OBJECTIVE

Angiogenesis is an important component of tissue regeneration. As Inflammatory bowel disease (IBD) involves inflammation, ulceration, and regeneration of the intestinal mucosa, angiogenesis may be an integral part of IBD pathology. This study investigated the role of vascular endothelial growth factor in IBD.

METHODS

The VEGF plasma (pVEGF) and serum (sVEGF) levels were assessed in patients with ulcerative colitis (UC; n=50) or Crohn's disease (CD; n=44) and in healthy controls (n=23). The immunohistochemical expression of VEGF was also assessed in surgical material from 11 patients with active IBD.

RESULTS

Overall the sVEGF levels ranged from 30-899 pg/ml (median 200 pg/ml) and were significantly higher than the pVEGF levels (range 20-80 pg/ml, median 30 pg/ml). pVEGF levels were significantly lower in patients with active and quiescent CD than in healthy controls. Despite the lower pVEGF levels noted also for patients with UC, the difference was not significant. sVEGF levels were also reduced in patients with IBD, but the difference was not significant. No association of pEGF/sVEGF with beta-thromboglobulin and platelet factor 4 levels (markers of platelet activation) was noted. On immunohistochemistry VEGF was not expressed in the inflammatory component (lymphocytes and macrophages), the fibroblasts, or the muscular layer of the intestinal wall. The intestinal epithelium was negative in CD, while a cytoplasmic reactivity was noted in UC and normal controls.

CONCLUSIONS

As VEGF is a vascular and epithelial cell survival factor, the defective VEGF response ability, confirmed here for patients with CD, may be a key element in the pathology of the disease. The pathology of UC, however, seems not to be VEGF dependent.

The purpose of this work was to study changes in serum levels of interleukins, <em>growth</em> <em>factors</em> and angiogenin during different stages of endometrial cancer progression. Serum levels were assayed by enzyme-linked immunosorbant assay in 59 women with stages I-IV of endometrial cancer (study subjects: stage I, n = <em>20</em>; stage II, n = 8; stage III, n = 5; stage IV, n = 6) and compared to the serum levels in <em>20</em> women without cancer as control subjects. Patients with endometrial cancer had varied serum levels of interleukins and <em>growth</em> <em>factors</em>. There was a significant increase in serum levels of angiogenin in all stages of tumor progression. Levels of interleukin-8 (IL-8), IL-10 and transforming <em>growth</em> <em>factor</em> beta (TGF beta) were significantly elevated in patients with stages I and II carcinoma. The serum levels of tumor necrosis <em>factor</em> alpha (TNF alpha), granulocyte/macrophage-colony-stimulating <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (BFGF), IL-7 and IL-2 were significantly elevated in patients with stages II and III carcinoma and the serum level of tumor necrosis <em>factor</em> beta (TNF beta) was slightly elevated in patients with stage II carcinoma only. The serum levels of IL-1 alpha, IL-1 beta and IL-6 were not elevated in endometrial cancer patients in any of the clinical stages. The results showed that progression of endometrial cancer is associated with increased serum levels of cytokines, <em>growth</em> <em>factors</em> and angiogenin, which possibly amplify angiogenesis during different clinical stages.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor <em>growth</em>, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-<em>20</em>. ©<em>20</em>17 AACR.

BACKGROUND

Fufang e׳jiao jiang (FEJ), which has been widely used in clinic to replenish qi (vital energy) and nourish blood, is a famous traditional Chinese medicine formula made up of Colla corii asini (donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus.), Radix codonopsis pilosulae (the root of Codonopsis pilosula (Franch.) Nannf.), Radix ginseng rubra (the steamed and dried root of Panax ginseng C.A. Mey.), Fructus crataegi (the fruit of Crataegus pinnatifida Bunge) and Radix rehmanniae preparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey.). The present study aimed to investigate the hematopoietic effects of FEJ on myelosuppressed mice induced by radiotherapy and chemotherapy systematically and to explore the underlying hematopoietic regulation mechanisms.

METHODS

The myelosuppressed mouse model was induced by (60)Co radiation, cyclophosphamide and chloramphenicol. FEJ was then administered by i.g. at the dosages of 5, 10, or <em>20</em> mL/kg·d for 10d. The numbers of blood cells from peripheral blood and bone marrow nucleated cells (BMNC) were counted. Body weight and the thymus and spleen indices were also measured. The numbers of hemopoietic progenitor cells and colony-forming unit-<em>fibroblast</em> (CFU-F) were measured in vitro. The ratio of hematopoietic stem cells (HSC) in BMNC, cell cycle and apoptosis of BMNC were determined by flow cytometry. The histology of femoral bone was examined by H&E staining. The levels of transforming <em>growth</em> <em>factor</em>-β (TGF-β), tumor necrosis <em>factor</em>-α (TNF-α), erythropoietin (EPO), granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6) in serum were measured by ELISA. IL-1β, IL-3, IL-6 mRNA levels in spleen were detected by real-time quantitative PCR (RT-qPCR). In addition, bone marrow stromal cells (BMSC) were cultured in vitro followed by treatment with different doses of FEJ (2.5, 5, 10 μL/mL) for 48 h. Then the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were determined by ELISA and RT-qPCR, respectively.

RESULTS

FEJ could significantly increase the numbers of peripheral blood cells and BMNC, and reverse the loss of body weight and the atrophy of thymus and spleen in a dose-dependent manner. The quantities of hemopoietic progenitor cells and CFU-F in bone marrow were also significantly increased in a dose-dependent manner after FEJ administration. A high-dose FEJ of <em>20</em> mL/kg·d could significantly increase the ratio of HSC in BMNC, promote bone marrow cells entering the proliferative cycle phase (S+G2/M) and prevent cells from proceeding to the apoptotic phase. FEJ could also improve the femoral bone marrow morphology. Furthermore, FEJ could increase the levels of GM-CSF and IL-3 and reduce the level of TGF-β in serum, and enhance the expressions of IL-1β and IL-3 mRNA in spleen. Lastly, the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were elevated after treatment with FEJ.

CONCLUSIONS

FEJ was clearly confirmed to promote the recovery of bone marrow hemopoietic function in a myelosuppressed mouse model, which may be attributed to (i) improving bone marrow hematopoietic microenvironment; (ii) facilitating the cell proliferation and preventing BMNC from apoptosis; (iii) stimulating the expressions of IL-1β, IL-3, IL-6, SCF and GM-CSF and inhibiting the expression of TGF-β.

Granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) is a small glycoprotein <em>growth</em> <em>factor</em> which stimulates the production and function of neutrophils, eosinophils and monocytes. GM-CSF can be produced by a wide variety of tissue types, including <em>fibroblasts</em>, endothelial cells, T cells, macrophages, mesothelial cells, epithelial cells and many types of tumor cells. In most of these tissues, inflammatory mediators, such as interleukin 1, interleukin 6, tumor necrosis <em>factor</em> or endotoxin, are potent inducers of GM-CSF gene expression, which occurs at least partly by post-transcriptional stabilization of the GM-CSF mRNA. The biological effects of GM-CSF are mediated through binding to cell surface receptors, which appear to be widely expressed by hematopoietic cells and also by some non-hematopoietic cells, such as endothelial cells. Receptor expression is characterized by low number (<em>20</em>-<em>20</em>0/cell) and high affinity (Kd = <em>20</em>-100 pM). At least two different functional classes of GM-CSF receptor have been identified. The neutrophil GM-CSF receptor exclusively binds GM-CSF, while interleukin 3 competes for binding of GM-CSF to a second class of receptors detected on some leukemic cell lines, such as KG1 and MO-7E. Signal transduction involves activation of a tyrosine kinase and possibly G protein-coupled stimulation of Na+/H+ exchange. The exact relationship of the two receptors needs further clarification.

OBJECTIVE

Serum concentrations of the hepatokine fibroblast growth factor (FGF) 21 are elevated in obesity, type-2 diabetes, and the metabolic syndrome. We asked whether FGF21 levels differ between subjects with metabolically healthy vs. unhealthy obesity (MHO vs. MUHO), opening the possibility that FGF21 is a cross-talker between liver and adipose tissue in MUHO. Furthermore, we studied the effects of chronic FGF21 treatment on adipocyte differentiation, lipid storage, and adipokine secretion.

METHODS

In 20 morbidly obese donors of abdominal subcutaneous fat biopsies discordant for their whole-body insulin sensitivity (hereby classified as MHO or MUHO subjects), serum FGF21 was quantified. The impact of chronic FGF21 treatment on differentiation, lipid accumulation, and adipokine release was assessed in isolated preadipocytes differentiated in vitro.

RESULTS

Serum FGF21 concentrations were more than two-fold higher in MUHO as compared to MHO subjects (457 ± 378 vs. 211 ± 123 pg/mL; p < 0.05). FGF21 treatment of human preadipocytes for the entire differentiation period was modestly lipogenic (+15%; p < 0.05), reduced the expression of key adipogenic transcription factors (PPARG and CEBPA, -15% and -40%, respectively; p < 0.01 both), reduced adiponectin expression (-20%; p < 0.05), markedly reduced adiponectin release (-60%; p < 0.01), and substantially increased leptin (+60%; p < 0.01) and interleukin-6 (+50%; p < 0.001) release.

CONCLUSIONS

The hepatokine FGF21 exerts weak lipogenic and anti-adipogenic actions and marked adiponectin-suppressive and leptin and interleukin-6 release-promoting effects in human differentiating preadipocytes. Together with the higher serum concentrations in MUHO subjects, our findings reveal FGF21 as a circulating factor promoting the development of metabolically unhealthy adipocytes.

The serum levels of several cytokines were determined in 94 patients with Adamantiades-Behçet's disease (ABD), aged 36.1+/-11.0 years, during the active stage (n = 75) and the inactive stage (n = 19) of the disease. A group of 75 healthy individuals matched for age and sex served as controls. Cytokine levels were determined using commercially available ELISA kits. Of the 75 patients with active disease and 19 with inactive disease, 38 (51%) and 4 (21%), respectively, and 23 healthy controls (31%) were found to have detectable levels of interleukin 8 (IL-8) in their serum (P < 0.05). Also, increased IL-8 serum levels were found in patients with active disease (median 12 pg/ml, P = 0.010) compared to patients with inactive disease (< or = 10 pg/ml) and to healthy controls (< or = 10 pg/ml). In particular, patients with oral aphthous ulcers (n = 51, 34 pg/ml) and neurological features (n = 4, 71 pg/ml) exhibited increased IL-8 levels. In contrast, there was no correlation between disease activity and the serum levels of IL-1alpha, IL-1beta, tumor necrosis <em>factor</em> alpha (TNF-alpha), soluble intercellular adhesion molecule-1 or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In a second set of experiments, the involvement of dermal microvascular endothelial cells in IL-8 secretion was investigated. Immortalized human dermal microvascular endothelial cells (HMEC-1 cells) were maintained for 4 h in vitro with serum from 18 ABD patients or with IL-1beta, a known stimulator of IL-8 synthesis, TNF-alpha or their combination at five- to tenfold higher concentrations than those found in the serum of ABD patients. Increased IL-8 secretion was found after incubation with ABD patients' serum (median <em>20</em> pg/ml), but IL-1beta, TNF-alpha and IL-1beta + TNF-alpha failed to induce IL-8 secretion by HMEC-1 cells (< or = 1-1.2 pg/ml) in biologically relevant concentrations. Our study showed increased IL-8 serum levels in ABD patients with active oral and neurological manifestations. Human microvascular endothelial cells may, at least partially, be responsible for the enhanced IL-8 secretion in the active stage of the disease.

OBJECTIVE

Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes.

METHODS

The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen.

RESULTS

We found that combination of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over <em>20</em>-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive.

CONCLUSIONS

The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells.

Bevacizumab is a humanized recombinant monoclonal antibody that neutralizes vascular endothelial <em>growth</em> <em>factor</em>, an agent with proangiogenic effects in melanoma. Interferon alpha (IFN-α) has antiangiogenic properties through its ability to downregulate basic-<em>fibroblast</em> <em>growth</em> <em>factor</em> levels. We hypothesized that the coadministration of these agents would lead to tumor regression. Patients with metastatic melanoma received bevacizumab 15 mg/kg intravenously on day 1 of the 2-week cycle. IFN-α was administered thrice weekly at 5 MU/m subcutaneously during cycle 1 and was increased to 10 MU/m during cycle 2. Patients were restaged every 6 cycles. Patients with stable disease or a response continued with therapy. Baseline serum vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em> were measured. Twenty-five patients were accrued. Mean age was 58.4 years. Eleven patients required IFN-α dose reductions due to toxicity. Common grade 3 toxicities associated with IFN-α included fatigue and myalgia. Bevacizumab administration was associated with grade 2-3 proteinuria in 6 patients. Grade 4 adverse events were pulmonary embolus (1), myocardial infarction (1), and stroke (1). Six patients had a partial response, and 5 patients exhibited stable disease that lasted more than 24 weeks (range: 30 to 122 wk). Median progression-free survival and overall survival were 4.8 and 17 months, respectively. Significantly lower <em>fibroblast</em> <em>growth</em> <em>factor</em> levels were observed in patients with a partial response compared to those with stable or progressive disease (P=0.040). Administration of bevacizumab with IFN led to a clinical response in 24% of patients with stage IV melanoma and stabilization of disease in another <em>20</em>% of patients. This regimen has activity in advanced melanoma.

The aim of the study is to characterize the signal transduction leading to interstitial fibrosis in the pathogenesis of atrial fibrillation (AF) and atrial remodeling. Samples of the left atrial appendage (LA) from patients with AF showed higher collagen content (73 ± 5 vs. 38 ± 2 μg/mg protein) and 2.5-fold increased collagen crosslinking compared to patients with sinus rhythm (SR). Affymetrix-assays, RT-PCR and western Blot analysis revealed that LA of AF patients are characterized by increased lysyl oxidase (LOX) mRNA (218 ± 42%) and protein (253 ± 11%) expression. This was associated with increased expression of connective tissue <em>growth</em> <em>factor</em> (CTGF), fibronectin and Rac1 activity compared to SR. In neonatal cardiac <em>fibroblasts</em>, the Rac1 specific small molecule inhibitor NSC23766 prevented angiotensin II (AngII) induced upregulation of LOX (214 ± 16%) expression. Inhibition of CTGF by siRNA transfections completely inhibited AngII induced LOX expression. The LOX specific small molecule inhibitor BAPN prevented AngII and CTGF induced fibronectin expression. Left atria of transgenic mice with cardiac overexpression of Rac1 (RacET) that develop AF at high age exhibited upregulation of CTGF as well as LOX (187 ± 7%) and fibronectin (627 ± 146%) expression. Atria of RacET showed increased collagen content (28 ± 2 μg/mg protein) and crosslinking (10 ± 0.7) compared to wildtypes (<em>20</em> ± 0.4 μg/mg protein; 5 ± 0.9). Left atrial myocardium of patients with atrial fibrillation is characterized by increased lysyl oxidase and fibronectin expression as well as collagen cross-linking. In cardiac <em>fibroblasts</em>, Rac1 GTPase mediates upregulation of fibronectin via LOX and CTGF. Inhibition of this signaling pathway may therefore represent a target for the prevention of fibrotic atrial remodeling.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> is a heparin-binding protein known to stimulate angiogenesis and promote wound healing in tissues. Since Crohn's disease is characterized in part by submucosal vascular proliferation, we sought to determine whether serum basic <em>fibroblast</em> <em>growth</em> <em>factor</em> is elevated in children with Crohn's disease and whether serum levels reflect disease activity. Sera were obtained from 64 children with Crohn's disease, 44 children with ulcerative colitis, <em>20</em> children with functional abdominal pain, and 29 from children with documented inflammatory disease evaluated in our gastroenterology program. Disease activity indices and clinical data were gathered prospectively for the inflammatory bowel disease patients. Serum basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels were measured by enzyme-linked immunosorbent assay. Although the mean basic <em>fibroblast</em> <em>growth</em> <em>factor</em> level did not significantly differ between children with Crohn's disease and other conditions, there was a strong (r = 0.53, P < 0.001) correlation between basic <em>fibroblast</em> <em>growth</em> <em>factor</em> level and disease activity. The relationship of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> with disease activity persisted even after adjusting for other covariates (including age, sex, hematocrit, albumin, and sedimentation rate) in a multivariate linear regression model. There was also a statistically significant, although less strong correlation (r = 0.33, P = 0.03) between basic <em>fibroblast</em> <em>growth</em> <em>factor</em> level and disease activity in ulcerative colitis. While basic <em>fibroblast</em> <em>growth</em> <em>factor</em> is not a specific marker for Crohn's disease, serum levels reflect disease activity. Therefore, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> release may be important in mediating the angiogenesis and wound healing seen in Crohn's disease.

BACKGROUND

Adipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.

METHODS

Normal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (<em>20</em>%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible <em>factor</em> (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these <em>growth</em> <em>factors</em> on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF-1α-binding hypoxia responsive element in FGF-2 was performed. HIF-1α was knocked down by siRNA, and FGF-2 expression was investigated.

RESULTS

ASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5-15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF-1α expression. The levels of VEGF and FGF-2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF-2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF-1α to FGF-2 in hypoxia. HIF-1α knockdown by siRNA partially inhibited the FGF-2 expression of ASCs induced by hypoxia.

CONCLUSIONS

ASC proliferation was enhanced by hypoxia. HIF-1α activation, FGF-2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.

Up to <em>20</em>% of patients with pilocytic astrocytoma (PA) experience a poor outcome. BRAF alterations and <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) point mutations are key molecular alterations in Pas, but their clinical implications are not established. We aimed to determine the frequency and prognostic role of these alterations in a cohort of 69 patients with PAs. We assessed KIAA1549:BRAF fusion by fluorescence in situ hybridization and BRAF (exon 15) mutations by capillary sequencing. In addition, FGFR1 expression was analyzed using immunohistochemistry, and this was compared with gene amplification and hotspot mutations (exons 12 and 14) assessed by fluorescence in situ hybridization and capillary sequencing. KIAA1549:BRAF fusion was identified in almost 60% of cases. Two tumors harbored mutated BRAF. Despite high FGFR1 expression overall, no cases had FGFR1 amplifications. Three cases harbored a FGFR1 p.K656E point mutation. No correlation was observed between BRAF and FGFR1 alterations. The cases were predominantly pediatric (87%), and no statistical differences were observed in molecular alterations-related patient ages. In summary, we confirmed the high frequency of KIAA1549:BRAF fusion in PAs and its association with a better outcome. Oncogenic mutations of FGFR1, although rare, occurred in a subset of patients with worse outcome. These molecular alterations may constitute alternative targets for novel clinical approaches, when radical surgical resection is unachievable.

The conversion of C19 steroids to estrogens occurs in a number of tissues and is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom). Previously, conversion of radiolabeled androstenedione to estrone has been demonstrated in uterine leiomyomas. By use of reverse transcription followed by polymerase chain reaction amplification of total RNA together with a rat cRNA as an internal standard, we detected and quantified P450arom transcripts in total RNA isolated from 32 of the 35 leiomyoma (91%) and from 18 of the 24 adjacent myometrial (75%) tissue samples from 26 women. P450arom transcripts were not detectable in myometrial tissues from disease-free uteri (n = 8). P450arom transcript levels in leiomyomas were similar to those in adipose tissue (normalized to total RNA) and were 1.5- to 25-fold higher than those in adjacent myometrial tissues. We did not find any correlation between P450arom transcript levels and leiomyoma size, histopathology, uterine weight, or patient age. In leiomyoma smooth muscle cells in culture (n = 4) and tissue explants (n = 4), aromatase activity was stimulated by dibutyryl cAMP, and this effect was potentiated by a phorbol ester. These increases in aromatase expression were accompanied by comparable increases in the levels of translatable P450arom mRNA. Treatment with dexamethasone or platelet-derived <em>growth</em> <em>factor</em> did not stimulate aromatase expression. Consistently higher levels of aromatase activity and P450arom transcripts were found in the leiomyoma tissues than in smooth muscle cells in culture (2- to <em>20</em>-fold). Reverse transcription-polymerase chain reaction analysis of untranslated 5'-termini of mRNA species in leiomyomas revealed the use of primarily promoter II (the ovarian-type promoter) for CYP19 gene transcription. Leiomyomas also contain some transcripts with untranslated exon I.4 (previously found in adipose stromal cells and skin <em>fibroblasts</em>). Placental-type promoter-specific 5'-ends were not present in leiomyomas. We conclude that aromatase expression in leiomyomas is regulated by the rate of CYP19 gene transcription, which is, in turn, regulated by the use of tissue-specific promoters. These findings are consistent with the hypothesis that localized estrogen biosynthesis may be of pathological significance in the promotion of leiomyoma <em>growth</em>.

We recently reported the first evidence of placental endoplasmic reticulum (ER) stress in the pathophysiology of human intrauterine <em>growth</em> restriction. Here, we used a mouse model to investigate potential underlying mechanisms. Eif2s1(tm1RjK) mice, in which Ser51 of eukaryotic initiation <em>factor</em> 2 subunit alpha (eIF2α) is mutated, display a 30% increase in basal translation. In Eif2s1(tm1RjK) placentas, we observed increased ER stress and anomalous accumulation of glycoproteins in the endocrine junctional zone (Jz), but not in the labyrinthine zone where physiological exchange occurs. Placental and fetal weights were reduced by 15% (97 mg to 82 mg, p < 0.001) and <em>20</em>% (1009 mg to 798 mg, p < 0.001), respectively. To investigate whether ER stress affects bioactivity of secreted proteins, mouse embryonic <em>fibroblasts</em> (MEFs) were derived from Eif2s1(tm1RjK) mutants. These MEFs exhibited ER stress, grew 50% slower, and showed reduced Akt-mTOR signalling compared to wild-type cells. Conditioned medium (CM) derived from Eif2s1(tm1RjK) MEFs failed to maintain trophoblast stem cells in a progenitor state, but the effect could be rescued by exogenous application of FGF4 and heparin. In addition, ER stress promoted accumulation of pro-Igf2 with altered glycosylation in the CM without affecting cellular levels, indicating that the protein failed to be processed after release. Igf2 is the major <em>growth</em> <em>factor</em> for placental development; indeed, activity in the Pdk1-Akt-mTOR pathways was decreased in Eif2s1(tm1RjK) placentas, indicating loss of Igf2 signalling. Furthermore, we observed premature differentiation of trophoblast progenitors at E9.5 in mutant placentas, consistent with the in vitro results and with the disproportionate development of the labyrinth and Jz seen in placentas at E18.5. Similar disproportion has been reported in the Igf2-null mouse. These results demonstrate that ER stress adversely affects placental development, and that modulation of post-translational processing, and hence bioactivity, of secreted <em>growth</em> <em>factors</em> contributes to this effect. Placental dysmorphogenesis potentially affects fetal <em>growth</em> through reduced exchange capacity.

In the course of experiments to identify and characterize the <em>factors</em> that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, <em>20</em>, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription <em>factors</em> in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like <em>factor</em> 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, <em>growth</em> <em>factor</em> receptors associated with EMT were analyzed. Upregulation of the <em>growth</em> <em>factor</em> receptor transcripts, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1), platelet-derived <em>growth</em> <em>factor</em> receptor, alpha polypeptide (PDGFRA), platelet-derived <em>growth</em> <em>factor</em> receptor, beta polypeptide (PDGFRB), and transforming <em>growth</em> <em>factor</em>, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.

Insulin-like <em>growth</em> <em>factors</em> (IGFs) and their specific regulatory binding proteins (IGFBPs) are postulated to play a key role in bone metabolism. To date, IGFBP-2 through -6 have been characterized in bone cell systems. In this study we focused on IGFBP-1. Primary cultures of normal human osteoblasts derived from trabecular bone (hOB cells) expressed low levels of IGFBP-1 messenger RNA (mRNA), as determined by Northern analyses. Treatment of hOB cells with 1 microM cortisol or 100 nM dexamethasone for <em>20</em> h stimulated IGFBP-1 mRNA expression 5-fold and increased levels of immunoassayable IGFBP-1 in the conditioned medium 3-fold. Estradiol and progesterone had no effect. IGFBP-1 expression was not observed in U-2, TE-85, or MG-63 human osteosarcoma cell lines or in normal human <em>fibroblasts</em>. Insulin (1-100 nM) potently inhibited both basal and glucocorticoid-stimulated IGFBP-1 expression in hOB cells. Insulin had little or no effect on steady state levels of the other IGFBP mRNA. A monoclonal antibody to the insulin receptor blocked insulin binding to insulin receptors and completely prevented insulin-induced suppression of IGFBP-1. In summary, we have documented IGFBP-1 mRNA and protein expression in normal nontransformed human osteoblastic cells. This expression was stimulated by glucocorticoids and inhibited by insulin in a manner similar to IGFBP-1 regulation in hepatocytes. Insulin acts through insulin receptors on hOB cells. We postulate that IGFBP-1 produced by osteoblasts in vivo can modulate local actions of IGF on bone formation in response to changes in glucocorticoid and insulin concentrations.

Platelet-derived <em>growth</em> <em>factor</em> AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and <em>20</em>%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of <em>growth</em> <em>factor</em>-induced transmodulation of receptor expression during atherogenesis.

The effects of dermal <em>fibroblasts</em> on keratinocyte out<em>growth</em> on collagen substrata was studied using an in vitro keratinocyte-collagen gel composite model. Skin <em>fibroblasts</em> were seeded inside collagen gels, which remained attached to the cell culture plastic substratum. <em>Fibroblasts</em> incorporated in collagen gels were either kept viable throughout the study, or were lysed hypotonically with water at different time intervals (2 hours and 5 days). Results show that very little keratinocyte out<em>growth</em> occurred on either plain collagen gels or gels that had previously contained viable <em>fibroblasts</em> for 2 hours. A 3- to 4-fold increase in keratinocyte out<em>growth</em> occurred on collagen gels that had previously contained viable <em>fibroblasts</em> for 5 days. A striking increase (<em>20</em>-fold) in keratinocyte out<em>growth</em> was observed on collagen gels that contain viable <em>fibroblasts</em>. The effect of <em>fibroblast</em> diffusible <em>factors</em> on keratinocyte out<em>growth</em> was further studied with a co-culture system using Millicell inserts. It was found that the co-culture of <em>fibroblasts</em> with the composite enhanced keratinocyte out<em>growth</em> on collagen gels that had previously contained viable <em>fibroblasts</em> for 5 days. Among all, however, the keratinocyte out<em>growth</em> was far better on gels containing viable <em>fibroblasts</em>. Addition of keratinocyte <em>growth</em> <em>factor</em> or its neutralizing antibody did not affect keratinocyte out<em>growth</em>. These results suggest that dermal <em>fibroblasts</em> can activate keratinocyte out<em>growth</em> on collagen matrices through some diffusible <em>factors</em> other than keratinocyte <em>growth</em> <em>factor</em>, and epithelial-mesenchymal interactions exert some special effects on keratinocyte out<em>growth</em> on collagen gels.

To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (<em>20</em>% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P < 0.01) and in vitro angiogenesis (2.4 times, P < 0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.

We investigated the relative roles of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and transforming <em>growth</em> <em>factor</em> beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a three-dimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC50 of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10-<em>20</em> ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3-0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a <em>fibroblast</em>-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/<em>20</em> and 19/<em>20</em> AML supernatents respectively. The degree of in vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic <em>factors</em> produced by AML cells. The expression of different angiogenic <em>factors</em> was studied using reverse transcription polymerase chain reaction. Cells from 17/<em>20</em> AML patients showed wide variation in spontaneous vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression, 4/19 expressed varied spontaneous blastic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the <em>growth</em> advantage of the malignant counterpart as a result of the paracrine production of <em>growth</em> <em>factors</em> produced by the surrounding endothelial cells.

A characteristic feature of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) family is the structural diversity generated by alternative splicing. The FGFR3 gene encodes two splice variants because of the mutually exclusive use of the exons IIIb and IIIc. In the present study we examined the expression of the two different splice forms IIIb and IIIc of FGFR3 in developing mouse embryos (12 days p.c., 14 days p.c., <em>20</em> days p.c.). The overall level of the IIIc exon splice product surpassed that of the IIIb exon form. The IIIc mRNA was detected in the developing brain and in the spinal cord. Outside the nervous system very strong expression was observed in the vertebra and in all other bony structures. In contrast, the IIIb splice form was restricted to epithelial structures with no expression detected in the central nervous system and bone.

BACKGROUND

Biological therapies are a new breakthrough in the treatment of psoriasis and psoriatic arthritis (PsA). Among these, tumour necrosis factor (TNF)-alpha antagonists such as infliximab and etanercept are the most promising as TNF is considered to be essential in driving cytokine cascade at sites of cutaneous and synovial inflammation in this disease.

OBJECTIVE

To evaluate the time-related response of serum cytokine release during infliximab monotherapy and assess serum cytokine levels in order to provide a fast, minimally invasive tool to monitor and/or predict efficacy of anti-TNF-alpha therapy.

METHODS

Twenty patients affected by PsA with Psoriasis Area and Severity Index (PASI) score between 0.4 and 42.8 were treated with infliximab for 30-42 weeks. The assessment of arthritis severity was performed using the American College of Rheumatology (ACR) criteria and ultrasonography evaluation. The treatment schedule consisted of infliximab (5 mg kg(-1) intravenously) at 0, 2 and 6 weeks and every 12 weeks on an individual basis determined by therapeutic results and adverse events reported. At baseline and before every infusion blood samples were taken to assess serum cytokine levels [TNF-alpha, interleukin (IL-6), E-selectin, vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), matrix metalloproteinase (MMP-2)].

RESULTS

Eighteen of 20 psoriatic patients achieved>> 50% improvement and 14 of 20 patients attained>> 75% improvement in the PASI score at 10 weeks. All arthritic patients achieved>> 50% improvement (ACR-50) and 16 of 20 patients attained>> 75% improvement (ACR-75) at 10 weeks. TNF-alpha did not decrease immediately during the first part of the study. A significant decrease was detected at week 12 (P < 0.01). In contrast, IL-6, VEGF, FGF and E-selectin showed significant decreases after early infliximab infusions. PASI was not correlated with TNF-alpha in the serum but was significantly correlated with FGF, VEGF and MMP-2. Treatment was well tolerated and there were no significant adverse events in most patients, other than an urticarial reaction and an autoimmune hepatitis.

CONCLUSIONS

Monotherapy with infliximab has to be considered an efficacious and safe treatment for PsA in comparison with traditional disease-modifying antirheumatic drugs. The resolution of cutaneous and synovial symptoms is not related to TNF-alpha serum levels in the initial phases. Apoptosis may play an important role in the modulation of the inflammatory response.