18 = 543 pg/mL, sensitivity 100%, specificity 72.2%, accuracy 93.8%).</AbstractText><AbstractText>Determination of FGF-2 and IL-18 levels in combination may represent a biomarker for the differential diagnosis of AOSD from sepsis, based on the measurement of multiple cytokines.</AbstractText>

Alpha-Klotho is a member of the Klotho family consisting of two other single-pass transmembrane proteins: βKlotho and γKlotho; αKlotho has been shown to circulate in the blood. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)23 is a member of the FGF superfamily of <em>22</em> genes/proteins. αKlotho serves as a co-receptor with FGF receptors (FGFRs) to provide a receptacle for physiological FGF23 signaling including regulation of phosphate metabolism. The extracellular domain of transmembrane αKlotho is shed by secretases and released into blood circulation (soluble αKlotho). Soluble αKlotho has both FGF23-independent and FGF23-dependent roles in phosphate homeostasis by modulating intestinal phosphate absorption, urinary phosphate excretion, and phosphate distribution into bone in concerted interaction with other calciophosphotropic hormones such as PTH and 1,25-(OH)₂D. The direct role of αKlotho and FGF23 in the maintenance of phosphate homeostasis is partly mediated by modulation of type II Na⁺-dependent phosphate co-transporters in target organs. αKlotho and FGF23 are principal phosphotropic hormones, and the manipulation of the αKlotho-FGF23 axis is a novel therapeutic strategy for genetic and acquired phosphate disorders and for conditions with FGF23 excess and αKlotho deficiency such as chronic kidney disease.

OBJECTIVE

To compare the effect of clomiphene citrate (CC) and letrozole on endometrial receptivity in women with polycystic ovary syndrome (PCOS).

METHODS

A randomized controlled trial.

METHODS

University teaching hospital.

METHODS

Ten anovulatory women with PCOS and 5 fertile ovulatory women.

METHODS

Patients received 2.5 mg of letrozole on cycle days 3-7 (5 patients, 1 cycle) or 50 mg of CC on cycle days 5-9 (5 patients, 1 cycle).

METHODS

Serum estrogen (E) and progesterone (P) endometrial protein and messenger RNA (mRNA) expression of leukemia inhibitory <em>factor</em> (LIF), dickkhopf homolog 1 (DKK-1), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>22</em> (FGF-<em>22</em>), and endometrial mRNA expression of LIF/GP130 receptor (LIFR).

RESULTS

No statistically significant differences were observed between groups compared with fertile ovulatory women when serum E and P were examined, or between body mass index (BMI), and cycle day at time of biopsy. Letrozole increased mRNA expression of LIF, DKK1, LIFR, and FGF-<em>22</em>, whereas CC only increased endometrial mRNA expression of LIF. Letrozole mRNA expression directly translated into increased protein expression of like genes in the endometrium. The CC protein expression of DKK-1 was significantly decreased compared with controls.

CONCLUSIONS

Letrozole positively influences a number of markers of endometrial receptivity compared with CC.

There is growing need for a reliable assay for measuring fibroblast growth factor 23 (FGF23), a regulator of phosphorus and vitamin D. In this work, we analyze and compare the performance of three available assays, including the effect of temperature and time. This knowledge will allow for better understanding of FGF23 in the future.

Intact and C-terminal FGF23 (iFGF23 and cFGF23) concentrations are important in the diagnosis of hypo- and hyperphosphatemic diseases. The effects of temperature, storage, and specimen handling on FGF23 levels are not well known. We investigated the effects of various factors on plasma and serum measurement of FGF23 using three different assays.

Serum and plasma FGF23 were measured using three commercially available ELISA assays-two measuring iFGF23 and one measuring cFGF23. Samples from subjects with known FGF23 disorders were stored at 4, 22, and 37 °C and analyzed at different intervals up to 48 hours (h). A subset of samples underwent repeated freeze-thaw cycles, and samples frozen at -80 °C for up to 60 months were reanalyzed. The effect of adding a furin convertase inhibitor on FGF23 degradation was investigated using samples stored at 37 °C for 48 h. Intact FGF23 levels were measured from plasma samples of four different groups to test the correlation of the two assays.

Plasma FGF23 levels were stable when stored at 4 and 22 °C for 48 h. Both plasma and serum FGF23 levels demonstrated relative stability after five freeze-thaw cycles. Long-term storage at -80 °C for 40 months induced some variability in FGF23 levels. The addition of a furin inhibitor did not affect FGF23 degradation. Intact FGF23 levels showed good correlation only at the upper limit of the assay range when comparing the two assays.

Sample type, handling, and choice of assay are factors that affect FGF23 levels and should be considered when measuring this hormone.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF), hepatocyte <em>growth</em> <em>factor</em> (HGF) and their receptors, FGFR and c-Met, are essential components of the regulatory networks between the epithelium and mesenchyme in embryonic lung, but their respective roles in tumour <em>growth</em> are not clear. We performed allelotyping at loci containing the candidate genes FGFR-1-2-3-4, FGF-1-2-7-10, c-Met and HGF in 36 non-small cell lung cancer (NSCLC) (20 squamous-cell carcinomas (SQC) and 16 adenocarcinomas (ADC)), by surrounding each locus with two microsatellites (MS), as close as possible to the genes of interest. Unexpectedly, SQC and ADC were frequently altered at all of these loci, and SQC showed more simultaneously altered loci. In ADC, alterations at the 15q13-<em>22</em> locus (FGF7 candidate gene) were significantly more frequent. Thus, these loci showed different patterns of molecular alterations between SQC and ADC. Finally, alterations at loci containing FGFR and HGF candidate genes were inversely correlated to the lymph node status in SQC and ADC, respectively.

Lymphocytic pleurisy is commonly observed in tuberculosis and cancer. Noninvasive biomarkers are needed to distinguish tuberculous pleural effusion (TPE) from malignant pleural effusion (MPE) because current clinical diagnostic procedures are often invasive. We identified immune response biomarkers that can discriminate between TPE and MPE. Fourteen pleural effusion biomarkers were compared in <em>22</em> MPE patients and five TPE patients. Of the innate immunity biomarkers, the median levels of interleukin (IL)-1β and interferon-induced protein-10 (IP-10) were higher in TPE patients than in MPE patients (P<0.05 and P<0.01, respectively). Of the adaptive immunity biomarkers, the median levels of IL-13 and interferon-γ (IFN-γ) were higher in TPE patients than in MPE patients (P<0.05). In addition, the levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were higher in MPE patients than in TPE patients (P<0.05). Receiver operator characteristic analysis of these biomarkers was performed, resulting in the highest area under the curve (AUC) for IP-10 (AUC =0.95, 95% confidence interval, P<0.01), followed by IL-13 (AUC =0.86, 95% confidence interval, P<0.05). Our study shows that five biomarkers (IL-1β, IP-10, IFN-γ, IL-13, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) have a potential diagnostic role in differentiating TPE from MPE, particularly in lung cancer-related MPE.

BACKGROUND

We aimed to define the relative importance of renal and endocrine changes in tenofovir disoproxil fumarate (TDF)-related bone toxicity.

METHODS

In a study of daily TDF/emtricitabine (FTC) preexposure prophylaxis (PrEP) in human immunodeficiency virus (HIV)-uninfected young men who have sex with men, we measured changes from baseline in blood and urine markers of the parathyroid hormone (PTH)-vitamin D-fibroblast growth factor 23 (FGF23) axis, creatinine, and renal tubular reabsorption of phosphate (TRP). We explored the relationship of those variables to changes in bone mineral density (BMD). Tenofovir-diphosphate (TFV-DP) in red blood cells was used to categorize participants into high and low drug exposure groups.

RESULTS

There were 101 participants, median age 20 years (range 15 to 22). Compared with low drug exposure, high-exposure participants showed increase from baseline in PTH and decline in FGF23 by study week 4, with no differences in creatinine, phosphate, or TRP. At 48 weeks, the median (interquartile range) percent decline in total hip BMD was greater in those with high- compared to low- exposure (-1.59 [2.77] vs +1.54 [3.34] %, respectively; P = .001); in high-exposure participants, this correlated with week 4 TFV-DP (inversely; r = -0.60, P = .002) and FGF23 (directly; r = 0.42; P = .039) but not other variables.

CONCLUSIONS

These findings support the short-term renal safety of TDF/FTC PrEP in HIV-seronegative young men and suggest that endocrine disruption (PTH-FGF23) is a primary contributor to TDF-associated BMD decline in this age group.

BACKGROUND

NCT01769469.

OBJECTIVE

Bladder outlet obstruction can lead to the deposition of extracellular matrix and a resultant decrease in bladder wall compliance. Platelet-derived growth factor (PDGF) is a potent mitogen for fibroblasts and can increase the deposition of extracellular matrix. We attempt to determine if the expression of PDGF-BB and its receptor are altered in human bladder fibroblasts and bladder smooth muscle cells when exposed to hydrostatic pressures in the physiologic range.

METHODS

Cultured human bladder fibroblasts and smooth muscle cells were evaluated in vitro by using a novel device that controls for hydrostatic pressure. Cells were exposed to pressures of 20 and 40 cmH(2)O for up to 72 h. Western blot analyses and RT-PCR were performed to evaluate expression of both PDGF-BB and PDGF-BB receptor.

RESULTS

PDGF-BB and its receptor increased up to 22-fold and 8-fold, respectively, when human bladder fibroblasts were exposed to 40 cmH(2)O sustained hydrostatic pressure, while at 20 cmH(2)O the effect was minimal until 72 h. mRNA for the PDGF-BB receptor in human bladder fibroblasts increased in comparison to control. Western blot analyses demonstrated that exposure of human bladder smooth muscle cells to a sustained hydrostatic pressure of 20 and 40 cmH(2)O for up to 72 h did not alter expression of either PDGF-BB or its receptor.

CONCLUSIONS

Both PDGF-BB and its receptor in human bladder fibroblasts were upregulated in a time- and pressure-dependent manner after as little as 24 h exposure to pressures of < or =40 cmH(2)O. Our results provide support for a potential role of both PDGF-BB and its receptor in bladder fibrosis secondary to increased intravesical pressure. Newer selective PDGF receptor antagonists may prove beneficial in preventing bladder wall fibrosis in patients with either anatomic or functional bladder outlet obstruction.

Although a number of ablative-laser techniques based on CO(2) and Er: YAG laser devices have been successfully developed and used in the clinical setting, the bio-molecular processes influencing wound healing after exposure to laser energy are not well elucidated. In this study, we aim to assess the impact of the mechanism of injury on the secretion of transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in various stages of wound healing, in wounds created with a CO(2) laser and scalpel. Ten Wistar rats were used to determine the levels of <em>growth</em> <em>factor</em> proteins TGF-beta1 and bFGF after CO(2) laser- and scalpel-induced skin injury. Tissue was excised on day 0 for untreated skin (control sites), and on days 1, 10, 30, and 90 following laser and scalpel surgery. Specimens were processed for histopathological analysis and for determining the concentration of <em>growth</em> <em>factors</em> by a Western blot technique. The concentration of TGF-beta1 increased markedly, at day 1 postinjury, from a baseline of 130+/-16 mm(2) (mean surface area of blotted-protein lanes) to 261+/-23 mm(2) and 394+/-<em>22</em> mm(2) for laser-inflicted injury and scalpel wounds, respectively; the latter values were found to differ significantly (p<0.001). The concentration of b-FGF on day 10 postinjury differed significantly (p<0.001) between the laser sites (553+/-45 mm(2)) and the corresponding scalpel sites (418+/-41 mm(2)). Laser energy alters local tissue secretion of TGF-beta1 and bFGF of skin injuries created with the CO(2) laser compared with wounds created with a scalpel. These differences might have an impact on various aspects of wound healing of skin injuries created by a laser.

Myostatin (MSTN), a member of transforming <em>growth</em> <em>factor</em>-beta superfamily is a negative regulator of the skeletal muscle <em>growth</em>. It suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. In the present investigation silencing of MSTN gene in caprine <em>fibroblast</em> cell line was evaluated using four different shRNA expressing constructs. Variation in the efficiency of silencing (<em>22</em>-92%) was obtained among different constructs. It was observed that sh1 and sh4 constructs downregulated the MSTN gene expression by reducing 92.4 and 80.5% (P<0.05) level of downregulation MSTN mRNA, respectively. On the contrary, the sh3 construct significantly upregulated the MSTN mRNA level (P<0.05). These two promising constructs (sh1 and sh4) need to be further tested for interferon (IFN) response before their use in long term stable expression of anti-MSTN shRNA in muscle cells to improve chevon production.

Extracts of skeletal muscle contain chromatographically distinct molecules that enhance the cholinergic development of cultured embryonic rat spinal cord neurons. We have recently purified a 20-<em>22</em> kilodalton anionic polypeptide choline acetyltransferase (ChAT) development <em>factor</em> (CDF) from rat skeletal muscle extracts that stimulates the development of ChAT activity in rat spinal cord cultures. The maximum increase in the level of ChAT activity achieved by this <em>factor</em>, however, is less than that achieved by the addition of the crude extract. We now show that muscle extract also contains mitogenic activity that is immunologically related to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and also that recombinant bFGF stimulates ChAT development in rat spinal cord cultures. bFGF, however, differs from CDF in its physiochemical, chromatographic, and immunological properties and by its action on nonneuronal cells. Individually, CDF and bFGF each enhance the level of ChAT activity in rat spinal cord cultures two- to threefold after 2 days of treatment. However, their combined actions result in a five- to sixfold enhancement of ChAT activity, suggesting that they are affecting cholinergic development through different means. The demonstration that extracts of rat skeletal muscle contain two biochemically and immunologically distinct polypeptides, with additive effects on cultured embryonic spinal cord neurons, provides additional evidence for the involvement of multiple target-derived neurotrophic <em>factors</em> in the regulation of cholinergic development.

The ligand binding and biological responsiveness of cultured <em>fibroblast</em> monolayers were measured for porcine insulin and for murine epidermal <em>growth</em> <em>factor</em>--urogastrone in skin <em>fibroblast</em> cultures derived from young (<em>22</em> to 31 years) and old (65 to 80 years) normal male volunteer donors. The receptor characteristics of the cells studied from the two groups of donors were not found to differ. In contrast with previous data, suggesting that there may be a genetically programmed aging of the insulin binding system, it is concluded that for both insulin and epidermal <em>growth</em> <em>factor</em>, receptor characteristics do not change with adult donor age.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown by some to promote angiogenesis and myocardial salvage in experimentally induced acute myocardial infarction. Although these findings have spurred much clinical interest, they are not universally observed, and the true efficacy of bFGF remains unclear. The authors used a rabbit model of acute myocardial infarction to further elucidate the effects of bFGF on acutely infarcted myocardium containing few collaterals. Myocardial infarction was evoked by ligation of the left coronary artery. Prior to ligation, either 100 microg of bFGF (bFGF group; n = 15) or physiological saline (control group; n = <em>22</em>) was injected into the myocardium supplied by the ligated artery. With use of nonradioactive colored microspheres, regional blood flow (Qm) was measured before, immediately after, and 4 weeks after coronary artery ligation. Infarct and border zone sizes were measured in cross-sectional slices of the resected hearts, and the amount of viable myocardium (myocardium score) and the extent of fibrosis were histologically determined in each area. Four weeks after ligation, Qm values in the infarcted area did not significantly differ between the bFGF and control groups (0.54 +/- 0.36 vs 0.48 +/- 0.30 mL/min/g); in the border zone, Qm tended to be higher in the bFGF group (3.39 +/- 2.68 vs 1.47 +/- 0.80 mL/min/g), but the difference was not significant; finally in the noninfarcted area, Qm was significantly (p < 0.05) higher in the bFGF group (6.06 +/- 3.85 vs 2.09 +/- 0.82 mL/min/g). There was no significant difference in the amount of viable myocardium or the extent of fibrosis in the infarcted areas of the two groups. In the border zone, however, the amount of viable myocardium was significantly (p < 0.005) larger in the bFGF group (61.8 +/- 8.5% vs 35.8 +/- 20.3% of the visual field). Likewise, as graded on a scale from 0 to 5, the extent of fibrosis was significantly (p < 0.005) less in the bFGF group (2.1 +/- 0.5 vs 3.3 +/- 0.8). In conclusion, injection of bFGF into acutely infarcted myocardium increased blood flow to the noninfarcted area and salvaged the myocardium in the border zone.

Genetically engineered mice deficient in the expression of type III nitric oxide synthase (NOS) [endothelial NOS (eNOS)] were used to decipher the importance of nitric oxide (NO)-dependent augmentation of regional cerebral blood flow (rCBF) to infarct volume reduction following basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) infusion during acute middle cerebral artery (MCA) occlusion. We have shown previously that intravenously administered bFGF reduces infarct volume following MCA occlusion in rats and that bFGF dilates cerebral pial arterioles by NO-dependent mechanisms. Halothane-anesthetized eNOS knockout and wild-type mice were subjected to permanent MCA occlusion by intraluminal filament for 24 h. bFGF (100 microg x kg(-1) x h(-1)) was infused intravenously for 2 h, beginning 15 min after the onset of occlusion. Infarct volume was reduced from 119 +/- 8 to 93 +/- 4 mm3 (<em>22</em>% reduction, P < 0.05) or from 102 +/- 9 to 77 +/- 6 mm3 (24% reduction, P < 0.05) in eNOS knockout or wild-type mice, respectively (means +/- SE; n = 10 per group), and neurological deficits were also significantly reduced. Although bFGF infusion caused a 27% increase in rCBF and a 17% reduction in vascular resistance in the infarct margin of wild-type animals as measured by laser Doppler flowmetry, bFGF did not enhance rCBF in the infarct margin of eNOS mutant mice. These data indicate that intravenous bFGF reduces infarct volume following focal ischemia by mechanisms that are largely blood flow independent.

BACKGROUND

Being a potent promoter of endothelial and smooth muscle cell proliferation, basic fibroblast growth factor (bFGF) is presumed to play a key role in coronary collateral development and atherogenesis.

OBJECTIVE

To characterize serum bFGF levels in patients with ischemic heart disease.

METHODS

The study population consisted of patients with angina (n=33) and after uncomplicated myocardial infarction (n=12). The number of significantly stenosed >> or = 50%) vessels and angiographic coronary collateral score were noted. Blood was drawn immediately prior to elective coronary angiography in study patients for bFGF levels. Twenty healthy, age-matched subjects served as control for serum bFGF.

RESULTS

Serum bFGF levels were undetectable in all 20 control subjects, but were detectable in 15/33 (45%) patients with angina and 3/12 (25%) post-infarction patients, respectively (P=0.002). Serum bFGF levels were detectable in 13/23 (57%) patients with 0- or 1-vessel disease, as compared with 5/22 (23%) patients with 2- or 3-vessel disease (P<0.05). Detectable serum bFGF levels were not in correlation with coronary collateral score (P=1).

CONCLUSIONS

Serum levels of bFGF are elevated in patients with ischemic heart disease, particularly in those with minimal coronary artery disease. We postulate that detectable serum bFGF levels reflect active atherogenesis rather than myocardial collateral development.