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.

Wound healing is primarily controlled by the proliferation and migration of keratinocytes and <em>fibroblasts</em> as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and <em>fibroblasts</em> and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and <em>fibroblasts</em> were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with <em>fibroblasts</em> proliferated significantly faster than those not in contact with <em>fibroblasts</em>, but in the late stage (11(th) to <em>15</em>(th) day), keratinocyte <em>growth</em> slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with <em>fibroblasts</em> in direct contact, but not in the transwells. Furthermore, the effects of the <em>fibroblasts</em> on keratinocyte migration and <em>growth</em> at early culture stage correlated with heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected <em>fibroblasts</em> exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with <em>fibroblasts</em> stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

METHODS

Human lumbar anulus tissue and cultured human lumbar anulus cells were used in retrospective studies of the immunocytochemical localization of the vitamin D receptor (VDR) in disc tissue, and of the in vitro effects of the active metabolite of vitamin D, 1,25(OH)2D3, on anulus cell proliferation, cytokine, and proteoglycan (PG) production. 24,25-D3 was also analyzed. Studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens and from control donors.

OBJECTIVE

To determine if human anulus cells express the VDR in vivo, and to test the effect of in vitro exposure to 1,25(OH)2D3 and 24,25-D3 on anulus cell proteoglycan and cytokine production in 3-dimensional culture.

BACKGROUND

Intragenic polymorphisms in the VDR gene have been associated with disc degeneration. 1,25(OH)2D3 has well-recognized effects on calcium homeostasis and bone mineralization, and is a negative growth regulator of a variety of normal and tumor cells. Its effects on human disc cells, however, are unexplored.

METHODS

Immunocytochemistry was performed on human lumbar disc anulus tissue from 19 subjects; human disc cells were cultured to test the effect of 1,25(OH)2D3 on proliferation of anulus cells from 5 subjects. A paired experimental design was used to determine proteoglycan production in control or 1,25(OH)2D3-treated cells, or in control or 24,25-D3-treated cells using the dimethylmethylene blue assay. A paired experimental design was also used to identify differences in cytokine production in conditioned media from control or 1,25(OH)2D3-treated cells, or in control or 24,25-D3-treated cells using ELISA assays.

RESULTS

Immunocytochemistry documented expression of the VDR in anulus cells. Young donor discs (aged newborn, 15 years) showed positive localization in all cells of the outer anulus, and some inner anulus cells. In adults (mean age, 38.9 years), some, but not all anulus cells, showed positive localization. Exposure to 10M 1,25(OH)2D3 in monolayer significantly reduced cell proliferation in vitro (P = 0.03). PG production in 3-dimensional was unchanged from control in both 1,25(OH)2D3- and 24,25-D3-treated cells. Cytokine production differed, however. 1,25(OH)2D3-treated cells showed significantly decreased production of vascular endothelial <em>growth</em> factor (VEGF) (P = 0.01), monocyte chemoattractant protein-1 (MCP-1) (P = 0.0006), angiogenin (P = 0.002), and thrombopoietin (P = 0.03) compared with controls. 24,25-D3-treated cells showed significantly elevated vascular endothelial <em>growth</em> factor-D (P = 0.01), beta-fibroblast <em>growth</em> factor (0.03), and significantly decreased interleukin-8, interferon-gamma, leptin, MCP-1, and TIMP-2 (tissue inhibitor of metalloproteinases-2) compared with controls (P <or= 0.01).

CONCLUSIONS

Data suggest that 1,25(OH)2D3 and 24,25-D3 may play roles as regulators of cell proliferation and production of specific cytokines in the lumbar anulus.

BACKGROUND

We previously described increased levels of <em>growth</em> and differentiation <em>factor</em> <em>15</em> (GDF-<em>15</em>) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-<em>15</em> as a biomarker for mitochondrial diseases affecting children and compared it to <em>fibroblast</em>-<em>growth</em> <em>factor</em> 21 (FGF-21). To investigate the mechanism of GDF-<em>15</em> induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction.

METHODS

We analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-<em>15</em> and FGF-21 circulating levels were determined by ELISA.

RESULTS

Our results showed that in children with mitochondrial diseases GDF-<em>15</em> levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-<em>15</em> was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-<em>15</em> for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-<em>15</em> and or FGF-21 correctly identified a larger proportion of patients than elevated levels of GDF-<em>15</em> or FGF-21 alone. GDF-<em>15</em>, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both <em>factor</em>s significantly correlated.

CONCLUSIONS

Our data indicate that GDF-<em>15</em> is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-<em>15</em> and FGF-21 improves the disease detection ability of either <em>factor</em> separately. Finally, we demonstrate for the first time that GDF-<em>15</em> is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-21 levels.

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 <em>15</em> 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 20% of patients. This regimen has activity in advanced melanoma.

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 (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors 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 20% 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 KIAA<em>15</em>49:BRAF fusion by fluorescence in situ hybridization and BRAF (exon <em>15</em>) 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. KIAA<em>15</em>49: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 KIAA<em>15</em>49: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.

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 <em>15</em>% (97 mg to 82 mg, p < 0.001) and 20% (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.

BACKGROUND

The 2009 KDIGO (Kidney Disease: Improving Global Outcomes) chronic kidney disease-mineral and bone disorder clinical practice guideline suggests correcting 25-hydroxyvitamin D3 (25[OH]D) levels<30ng/mL in patients treated with maintenance hemodialysis, but does not provide a specific treatment protocol.

METHODS

2-center, double-blind, randomized, 13-week, controlled trial followed by a 26-week open-label study.

METHODS

55 adult maintenance hemodialysis patients with 25(OH)D levels<30ng/mL were recruited from June 2008 through October 2009.

METHODS

Cholecalciferol, 25,000IU, per week orally versus placebo for 13 weeks, then 26 weeks of individualized cholecalciferol prescription based on NKF-KDOQI (National Kidney Foundation-Kidney Disease Outcomes Quality Initiative) guidelines.

RESULTS

Primary end point was the percentage of patients with 25(OH)D levels≥30ng/mL at 13 weeks. Secondary outcomes included the percentage of patients with normal calcium, phosphorus, and intact parathyroid hormone (iPTH) blood levels. Safety measures included incidence of hypercalcemia and hypervitaminosis D.

METHODS

Blood calcium and phosphate were measured weekly; iPTH, 25(OH)D, 1,25-dihydroxyvitamin D3 (1,25[OH]2D), and bone turnover markers, trimonthly; fetuin A and fibroblast growth factor 23 (FGF-23) serum levels and aortic calcification scores were determined at weeks 0 and 39.

RESULTS

The primary end point significantly increased in the treatment group compared with the placebo group (61.5% vs 7.4%; P<0.001), as well as 1,25(OH)2D levels (22.5 [IQR, 15-26] vs 11 [IQR, 10-15]pg/mL; P<0.001) and the proportion of patients achieving the target calcium level (76.9% vs 48.2%; P=0.03). Incidence of hypercalcemia and phosphate and iPTH levels were similar between groups. The second 26-week study phase did not significantly modify the prevalence of 25(OH)D level≥30ng/mL in patients issued from the placebo group.

CONCLUSIONS

Small size of the study population.

CONCLUSIONS

Oral weekly administration of 25,000IU of cholecalciferol for 13 weeks is an effective, safe, inexpensive, and manageable way to increase 25(OH)D and 1,25(OH)2D levels in hemodialysis patients. Further evaluation of clinical end points is suggested.

Important characteristics of chronic obstructive pulmonary disease (COPD) include airway and vascular remodeling, the molecular mechanisms of which are poorly understood. We assessed the role of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) in pulmonary vascular remodeling by examining the expression pattern of FGF-1, FGF-2, and the FGF receptor (FGFR-1) in peripheral area of lung tissues from patients with COPD (FEV(1) < or = 75%; n = <em>15</em>) and without COPD (FEV(1)>> or = 85%; n = 13). Immunohistochemical staining results were evaluated by digital video image analysis as well as by manual scoring. FGF-1 and FGFR-1 were detected in vascular smooth muscle (VSM), airway smooth muscle, and airway epithelial cells. FGF-2 was localized in the cytoplasm of airway epithelium and in the nuclei of airway smooth muscle, VSM, and endothelial cells. In COPD cases, an unequivocal increase in FGF-2 expression was observed in VSM (3-fold, P = 0.001) and endothelium (2-fold, P = 0.007) of small pulmonary vessels with a luminal diameter under 200 micro m. In addition, FGFR-1 levels were elevated in the intima (1.5-fold, P = 0.05). VSM cells of large >> 200 micro m) pulmonary vessels showed increased staining for FGF-1 (1.6-fold, P < 0.03) and FGFR-1 (1.4-fold, P < 0.04) in COPD. Pulmonary vascular remodeling, assessed as the ratio of alpha-smooth muscle actin staining and vascular wall area with the lumen diameter, was increased in large vessels of patients with COPD (P = 0.007) and was inversely correlated with FEV(1) values (P < 0.007). Our results suggest an autocrine role of the FGF-FGFR-1 system in the pathogenesis of COPD-associated vascular remodeling.

<em>Growing</em> axons in the peripheral nervous system (PNS) encounter a variety of cellular and extracellular substrates. Since it is difficult to sort out the possible contributions of these diverse components of the extracellular environment to axonal guidance in vivo, I have developed an in vitro system to study neurite out<em>growth</em> on two classes of cells which may provide as substrates for <em>growing</em> axons during development or regeneration: glial cells, e.g., astrocytes and Schwann cells, and nonglial cells, e.g., <em>fibroblasts</em>. Although neurites from sympathetic and spinal sensory ganglia explants grew onto preformed monolayers of both glial and nonglial cells, glial cells were a markedly better substrate. On the glial cells the neurites extended at a rate of 25 to 30 micron/hr and traveled singly or in fine fascicles; their <em>growth</em> cones displayed long filopodia and migrated on the upper surface of the monolayer cells. Conditioned media experiments suggested that neurite out<em>growth</em> on glial cell monolayers was not mediated by soluble secreted <em>factors</em>. These results indicate that the glial cell surface is an attractive substrate for neurite out<em>growth</em>. In contrast, on nonglial cells the rate of out<em>growth</em> was only 10 to <em>15</em> micron/hr, large neurite fascicles were common, and the <em>growth</em> cones migrated beneath the monolayer cells in contact with the underlying artificial substrate. This location of the <em>growth</em> cone, coupled with the observation that conditioned medium from these cells promoted neurite out<em>growth</em> only when bound to artificial substrates, suggests that secreted substrate-associated components may be an important determinant of neurite out<em>growth</em> on nonglial cell monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

Submucosal fibrosis due to excessive accumulation of collagens is an important histologic feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). Heat shock protein 47 (HSP47), a collagen-binding protein, plays an important role in the biosynthesis of procollagens. In the present study, we examined the role of HSP47 in conjunctival scarring in patients with OCP.

METHODS

Biopsy specimens of the conjunctiva of <em>15</em> patients with OCP and 5 normal subjects were studied for the expression of HSP47, transforming <em>growth</em> <em>factor</em> (TGF)-beta1, type I collagen, and type III collagen. The role of TGF-beta1 on the induction of HSP47 and type I collagen by conjunctival <em>fibroblasts</em> was studied by immunostaining, Western blot analysis, and quantitative real-time PCR.

RESULTS

Compared with the control, increased accumulations of type I and type III collagens were detected by immunohistochemistry in fibrotic conjunctiva of patients with OCP. Weak and sparse expression of HSP47 was detected in the epithelial cells and stromal fibroblasts in control conjunctival tissues. In contrast to the control, the expression of HSP47 was markedly increased in the stromal fibroblasts in conjunctival tissues obtained from patients with OCP, as detected by immunohistochemistry. By quantitative real-time PCR, compared with control conjunctival tissues, a 3.4-fold increase in the expression of HSP47 was noted in the conjunctival tissues obtained from patients with OCP. Similar to conjunctival tissues, fibroblasts isolated from conjunctiva of patients with OCP exhibited 4.8-fold increase in the expression of HSP47, compared with control fibroblasts. When conjunctival fibroblasts were treated with various concentration of TGF-beta1, upregulation in the expression of HSP47 and type I collagen was detected.

CONCLUSIONS

This study demonstrated increased expression of HSP47 and TGF-beta1 by conjunctival fibroblasts in biopsy specimens obtained from patients with OCP. TGF-beta1 induced the expression of HSP47 and type I collagen by conjunctival fibroblasts. Increased levels of TGF-beta1 and HSP47 may regulate increased synthesis, assembly, and production of collagens and thereby could significantly contribute to the process of conjunctival scarring in patients with OCP.

Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic <em>factor</em> <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the <em>15</em>-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.

OBJECTIVE

To determine changes in expression of transforming growth factor β-2 (TGF-β2) and basic fibroblast growth factor (bFGF) in scleral desmocytes from anterior and posterior portions of experimentally-induced myopic eyes of guinea pigs.

METHODS

Three groups (n = 10) of 2-week-old guinea pigs were used to develop concave lens-induced myopia (LIM) in one eye via the out-of-focus method for 6, 15, or 30 days respectively, while the other eye in each guinea pig served as the self-control (SC). After myopia induction, lenses were removed, and scleral fibroblasts were cultured and passaged twice. TGF-β2 and bFGF expression levels of scleral desmocytes in LIM and SC groups were compared by immunocytochemistry, quantitative real-time PCR (qRT-PCR) and Western blot analyses.

RESULTS

The TGF-β2 expression of the anterior portion of the sclera in the LIM group was significantly higher at 15 days, and at its highest at 30 days after myopia induction compared with the SC group (P < 0.05). The TGF-β2 staining of the posterior sclera in the LIM group began to rise significantly at 6 days, peaked at 15 days and remained significantly higher than that of the anterior part, as well as the SC group, even at 30 days after myopia induction (P < 0.05). BFGF levels in scleral desmocytes from the anterior and posterior regions in the LIM group were both significantly lower than those of the SC group at all time points after myopia induction (P < 0.05). Furthermore, as the myopia progressed, bFGF expression in the anterior and posterior sclera in the LIM group gradually and statistically significantly decreased compared with the SC group (P < 0.05); however, no significant differences were observed between the anterior and posterior parts in the LIM group at any time after myopia induction (P>> 0.05). All these results were consistent at the mRNA and protein levels.

CONCLUSIONS

During myopia development in lens-induced guinea pigs, the increase in TGF-β2 activity of scleral desmocytes initiated at the posterior pole. Along with the induction time, the TGF-β2 activity in all scleral desmocytes became elevated. By contrast, the bFGF activity showed a general decline in all scleral desmocytes, rather than mainly in the posterior pole. These results imply that expression of TGF-β2 in scleral desmocytes plays a direct role, while that of bFGF exerts an indirect role in myopia development.

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. <em>Fibroblast</em> feeder layers are used to maintain the undifferentiated <em>growth</em> of hES cells and an examination of the conditioned media (CM) of human neonatal <em>fibroblasts</em> (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin <em>fibroblast</em> line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated <em>growth</em> of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into <em>15</em> functional groups. Proteins identified in the extracellular matrix and differentiation and <em>growth</em> <em>factor</em> functional categories were considered most likely to be involved in the maintenance of hES cell <em>growth</em>, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Wnt signaling and inhibition of bone morphogenetic proteins.

Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. To elucidate the mechanism underlying this process, we investigated the effect of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, on the smooth muscle cell cycle. When G0 cells were stimulated with fetal bovine serum and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, DNA synthesis assessed by [3H]thymidine incorporation started about <em>15</em> h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 microM. This inhibition was attenuated when SNAP was added after 9-12 h. SNAP inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the retinoblastoma protein, both of which usually increased from about 9 h, whereas it did not inhibit the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, which normally increased from 0-3 h. Although SNAP reduced the mRNA levels of cyclins E and A, it neither reduced their protein levels nor impaired their association with Cdk2. SNAP did not reduce the mRNA levels of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally elevated from 0-3 h. The mRNA and protein levels of the Cdk inhibitor p21 were high in the early G1 phase, peaking at 3 h and then rapidly decreasing after 6 h. In the presence of SNAP, however, p21 expression was enhanced, and moreover, the later decrease disappeared. SNAP also increased the amount of Cdk2-associated p21. These results suggested that nitric oxide inhibits the G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the Cdk2 inhibition.

The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which <em>factors</em> regulate <em>growth</em> and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and thrombin. IL-4 (5, <em>15</em>, 50, and <em>15</em>0 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived <em>growth</em> <em>factor</em> (20 and 40 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 microM) and forskolin (10 microM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 microM), and forskolin (10 microM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and noggin are two unrelated ligands of two distinctly different signaling pathways that have a similar inhibitory effect on osteoblast differentiation. Because of their differences, we postulated that they probably acted at a different stage within the osteoprogenitor differentiation pathway. This study was performed on primary murine bone cell cultures under conditions where alkaline phosphatase (AP) and type I collagen expression (Col1a1) were observed by day 7 (preosteoblast stage), followed by bone syaloprotein (BSP) at day 11 (early osteoblast) and osteocalcin (OC) by day <em>15</em>-18 (mature osteoblast stage). FGF2 completely inhibited expression of AP and the mRNA transcript for Col1a1, while noggin showed only a partial inhibition of these markers of preosteoblast differentiation. However, the markers of differentiated osteoblasts (BSP and OC) were completely inhibited in both the FGF2 and noggin treated cultures, suggesting that noggin acts at later point in the osteoprogenitor differentiation pathway than FGF2. To further verify that the inhibition was occurring at a different stage of osteoblasts development, primary cultures derived from transgenic mice harboring segments of the collagen promoter driving green fluorescent protein (GFP) that activate at different levels of osteoblast differentiation were analyzed. Consistent with the endogenous markers, pOBCol3.6GFP and pOBCOL2.3GFP transgene activity was completely inhibited by continuous addition of FGF2, while noggin showed partial inhibition of pOBCol3.6GFP and complete inhibition of the pOBCol2.3GFP transgene. Upon removal of either agent, endogenous and GFP markers of osteoblast differentiation reappeared although at a different temporal pattern. This work demonstrates that FGF2 and noggin can reversibly modulate osteoblast lineage differentiation at different maturational stages. These agents may be useful to enrich for and maintain a population of osteoprogenitor cells at a defined stage of differentiation.

Differentiation and luteinization of granulosa cells are induced by gonadotrophic hormones and other substances elevating intracellular levels of cyclic AMP (cAMP). We have investigated the correlation between the potency of these substances to enhance steroidogenesis and to induce apoptosis in primary granulosa cell cultures obtained from rat preovulatory follicles. The cAMP analog, 8-Br cAMP, induced apoptosis in more than 90% of the cell population within <em>15</em> h of incubation at 37 degrees C in serum-free medium. The physiological stimulants of these cells, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which caused a moderate cAMP response in these cells, followed by a desensitization period, increased progesterone production by fourfold with no apparent effect on cell death. In contrast, forskolin, a potent activator of adenylate cyclase, stimulated both the cAMP and steroidogenic response by an order of magnitude greater than the gonadotropin stimulation, concomitantly with a pronounced increase in cell death (25%). Moreover, blocking of the cellular phosphodiesterase activity in forskolin-stimulated cells by isobutylmethylxanthine (IBMX), which maintains high levels of intracellular cAMP, led to further enhancement of cell death following 40 h of incubation (50%). Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and gonadotropin-releasing hormone (GnRH), which stimulated steroidogenesis in these cells in a cAMP-independent manner, did not promote cell death. Moreover, costimulation of the cells with forskolin and bFGF led to a substantial decrease in the incidence of apoptosis relative to forskolin alone. In order to examine whether the expression of tumor suppressor genes is involved in granulosa cell differentiation and apoptosis induced by cAMP, we examined the effect of cAMP in SV40 transformed granulosa cells, in which T-antigen expression is expected to block the activity of p53 as well as of the retinoblastoma gene product (pRB) and its related proteins. Cultures of three different cell lines established by SV40 transformation demonstrated resistance to 8-Br-cAMP- or forskolin plus IBMX-induced apoptosis, in contrast to the severe apoptotic response in primary cells. We suggest that stimulation of primary granulosa cells by high levels of cAMP catalyzes programmed cell death, while stimulation of the cells by gonadotropic hormones, which result in a moderate cAMP response, followed by desensitization to further stimulation, can prolong the lifespan of the luteinized granulosa cells. Moreover, one or more tumor suppressor proteins may mediate the cAMP generated signal leading to cell death.

Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome associated with tumors that secrete phosphaturic hormones, most notably <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23). The majority of tumors associated with this syndrome show stereotypical histological features and are now known as phosphaturic mesenchymal tumors (PMTs). We postulated that immunohistochemistry for somatostatin receptor 2A (SSTR2A) could be used to definitively identify PMTs or other tumors that cause TIO. Immunohistochemistry for FGF23 and SSTR2A was performed on <em>15</em> tumors from 14 patients with a definite diagnosis of TIO. All showed positive staining for both markers. While FGF23 staining was quite focal in some tumors, SSTR2A showed diffuse strong expression. In 40 control tumors not known to be associated with the clinical or biochemical features of TIO, FGF23 expression was found in 2 cases (one aneurysmal bone cyst and one osteosarcoma). SSTR2A expression was found in 9 control tumors (4 synovial sarcomas, 2 hemangiomas, 2 aneurysmal bone cysts and one osteosarcoma). Only one tumor (an aneurysmal bone cyst) showed positive staining for both FGF23 and SSTR2A. SSTR2A also commonly stained neoplastic and non-neoplastic endothelial cells. We conclude that neither FGF23 nor SSTR2A expression are specific for the diagnosis of PMT. However both stains are highly sensitive. Because of its diffuse strong expression and widespread availability, immunohistochemistry for SSTR2A is useful to confirm the diagnosis of PMT in an appropriate setting particularly if material is limited. Negative staining can serve as an excellent rule out test for this diagnosis.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF) has been shown to enhance the in vitro survival and neurite extension of various types of neurons including dorsal root ganglia (DRG) cells. Alpha-1 glycoprotein (alpha 1-GP), an acute phase reactant, has been reported to enhance the in vitro neuritic extensions of chick DRG cells. In the present study, we investigated the ability of synthetic nerve guidance channels, which release sustained controlled amounts of b-FGF and/or alpha 1-GP, to support the regeneration of a transected peripheral nerve over a <em>15</em> mm long gap, a distance that does not permit regeneration in conventional polymeric tubes. Tubes releasing bovine serum albumin (BSA), cytochrome C, BSA and b-FGF, BSA and denatured b-FGF, BSA and alpha 1-GP, or BSA, b-FGF, and alpha 1-GP were fabricated by a dip-molding technique. In vitro kinetic studies of protein release from these channels showed an initial burst during the 1st day, followed by a linear release for at least 2 weeks thereafter. In vitro studies indicated that the b-FGF released from the polymer was biologically active as assessed by the ability of channels releasing b-FGF to induce neurite extensions in PC12 cells. For in vivo studies, the various types of tubes were used as nerve guidance channels for the repair of a <em>15</em> mm nerve gap in the sciatic nerve of rats. Four weeks postimplantation, only the tubes releasing b-FGF or b-FGF and alpha 1-GP displayed regenerated cables bridging both nerve stumps, which contained nerve fascicles with myelinated and unmyelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)

The present investigation was performed to study the adsorption behavior of <em>growth</em> <em>factors</em> and their release characteristics from biodegradable implants in an in vitro study. We investigated the stability of <em>growth</em> <em>factors</em> administered on various scaffolds. We used porous tricalcium phosphate ceramics (alpha-TCP), a neutralized glass-ceramics (GB9N), a composite (polylactid/-glycolid/GB9N), and solvent dehydrated human bone as carriers. Block shaped scaffolds (sized: 7 x 7 x 10 mm) were loaded with 5 microg of either bone morphogenetic protein (rxBMP-4), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (rh-bFGF), or vascular endothelial <em>growth</em> <em>factor</em> (rh-VEGF) solved in <em>15</em>0 microL PBS. The <em>growth</em> <em>factors</em> were labeled with Iodine125 (I-125) for detecting the adsorbed and released amount of <em>growth</em> <em>factors</em> by counting the samples for total I-125 activity. We observed that the adsorption of these <em>growth</em> <em>factors</em> seems to depend on two different parameters: first on the nature of the tested material, and second on the <em>growth</em> <em>factors</em> on their own. The release kinetics of the <em>growth</em> <em>factors</em> from the biodegradable implants can be described as a two phase process-a very rapid release during the first hours by an elution of not adsorbed protein, followed by a specific release, which depends upon the chemical/physical interaction of the material and the <em>growth</em> <em>factor</em> used. Analyzing the eluted proteins on SDS-PAGEs rh-VEGF was degraded into a smaller fragment with a size of around <em>15</em> kDa, while rxBMP-4 and rh-bFGF showed a complete degradation into fragments smaller than 3 kDa after more than 3 days. Although this in vitro study suggests that biodegradable implants might be successfully used as carriers for osteogenic <em>growth</em> <em>factors</em>, the different release kinetics as well as the alteration of their molecular structure including loss of biological activity should be considered.

The effects of fetal bovine serum, insulin-like <em>growth</em> <em>factor</em>-I, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 on the regulation of the functional physical properties of adult bovine cartilage explants during an incubation period of 18-20 days was determined, and the relationship between the measured functional properties of the cartilage and the tissue composition was assessed. Cartilage disks were tested in the uniaxial radially confined configuration by the application of low amplitude oscillatory displacement and measurement of the resultant load and streaming potential. For the control cartilage terminated just after explant, the modulus was 0.39 +/- 0.28 MPa, the open circuit hydraulic permeability was 2.0 +/- 1.0 x 10(-<em>15</em>) m2/(Pa.sec), and the electrokinetic (streaming potential) coefficient was -2.3 +/- 0.6 mV/MPa. Incubation of cartilage in medium supplemented with serum or insulin-like <em>growth</em> <em>factor</em>-I resulted in maintenance of the modulus and electrokinetic coefficient, whereas incubation in basal medium or medium supplemented with <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 led to a marked decrease from control values in the modulus and the amplitude of the electrokinetic coefficient. All of the culture conditions examined resulted in an increase in permeability that was not statistically significant. The variation in the electromechanical properties of all the cartilage samples tested was related to the density of tissue proteoglycan and collagen (hydroxyproline). The modulus was correlated with both the density of tissue proteoglycan (+0.014 MPa/[mg/ml]) and the density of tissue hydroxyproline (+0.008 MPa/[mg/ml]). The electrokinetic coefficient was also correlated with the density of proteoglycan (-0.080 [mV/MPa]/[mg/ml]) and the density of hydroxyproline (+0.064 [mV/MPa]/[mg/ml]). These data indicate that the regulation of chondrocyte matrix metabolism by <em>growth</em> <em>factors</em> can significantly affect the physical properties and function of cartilage.

The role of alveolar macrophage activation and release of mediators remains unclear. In this study, this role is examined with respect to the effects of relatively selective inhibitors of arachidonate metabolism on the pathogenesis of pulmonary fibrosis. CBA/J mice were administered bleomycin (0.037 units) endotracheally to induce pulmonary fibrosis. Daily intraperitoneal injections of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA) inhibited pulmonary fibrosis in a dose-dependent manner (<em>15</em>-25 mg/kg body weight), as assessed by both lung collagen synthesis and total lung hydroxyproline content. The less specific inhibitor BW755c was also effective at a dose of 25 mg/kg. In contrast, the cyclooxygenase inhibitor, ibuprofen (<em>15</em> mg/kg), was completely ineffective. Correlated with this antifibrogenic activity of NDGA was the inhibition of several other parameters of bleomycin-induced pulmonary fibrosis. Bleomycin treatment caused a greater than threefold increase in the percentage of alveolar macrophages expressing Ia antigen (from 7.7% +/- 1.07% to 29.9% +/- 4.16% of total recoverable alveolar macrophages). NDGA, but not ibuprofen, inhibited this increase in a dose-dependent manner. Associated with this indication of macrophage stimulation was an increase in spontaneous macrophage production of <em>fibroblast</em> <em>growth</em> <em>factor</em> (MDGF) activity as a result of bleomycin instillation. This increase was also inhibited by NDGA treatment. In contrast, bleomycin treatment caused a reduction in alveolar macrophage interleukin-1 (IL-1) production, and NDGA treatment did not alter this reduction, which suggests that MDGF is separate from IL-1 in this case, and that MDGF played a more dominant role, at least in this model of pulmonary fibrosis. This antifibrogenic activity of NDGA was accomplished without any reduction in spontaneous macrophage prostaglandin (PG)E2 production, which suggests the selectivity (versus cyclooxygenase pathway) of NDGA inhibition and the relative lack of importance of macrophage-derived PGE2 in modulating fibrogenesis in this model. The results of this study have thus demonstrated the importance of alveolar macrophage stimulation and increased production of MDGF in the pathogenesis of bleomycin-induced pulmonary fibrosis. The data also suggest that both macrophage parameters are subject to regulation by arachidonate metabolites.