BACKGROUND

Blood biomarkers are needed to monitor anti-angiogenic treatments for cancer. The association of blood levels of microRNAs (miRs) implicated in angiogenesis with circulating endothelial cells (CEC) and with angiogenic proteins was examined in patients administered drugs with anti-angiogenic activity.

METHODS

Blood was collected from patients with uveal melanoma enrolled on an adjuvant therapy trial in which they were treated sequentially with dacarbazine and interferon-alfa-2b. Plasma levels of nine angioregulatory miRs, miR-<em>16</em>, 20a, 106a, 125b, 126, 146a, 155, 199a, and 221, were determined by quantitative real time polymerase chain reaction; CEC, by semi-automated immunomagnetic; and plasma angiogenic proteins, by enzyme linked immunosorbent assays.

RESULTS

Levels of miR-199a were positively correlated and miR-106a negatively correlated with CEC pre-therapy. Decreases in miR-126 and miR-199a and increases in miR-<em>16</em> and miR-106a were observed after interferon-alfa-2b, but not after dacarbazine. CEC also increased after treatment with interferon but not after treatment with dacarbazine. Levels of miRs did not correlate with levels of vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. Angiogenic proteins also did not change significantly with treatment.

CONCLUSIONS

Blood levels of specific angioregulatory miRs are associated with CEC, and changes in specific angioregulatory miRs parallel increases in CEC after treatment with interferon-alfa-2b. Blood levels of specific angioregulatory miRs are not associated with levels of angiogenic proteins. miRs warrant further evaluation as blood biomarkers of angiogenesis.

BACKGROUND

Fibroblast growth factor 19 (FGF19) improves glycemic control in diabetic animals and is secreted from the gastrointestinal tract after meals in response to bile acid stimulation.

OBJECTIVE

We sought to understand how ingestion of carbohydrates, protein or lipids affect both FGF19 and bile acid concentrations in human plasma, with the hypothesis that variation in the bile acid response to different macronutrients would predict differences in plasma FGF19 levels.

METHODS

This was a randomized, within-subjects crossover study.

METHODS

The study was conducted at a university clinical research center.

METHODS

There were 16 healthy human subjects included in the study.

METHODS

Isocaloric, isovolemic beverages composed primarily of carbohydrates, proteins, or lipids were provided to each participant on 3 separate occasions.

METHODS

The magnitudes of postprandial rises of plasma FGF19 and total bile acid levels were determined.

RESULTS

All beverages induced an initial transient decline of plasma FGF19 levels during the first 60 minutes after consumption. For FGF19, the ingestion of carbohydrate was associated with the fastest and highest increase of plasma levels, returning to baseline at 5 hours. By comparison, the protein beverage induced a modest but significant elevation of FGF19 levels that peaked at the end of the 6-hour sampling interval, whereas a lipid beverage was without effect. In contrast, total bile acid levels increased in plasma only in response to a high-lipid beverage, demonstrating a marked divergence between the FGF19 and bile acid response to lipid vs carbohydrate.

CONCLUSIONS

A bile acid-independent mechanism is implicated in the effect of meals to raise plasma FGF19 concentrations.

BACKGROUND

Since head and neck cancer is characterized by poor survival rates, there is a demand for novel therapeutic targets and prognostic biomarkers. An upcoming therapeutic target is the fibroblast growth factor receptor (FGFR) family. However, their prognostic role in head and neck cancer remains unclear.

OBJECTIVE

To systematically review current evidence on the prognostic value of FGFR family members in head and neck squamous cell carcinoma (HNSCC).

METHODS

A systematic search of PubMed, Embase, and the Cochrane Library was performed for publications up to 14 May 2014. Two reviewers screened all articles and included prognostic studies on the molecular biomarkers FGFR1-5 in any type of HNSCC. Relevant studies were assessed on risk of bias using the Quality in Prognostic Studies (QUIPS) tool. Data on FGFR aberrations and survival outcome were extracted from relevant studies. The prognostic value of FGFR aberrations was compared among studies.

RESULTS

The initial search yielded 1568 publications of which 12 fulfilled the inclusion criteria. Four studies reported FGFR1 gene amplification (9.3-17.4 %) and FGFR1 protein overexpression (11.8 %) in HNSCC. FGFR1 protein expression by cancer-associated fibroblasts correlated with poor survival outcome in one study (p < 0.01). Eight studies reported high rates of FGFR4 Gly388Arg polymorphisms (32.5-54.2 %) and FGFR4 protein overexpression (16-35 %), with varying correlations with survival. So far, no studies assessed the prognostic role of FGFR2, FGFR3, or FGFR5 in HNSCC.

CONCLUSIONS

Significant risk of bias has been identified among included studies. Therefore, cautious interpretation of the results is recommended.

CONCLUSIONS

In conclusion, evidence was found for prognostic value of FGFR1 expression in cancer-associated fibroblasts in HNSCC. Prognostic evidence on the other FGFR family members in HNSCC is limited and conflicting. This emphasizes the need for future well-conducted prognostic studies.

We previously reported that alcohol drinkers with and without cirrhosis showed a significant increase in fecal bile acid secretion compared to nondrinkers. We hypothesized this may be due to activation by alcohol of hepatic cyclic adenosine monophosphate responsive element-binding protein 3-like protein 3 (CREBH), which induces cholesterol 7α-hydroxylase (Cyp7a1). Alternatively, the gut microbiota composition in the absence of alcohol might increase bile acid synthesis by up-regulating Cyp7a1. To test this hypothesis, we humanized germ-free (GF) mice with stool from healthy human subjects (Ctrl-Hum), human subjects with cirrhosis (Cirr-Hum), and human subjects with cirrhosis and active alcoholism (Alc-Hum). All animals were fed a normal chow diet, and none demonstrated cirrhosis. Both hepatic Cyp7a1 and sterol 12α-hydroxylase (Cyp8b1) messenger RNA (mRNA) levels were significantly induced in the Alc-Hum and Ctrl-Hum mice but not in the Cirr-Hum mice or GF mice. Liver bile acid concentration was correspondingly increased in the Alc-Hum mice despite <em>fibroblast</em> <em>growth</em> <em>factor</em> 15, <em>fibroblast</em> <em>growth</em> receptor 4, and small heterodimer partner mRNA levels being significantly induced in the large bowel and liver of the Ctrl-Hum mice and Alc-Hum mice but not in the Cirr-Hum mice or GF mice. This suggests that the normal pathways of Cyp7a1 repression were activated in the Alc-Hum mice and Ctrl-Hum mice. CREBH mRNA was significantly induced only in the Ctrl-Hum mice and Alc-Hum mice, possibly indicating that the gut microbiota up-regulate CREBH and induce bile acid synthesis genes. Analysis of stool bile acids showed that the microbiota of the Cirr-Hum and Alc-Hum mice had a greater ability to deconjugate and 7α-dehydroxylate primary bile acids compared to the microbiota of the Cirr-Hum mice. <em>16S</em> ribosomal RNA gene sequencing of the gut microbiota showed that the relative abundance of taxa that 7-α dehydroxylate primary bile acids was higher in the Ctrl-Hum and Alc-Hum groups. Conclusion: The composition of gut microbiota influences the regulation of the rate-limiting enzymes in bile acid synthesis in the liver. (Hepatology Communications 2017;1:61-70).

OBJECTIVE

Can deep infiltrating endometriotic stromal cells (DES) sense changes in extracellular matrix (ECM) stiffness and respond to them?

CONCLUSIONS

Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of DES in vitro.

BACKGROUND

Deep infiltrating endometriosis (DIE) is characterized histologically by dense fibrous tissue. Tissue stiffening is a hallmark of fibrosis. Studies show that matrix stiffness is involved in the progression of numerous diseases, including cancer and fibrosis. However, no studies to date have investigated whether tissue stiffening could influence cell behavior in DIE. Previous in vitro studies typically analyzed cells grown on rigid plastic or glass substrates with stiffness in the gigapascal (gPa) range, which is much stiffer than that occurring in vivo. To investigate how changes in ECM stiffness affect the behavior of DES, it is critical to model in vivo tissue compliance conditions in vitro.

METHODS

For this laboratory study, paired endometrial and endometriotic samples from 40 patients who had histological evidence of DIE and endometrial samples from 23 patients without endometriosis were analyzed (uterine fibroma: n = 10, tubal infertility: n = 13).

METHODS

All participants were 20-37 years old and had regular menstrual cycles of 26-32 days. The abundance of F-actin, alpha smooth muscle actin (αSMA), Ki67, and procollagen type I in DES and endometrial stromal cells (EES) on polyacrylamide gel substrates of varying stiffness (2, 4, 8, <em>16</em> and/or 30 kPa) was determined by immunofluorescence confocal microscopy. mRNA level of type I collagen, matrix metalloproteinase-1 (MMP-1), MMP-14 and cyclin D1 was measured by real-time PCR. The cellular proliferation index (CPI), assessed as the percentage of Ki67-positive cells among the total number of nuclei stained by 4',6-diamidino-2-phenylindole (DAPI) was determined.

RESULTS

Increased matrix stiffness induced F-actin stress fiber formation in both EES and DES, whereas αSMA-containing stress fibers were induced only in DES. Furthermore, increased stiffness increased the CPI in both EES (<em>16</em> or 30 kPa versus 2 kPa, P < 0.05) and DES (<em>16</em> or 30 kPa versus 2, 4 or 8 kPa, P < 0.05). Increased stiffness increased the percentage of procollagen I-positive cells as well as mRNA levels of type I collagen in both EES and DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa) (P < 0.05). Increased stiffness also increased MMP-14 mRNA levels in EES (30 versus 2 kPa, P < 0.05), but decreased MMP-1 mRNA levels in DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa; P < 0.05). Treatment with transforming growth factor (TGF)-β1 further increased type I collagen mRNA levels in both EES and DES when compared with cells grown on a substrate of the same stiffness (2, 8 or 30 kPa, with versus without TGF-β1, P < 0.05). Treatment with TGF-β1 also increased MMP-1 (8 or 30 kPa, P < 0.05 versus no TGF-β1) and MMP-14 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-β1) in EES, but decreased MMP-1 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-β1) in DES. On a soft substrate (2 kPa), both EES and DES exhibited a small rounded morphology with diffuse labeling for F-actin. No F-actin-positive stress fibers were observed in either EES or DES grown on 2 kPa substrates. There were more Ki67-positive EES when grown on 2, 4 or 8 kPa compared with Ki67-positive DES (P < 0.05).

CONCLUSIONS

A tremendous gap exists between the present in vitro model and in vivo deep endometriotic tissues. Cell culture systems that more closely mimic the cellular complexity typical of in vivo endometriotic tissues are required to develop novel strategies for treatment of DIE. A disadvantage of polyacrylamide is its cytotoxicity but in the two-dimensional culture models used here, where cells are seeded above the polyacrylamide gel, this should not have a major impact. Finally, the soft substrates we used in vitro (2 and 4 kPa) may represent the elasticity of the endometrium in vivo, however, currently there are no data regarding tissue stiffness in DIE in vivo.

CONCLUSIONS

Hormonal suppressive therapy is not usually effective for treating DIE. Interrupting the mechanical interactions between endometriotic fibroblasts and aberrant ECM may be a novel strategy for treatment of DIE.

BACKGROUND

This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

OBJECTIVE

Endurance exercise training can ameliorate many cardiovascular and metabolic disorders and attenuate responses to inflammatory stimuli. The purpose of this study was to determine whether the angiogenic and pro-inflammatory cytokine response to acute endurance exercise differs between endurance-trained and sedentary young men.

METHODS

Ten endurance-trained and ten sedentary healthy young men performed 30 min of treadmill running at 75 % VO2max with blood sampling before and after exercise. Plasma concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and soluble VEGF receptor-1 (sFlt-1) were measured by multiplex ELISA.

RESULTS

Acute exercise increased IL-6 by 165 % (P < 0.05), IL-8 by 32 % (P < 0.05), PlGF by ~16 % (P < 0.05), sFlt-1 by 36 % (P < 0.001), and tended to increase bFGF by ~25 % (P = 0.06) in main effects analyses. TNF-α and VEGF did not change significantly with exercise in either group. Contrary to our hypothesis, there were no significant differences in TNF-α, IL-6, VEGF, bFGF, PlGF, or sFlt-1 between groups before or after acute exercise; however, there was a tendency for IL-8 concentrations to be higher in endurance-trained subjects compared to sedentary subjects (P = 0.06).

CONCLUSIONS

These results indicate that 30 min of treadmill running at 75 % VO2max produces a systemic angiogenic and inflammatory reaction, but endurance exercise training does not appear to significantly alter these responses in healthy young men.

The endometrium goes through a unique cycle of physiological angiogenesis during the normal menstrual cycle (MC). We studied whether there is a correlation between endothelial progenitor cells (EPCs) and plasma and endometrial levels of angiogenic <em>growth</em> <em>factors</em> during the MC. Ten healthy, regularly menstruating women provided blood samples and another <em>16</em> supplied endometrial biopsies. Blood samples were obtained over a single MC: twice in the proliferative and once in the secretory phase and at ovulation. Endometrial biopsies were provided in the proliferative or in the secretory phase. We assessed plasma levels of vascular endothelial and <em>fibroblast</em> <em>growth</em> <em>factors</em>, granulocyte and granulocyte-macrophage colony-stimulating <em>factors</em> and stromal cell-derived <em>factor</em>-1 (SDF-1) by ELISA; EPCs by a colony-forming unit (CFU) assay; immunostaining for endometrial SDF-1 by computer-assisted software; and endothelial cell (EC) markers by flow cytometry. In the proliferative phase, SDF-1 levels were significantly higher than during the secretory phase. EPC-CFUs correlated negatively to SDF-1 levels. Endometrial SDF-1 expression tended to be higher in the secretory than in the proliferative phase. Furthermore, vascular endothelial <em>growth</em> <em>factor</em> receptors and Tie-2 EPCs showed a cyclic pattern over the MC. Our results point to SDF-1 as a novel mediator of EPC trafficking during the MC.

To study the role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) in the development of sensory neurons, the cochleovestibular ganglion of the chicken embryo provides a well-characterized structure. This permits use of morphological markers in a cell culture preparation comparable to the normal embryo (normocytic). Otocysts were explanted from white leghorn embryos at Hamburger-Hamilton Stages 14-<em>16</em>, when ganglion cell precursors normally start migrating from the otic epithelium. The cultures were supplemented with either fetal bovine serum or human recombinant FGF-2 (in defined medium or serum) for 2 or 5 days. FGF-2 increased explant <em>growth</em>, neuroblast migration, and neurite out<em>growth</em> 2- to 10-fold in the first 2 days. Neuronal morphology appeared within 2-3 days with FGF-2 but required at least 4-5 days with serum. FGF-2 in defined medium stimulated early migration and differentiation, but without serum led to degeneration after 5 days. In serum, <em>growth</em> was later and slower but continued for at least 3 weeks. When explants were cultured in serum with a neutralizing antibody to FGF-2, but no FGF added, neuroblast migration and elongation were decreased by 2- to 4-fold, compared to serum alone. Immunocytochemistry demonstrated FGF receptor sites on the migrating ganglionic neuroblasts, on their processes and <em>growth</em> cones, and in the incipient ganglion and otic epithelium at Stages 15-17, both in the embryo and in vitro. The findings suggest that FGF-2 stimulates early migration and differentiation of ganglion cells by activating the receptors of neuroblasts or their precursors in the embryonic otocyst. However, other <em>factors</em> must sustain their later development.

Chinese herbal medicine (CHM) evolved through thousands of years of practice and was popular not only among the Chinese population, but also most countries in the world. Blumea balsamifera (L.) DC. as a traditional treatment for wound healing in Li Nationality Medicine has a long history of nearly 2000 years. This study was to evaluate the effects of total flavonoids from Blumea balsamifera (L.) DC. on skin excisional wound on the back of Sprague-Dawley rats, reveal its chemical constitution, and postulate its action mechanism. The rats were divided into five groups and the model groups were treated with 30% glycerol, the positive control groups with Jing Wan Hong (JWH) ointment, and three treatment groups with high dose (2.52 g·kg-1), medium dose (1.26 g·kg-1), and low dose (0.63 g·kg-1) of total flavonoids from B. balsamifera. During 10 consecutive days of treatment, the therapeutic effects of rates were evaluated. On day 1, day 3, day 5, day 7, and day 10 after treatment, skin samples were taken from all the rats for further study. Significant increases of granulation tissue, <em>fibroblast</em>, and capillary vessel proliferation were observed at day 7 in the high dose and positive control groups, compared with the model group, with the method of 4% paraformaldehyde for histopathological examination and immunofluorescence staining. To reveal the action mechanisms of total flavonoids on wound healing, the levels of CD68, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), transforming <em>growth</em> <em>factor</em>-β₁ (TGF-β₁), and hydroxyproline were measured at different days. Results showed that total flavonoids had significant effects on rat skin excisional wound healing compared with controls, especially high dose ones (p < 0.05). Furthermore, the total flavonoid extract was investigated phytochemically, and twenty-seven compounds were identified from the total flavonoid sample by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry/diode array detector (UPLC-Q-TOF-MS/DAD), including <em>16</em> flavonoid aglucons, five flavonoid glycosides (main peaks in chromatogram), five chlorogenic acid analogs, and 1 coumarin. Reports show that flavonoid glycoside possesses therapeutic effects of curing wounds by inducing neovascularization, and chlorogenic acid also has anti-inflammatory and wound healing activities; we postulated that all the ingredients in total flavonoids sample maybe exert a synergetic effect on wound curing. Accompanied with detection of four <em>growth</em> <em>factors</em>, the upregulation of these key <em>growth</em> <em>factors</em> may be the mechanism of therapeutic activities of total flavonoids. The present study confirmed undoubtedly that flavonoids were the main active constituents that contribute to excisional wound healing, and suggested its action mechanism of improving expression levels of <em>growth</em> <em>factors</em> at different healing phases.

BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) is common both in obese and overweight patients. Fibroblast growth factor 19 (FGF19), an intestinal hormone, could play a role in the complex pathogenesis of NAFLD. The aim of our study was to investigate responses of FGF19 and bile acid (BA) synthesis after a body weight-adjusted oral fat tolerance test (OFTT) in overweight and obese NAFLD patients.

METHODS

For this study, we recruited 26 NAFLD patients; 14 overweight (median BMI 28.3 kg/m2), 12 obese (35.3 kg/m2) and 16 healthy controls (24.2 kg/m2). All individuals received 1 g fat (Calogen®) per kg body weight orally. Serum concentrations of FGF19 were determined by ELISA. Concentrations of BAs and BA synthesis marker 7α-hydroxy-4-cholesten-3-one (C4) were measured by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively; all at 0 (baseline), 2, 4 and 6 h during the OFTT.

RESULTS

BMI correlated negatively with fasting FGF19 concentrations (rho = - 0.439, p = 0.004). FGF19 levels of obese NAFLD patients were significantly (p = 0.01) lower in the fasting state (median 116.0 vs. 178.5 pg/ml), whereas overweight NAFLD patients had significantly (p = 0.004) lower FGF19 concentrations 2 h after the fat load (median 163.0 vs. 244.5 pg/ml), and lowest values at all postprandial time points as compared to controls. Baseline BA concentrations correlated positively with FGF19 values (rho = 0.306, p = 0.048). In all groups, we observed BA increases during the OFTT with a peak at 2 h but no change in C4 levels in overweight/obese NAFLD patients.

CONCLUSIONS

Reduced basal gastrointestinal FGF19 secretion and decreased postprandial response to oral fat together with blunted effect on BA synthesis indicate alterations in intestinal or hepatic FXR signaling in overweight and obese NAFLD subjects. The precise mechanism of FGF19 signaling after oral fat load needs further evaluation.

BACKGROUND

We have registered the trial retrospectively on 30 Jan 2018 at the German clinical trials register ( http://www.drks.de /), and the following number has been assigned DRKS00013942 .

OBJECTIVE

Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs) have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs) and endometrial fibroblasts (eSFs).

METHODS

The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS)-induced state.

RESULTS

Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF)-A, stromal cell-derived factor-1 alpha (SDF)-1α, interleukin-1 receptor antagonist (IL-1RA), IL-6, interferon-gamma inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs.

CONCLUSIONS

Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed to result in loss of stem cell surface markers, minimal migration activity and a subtler cytokine profile likely contributing to normal endometrial function.

The biological effects of different wavelengths of light emitting diode (LED) light tend to vary from each other. Research into use of photobiomodulation for treatment of skin wounds and the underlying mechanisms has been largely lacking. We explored the histopathological basis of the therapeutic effect of photobiomodulation and the relation between duration of exposure and photobiomodulation effect of different wavelengths of LED in a Japanese big-ear white rabbit skin-wound model. Skin wound model was established in <em>16</em> rabbits (three wounds per rabbit: one served as control, the other two wounds were irradiated by red and blue LED lights, respectively). Rabbits were then divided into 2 equal groups based on the duration of exposure to LED lights (15 and 30 min/exposure). The number of wounds that showed healing and the percentage of healed wound area were recorded. Histopathological examination and skin expression levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), epidermal <em>growth</em> <em>factor</em> (EGF), endothelial marker (CD31), proliferating cell nuclear antigen (Ki67) and macrophagocyte (CD68) infiltration, and the proliferation of skin collagen fibers was assessed. On days <em>16</em> and 17 of irradiation, the healing rates in red (15 min and 30 min) and blue (15 min and 30 min) groups were 50%, 37.5%, 25% and 37.5%, respectively, while the healing rate in the control group was 12.5%. The percentage healed area in the red light groups was significantly higher than those in other groups. Collagen fiber and skin thickness were significantly increased in both red light groups; expression of EGF, FGF, CD31 and Ki67 in the red light groups was significantly higher than those in other groups; the expression of FGF in red (30 min) group was not significantly different from that in the blue light and control groups. The effect of blue light on wound healing was poorer than that of red light. Red light appeared to hasten wound healing by promoting fibrous tissue, epidermal and endothelial cell proliferation. An increase in the exposure time to 30 min did not confer any additional benefit in both red and blue light groups. This study provides a theoretical basis for the potential therapeutic application of LED light in clinical settings.

BACKGROUND

The poor glycemic status seems to be an important factor affecting implant complication rates, including peri-implant bone loss.

OBJECTIVE

This trial evaluated the influence of glycemic control of type 2 diabetes mellitus (T2DM) patients on implant stabilization and on the levels of bone markers in peri-implant fluid during the healing.

METHODS

Systemically healthy patients (SH,n = 19), better-controlled T2DM (BCDM,n = 16), and poorly controlled T2DM (PCDM,n = 16) indicated for implant therapy were recruited. The implant stability quotient (ISQ) was determined at implant placement, 3, 6, and 12 months. Levels of transforming growth factor- β (TGF-β), fibroblast growth factor (FGF), osteopontin (OPN), osteocalcin (OC), and osteoprotegerin (OPG) in the peri-implant fluid were quantified at 15 days, and 3, 6, and 12 months, using the Luminex assay.

RESULTS

OPG and OPN levels were higher in SH at 12 months than at15 days (p < .05), whereas OC and TGF-β were lower in PCDM at 12 months compared with the 15-day and 3-month follow-ups, respectively (p < .05). Inter-group analyses showed lower OPN levels in PCDM compared with SH at 12 months (p < .05). The ISQ was higher at 12 months when compared with baseline and 3 months in SH (p < .05), whereas no differences were observed during follow-up in diabetics, regardless of glycemic control (p>> .05). No difference in ISQ was observed among groups over time (p>> .05).

CONCLUSIONS

Poor glycemic control negatively modulated the bone factors during healing, although T2DM, regardless of glycemic status, had no effect on implant stabilization.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with very limited therapeutic options. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signals play important roles in mesothelioma cell <em>growth</em>. Several FGFs and FGF receptors (FGFRs) are predicted targets of the miR-15/<em>16</em> family, which is downregulated in MPM. The aim of this study was to explore the link between the miR-15/<em>16</em> family and the FGF axis in MPM. Expression analyses via RT-qPCR showed downregulation of the FGF axis after transfection with miR-15/<em>16</em> mimics. Direct interaction was confirmed by luciferase reporter assays. Restoration of miR-15/<em>16</em> led to dose-dependent <em>growth</em> inhibition in MPM cell lines, which significantly correlated with their sensitivity to FGFR inhibition. Treatment with recombinant FGF2 prevented <em>growth</em> inhibition and further reduced the levels of FGF/R-targeting microRNAs, indicating a vicious cycle between miR-15/<em>16</em> down- and FGF/FGFR signaling upregulation. Combined inhibition of two independent miR-15/<em>16</em> targets, the FGF axis and Bcl-2, resulted in additive or synergistic activity. Our data indicate that post-transcriptional repression of FGF-mediated signals contributes to the tumor suppressor function of the microRNA-15/<em>16</em> family. Inhibiting hyperactivated FGF signals and Bcl-2 might serve as a novel therapeutic combination strategy in MPM.

Thrombospondin-2 (TSP-2) is an endogenous negative regulator of vascularization in human cancer. TSP-2 regulates angiogenesis through binding and sequestration of the proangiogenic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2). However, it is unclear whether TSP-2 and FGF-2 are related to prognosis in non-small cell lung cancer (NSCLC). To study this issue, we measured serum (Elisa) levels of TSP-2 and FGF-2 in 40 NSCLC patients (before chemotherapy) and 22 healthy subjects. Both TSP-2 and FGF-2 concentrations were elevated in the NSCLC group compared with control (TSP-2: 26.72±8.00 vs. 18.64±5.50 ng/ml, p=0.002; FGF-2: 11.90±5.80 vs. 7.26±3.90 pg/ml, p=0.01). Receiver-operating characteristic (ROC) curves were applied to find the cut-off serum levels of TSP-2 and FGF-2 (NSCLC vs. healthy: TSP-2=15.09 ng/ml, FGF-2=2.23 pg/ml). Patients before treatment with the TSP-2 level<24.15 ng/ml had a median survival of 23.7 months, but those with TSP-2>24.15 ng/ml had only 9 months' median survival (p=0.007). Patients with FGF-2 level>11.21 pg/ml had significantly shorter survival than patients with FGF-2<11.21 pg/ml (7.5 months vs. <em>16</em> months, p=0.034). We conclude that NSCLC patients have higher serum concentrations of TSP-2 and FGF-2 than healthy people. High levels of TSP-2 and FGF-2 may predict worse survival.

The enteric nervous system (ENS) regulates gastrointestinal function via different subtypes of neurons, organized into fine-tuned neural circuits. It is not clear how cell diversity is created within the embryonic ENS; information required for development of cell-based therapies and models of enteric neuropathies. We aimed to identify proteins that regulate ENS differentiation and network formation.

We generated and compared RNA expression profiles of the entire ENS, ENS progenitor cells, and non-ENS gut cells of mice, collected at embryonic days 11.5 and 15.5, when different subtypes of neurons are formed. Gastrointestinal tissues from R26ReYFP reporter mice crossed to Sox10-CreERT2 or Wnt1-Cre mice were dissected and the 6 populations of cells were isolated by flow cytometry. We used histochemistry to map differentially expressed proteins in mouse and human gut tissues at different stages of development, in different regions. We examined enteric neuronal diversity and gastric function in Wnt1-Cre x Sox6fl/fl mice, which do not express the Sox6 gene in the ENS.

We identified 147 transcription and signaling <em>factors</em> that varied in spatial and temporal expression during development of the mouse ENS. Of the <em>factors</em> also analyzed in human ENS, most were conserved. We uncovered <em>16</em> signaling pathways (such as <em>fibroblast</em> <em>growth</em> <em>factor</em> and Eph/ephrin pathways). Transcription <em>factors</em> were grouped according to their specific expression in enteric progenitor cells (such as MEF2C), enteric neurons (such as SOX4), or neuron subpopulations (such as SATB1 and SOX6). Lack of SOX6 in the ENS reduced the numbers of gastric dopamine neurons and delayed gastric emptying.

Using transcriptome and histochemical analyses of the developing mouse and human ENS, we mapped expression patterns of transcription and signaling factors. Further studies of these candidate determinants might elucidate the mechanisms by which enteric stem cells differentiate into neuronal subtypes and form distinct connectivity patterns during ENS development. We found expression of SOX6 to be required for development of gastric dopamine neurons.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in areas of the brain, including the septal nucleus. Certain <em>growth</em> <em>factors</em> may promote the long-term survival of this subpopulation of neurons at risk. This study was undertaken to characterize <em>growth</em> <em>factors</em>' long-term effects on survival and development of neurons expressing the calcium-binding protein calbindin. In order to accomplish this, embryonic day <em>16</em> rat septal neurons were grown in bilaminar culture with astrocytes and in the absence of serum. These cultures were chronically treated with estrogen (Es), insulin-like <em>growth</em> <em>factors</em> I/II (IGF-I, IGF-II), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and nerve <em>growth</em> <em>factor</em> (NGF). Insulin-like <em>growth</em> <em>factor</em> II significantly increased the number of neurons immunoreactive for calbindin by 155%, suggesting either an increase in the survival of this subpopulation or an increase in the percentage of cells expressing calbindin. Chronic treatment with NGF, IGF-II, and Es significantly increased the number of primary neuritic processes on calbindin-positive neurons, whereas NGF and Es caused significant increases in the number of secondary processes and in the total lengths of the neuritic processes. Thus, effects of IGF-II, estrogen, and NGF on survival and maintenance of this neuronal subpopulation may be dependent on alterations in neurons which are immunopositive for calbindin.

The senile plaques of Alzheimer's disease contain a high concentration of beta-amyloid (betaA) protein, which may affect the glial population in the septal nucleus, an area of increased risk in AD. BetaA toxicity was measured in septal glia, via a dose-response experiment, by quantifying the effects of three different doses (0.1, 1, and 10 microM) of betaA on cell survival. Astrocytes from embryonic day-<em>16</em> rats were grown in serum-free media in a single layer culture. Cells were treated on day in vitro (DIV)1 and survival was determined on DIV3 to ascertain which concentration was most toxic. In a separate set of experiments, an attempt was made to protect glial cells from the degenerative effects of betaA, with treatments of <em>growth</em> <em>factors</em> and estrogen. BetaA (10 microM) treatment was administered on DIV1, on DIV2 the cells were treated with estrogen (EST, 10 nM), insulin-like <em>growth</em> <em>factors</em> (IGF1 and IGF2, each 10 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 5 ng/ml) or nerve <em>growth</em> <em>factor</em> (NGF, 100 ng/ml), and on DIV3 the cells were visualized and quantified by fluorescence microscopy with DAPI (4,6-diamidino-2-phenylindole). In addition to dose-response and glial protection, experiments were also conducted to determine whether toxic effects were due to apoptosis. Our results suggest that the survival of glial populations is significantly affected in all three concentrations (0.1, 1.0, and 10 microM) of betaA. Glial protection was evident in the presence of NGF, for it showed the significantly highest survival rate relative to the betaA treatment alone. Furthermore, toxic effects of betaA appear to be due primarily to apoptosis. Significant reversal of betaA-induced apoptosis was seen with bFGF and IGF1.

<em>Fibroblasts</em> are a key stromal cell in the tumor microenvironment (TME) and promote tumor <em>growth</em> via release of various <em>growth</em> <em>factors</em>. Stromal <em>fibroblasts</em> in cancer, called cancer-associated <em>fibroblasts</em> (CAF), are related to myo<em>fibroblasts</em>, an activated form of <em>fibroblast</em>. While investigating the role of stroma <em>fibroblasts</em> on radiation-related carcinogenesis, it was observed following long-term fractionated radiation (FR) that the morphology of human diploid <em>fibroblasts</em> changed from smaller spindle shapes to larger flat shapes. These cells expressed smooth muscle actin (α-SMA) and platelet-derived <em>growth</em> <em>factor</em> receptors, markers of myo<em>fibroblasts</em> and CAFs, respectively. Long-term FR induces progressive damage to the <em>fibroblast</em> nucleus and mitochondria via increases in mitochondrial reactive oxygen species (ROS) levels. Here, it is demonstrated that long-term FR-induced α-SMA-positive cells have decreased mitochondrial membrane potential and activated oxidative stress responses. Antioxidant N-acetyl cysteine suppressed radiation-induced mitochondrial damage and generation of myo<em>fibroblasts</em>. These results indicate that mitochondrial ROS are associated with the acquisition of myo<em>fibroblasts</em> after long-term FR. Mechanistically, mitochondrial ROS activated TGFβ signaling which in turn mediated the expression of α-SMA in radiation-induced myo<em>fibroblasts</em>. Finally, in vivo tumor <em>growth</em> analysis in a human tumor xenograft model system revealed that long-term FR-induced myo<em>fibroblasts</em> promote tumor <em>growth</em> by enhancing angiogenesis.Implications: Radiation affects malignant cancer cells directly and indirectly via molecular alterations in stromal <em>fibroblasts</em> such as activation of TGFβ and angiogenic signaling pathways. Mol Cancer Res; <em>16</em>(11); <em>16</em>76-86. ©2018 AACR.

Functional wild-type p53 is required for human diploid <em>fibroblasts</em> (HDF) to enter an irreversible <em>growth</em> arrest known as replicative senescence. Experimentally, abrogation of p53 function by expression of human papillomavirus type <em>16</em> E6 or disruption of a key downstream effector p21 by homologous recombination both extended HDF life span. However, although sufficient to extend life span, p21 down-regulation is not necessary, because expression of a dominant-negative mutant p53 (143(ala)) extends life span without apparently decreasing p21 expression. Given the importance of p53 in cellular senescence and the general assumption that p21 may be the sole mediator of its action in this process, we have investigated how abrogation of p53 function can overcome senescence without lowering expression of p21. We have found up-regulated levels of the cyclin-dependent kinase 2 (cdk2) protein in HDF expressing 143(ala) mutant p53 as compared to senescent controls, together with an increase in p21-free cdk2 which, in conjunction with cyclin E, is able to form an active kinase which can phosphorylate the retinoblastoma protein. However, forced overexpression of cdk2 in near-senescent HDF failed to restore cdk2-associated kinase activity. Our data suggest that p53-mediated senescence depends on <em>factor</em>(s) other than p21 which modulate formation of cyclin E-cdk2 complexes.

The objective of the study was to investigate transcriptomic profile of pig endometrium on Days 12 and <em>16</em> of pregnancy in comparison with the respective days of the estrous cycle. Labeled complementary DNA was hybridized to Porcine Long Oligo microarray containing 13,297 oligonucleotide probes, which represented complementary DNA and expressed sequence tags. Statistical analysis revealed 110 differentially expressed genes (DEGs) on Day 12 of pregnancy and 179 DEGs on Day <em>16</em> of pregnancy. In silico analysis of gene function and functionality networks revealed links between genes implicated in cell death and survival, protein synthesis, lipid metabolism, cellular movement, tissue development, and cell-to-cell signaling. On Day 12 of pregnancy, estrogen, transforming <em>growth</em> <em>factor</em> (TGF) β1, and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 2, and on Day <em>16</em> of pregnancy, epidermal <em>growth</em> <em>factor</em> (EGF), insulin, interleukin 11 (IL-11), and FGF family members were indicated as possible upstream regulators of several DEGs. Obtained results showed changes in global endometrial gene expression at the time of maternal recognition of pregnancy and embryo implantation. Additionally, these data revealed signaling molecules, which together with E2, may evoke molecular changes in the uterus, leading to successful pregnancy establishment.

OBJECTIVE

We examined the safety and efficacy of controlled-release basic fibroblast growth factor (b-FGF) for peripheral artery disease (PAD), compared with autologous bone marrow mononuclear cell implantation (BMCI).

BACKGROUND

We recently developed a b-FGF-incorporated biodegradable hydrogel that enables slow-releasing drug delivery system.

METHODS

PAD patients were divided into a b-FGF group (n=10) and BMCI group (n=15). Injection of gelatin hydrogel containing 600 μg b-FGF or BMCI (0.4-5.1×10(10) cell) was performed. Visual analog pain scale (VAS), (99m)technetium-tetrofosmin (Tc-TF) scintigraphy, transcutaneous oxygen tension (TcPO(2)), and ankle-brachial index (ABI) were evaluated before and 4 weeks after each treatment, and 2-year prognosis was determined.

RESULTS

VAS (b-FGF 67±15 to 4±5, p<0.01, BMCI 67±42 to 5±9 mm, p<0.01) and TcPO(2) (b-FGF 16±14 to 47±17, p<0.01, BMCI 13±13 to 37±21 mmHg, p<0.01) were significantly improved in both groups. Tc-TF and ABI were not changed. Prognosis was similar between the groups (b-FGF 91%, BMCI 80%, NS).

CONCLUSIONS

Controlled-release b-FGF is as safe as BMCI, and its efficacy appears to be comparable. Thus, this therapy may be an alternative to BMCI.

OBJECTIVE

The level of constitutive plasminogen activator inhibitor type-1 (PAI-1) expression in cultured human orbital fibroblasts is considerably lower than that found in dermal fibroblasts. This divergence in PAI-1 expression implies differences in the pericellular proteolytic environment and, therefore, in the turnover of extracellular matrix. In this article, the authors examine the effect of transforming growth factor-beta (TGF-beta) on PAI-1 expression in orbital fibroblasts.

METHODS

Human orbital and dermal fibroblasts were grown in culture. Confluent monolayers were treated with TGF-beta. PAI-1 in the extracellular matrix was quantitated by radiolabeling the cultures and electrophoresing the cellular material on SDS-PAGE. Medium content was determined by immunoprecipitation of [35S]PAI-1 with a rabbit, anti-human, polyclonal antibody. PAI-1 mRNA was determined by Northern hybridization.

RESULTS

TGF-beta increased PAI-1 levels in orbital fibroblasts in a dose-dependent manner, up to 35-fold. The induction was maximal after 16 hours of treatment. The increases in extracellular matrix PAI-1 paralleled those observed in the medium. The steady state levels of the mRNA encoding the protein were upregulated by TGF-beta up to 60-fold 8 hours after the addition of TGF-beta. The fractional increase in PAI-1 expression in orbital fibroblasts was consistently greater than that observed in dermal strains.

CONCLUSIONS

Exposure to TGF-beta consistently induces PAI-1 expression in orbital fibroblasts, cells that do not express the polypeptide constitutively at high levels. The effects are mediated at the pretranslational level and involve the upregulation of PAI-1 mRNA. These results suggest that TGF-beta may exert a profound regulatory influence on the pericellular proteolytic environment in orbital connective tissue.

OBJECTIVE

To evaluate the effects of tamoxifen on the growth and autocrine growth factor production of human dermal fibroblasts from the face.

METHODS

In vitro study of normal adult dermal fibroblast cells developed from surgical specimens in a serum-free model. Cell cultures were exposed to 5-, 8-, 12-, 16-, and 50-microg/mL concentrations of tamoxifen solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, 5, and 7 days after the initial exposure. Population doubling times were calculated, and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF) beta1.

RESULTS

Tamoxifen appears to delay cellular proliferation rates in a dose-dependent manner up to a concentration of 12 microg/mL. Higher concentrations, approaching 50 microg/mL, appear to have a toxic effect on cell growth. The analysis of growth factor production revealed decreased levels of bFGF and VEGF but no change in the levels of TGF-beta1.

CONCLUSIONS

The in vitro findings of delayed cell proliferation and decreased production of VEGF and bFGF in cells exposed to tamoxifen are consistent with previous in vivo reports of delayed wound healing but improved scar formation. The in vitro findings of growth factor modulation by tamoxifen provide cellular and molecular evidence supporting the clinical use of tamoxifen to ultimately improve scar formation.