Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and beta-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, beta-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Tenocyte responses to mechanical loading in vivo : A role for local insulin-like growth factor 1 signaling in early tendinosis in rats

Objective
To investigate tenocyte regulatory events during the development of overuse supraspinatus tendinosis in rats.

Methods
Supraspinatus tendinosis was induced by running rats downhill at 1 km/hour for 1 hour a day. Tendons were harvested at 4, 8, 12, and 16 weeks and processed for brightfield, polarized light, or transmission electron microscopy. The development of tendinosis was assessed semiquantitatively using a modified Bonar histopathologic scale. Apoptosis and proliferation were examined using antibodies against fragmented DNA or proliferating cell nuclear antigen, respectively. Insulin-like growth factor 1 (IGF-1) expression was determined by computer-assisted quantification of immunohistochemical reaction. Local IGF-1 signaling was probed using antibodies to phosphorylated insulin receptor substrate 1 (IRS-1) and ERK-1/2.

Results
Tendinosis was present after 12 weeks of downhill running and was characterized by tenocyte rounding and proliferation as well as by glycosaminoglycan accumulation and collagen fragmentation. The proliferation index was elevated in CD90+ tenocytes in association with tendinosis and correlated with increased local IGF-1 expression by tenocytes and phosphorylation of IRS-1 and ERK-1/2. Both apoptosis and cellular inflammation were absent at all time points.

Conclusion
In this animal model, early tendinosis was associated with local stimulation of tenocytes rather than with extrinsic inflammation or apoptosis. Our data suggest a role for IGF-1 in the load-induced tenocyte responses during the pathogenesis of overuse tendon disorders.

Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of α6β1 integrin

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial–mesenchymal interconversions leading to the disruption of cell–cell contact and modulation of cell–extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in α2, α6 and β1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of α6 and β1 integrin subunits. Neutralizing antibodies against α6 and β1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of α6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475–85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and α6β1 integrin-mediated signalling in ovarian carcinomas.

Expression of the disintegrin metalloprotease ADAM-10 in prostate cancer and its regulation by dihydrotestosterone, insulin like growth factor -1 and epidermal growth factor in the prostate cell model LNCaP

Purpose: The disintegrin metalloprotease ADAM-10 is a multidomain metalloprotease that is potentially significant in tumor progression due to its extracellular matrix-degrading properties. Previously, ADAM-10 mRNA was detected in prostate cancer (PCa) cell lines; however, the presence of ADAM-10 protein and its cellular localization, regulation, and role have yet to be described. We hypothesized that ADAM-10 mRNA and protein may be regulated by growth factors such as 5α-dihydrotestosterone, insulin-like growth factor I, and epidermal growth factor, known modulators of PCa cell growth and invasion.

Experimental Design: ADAM-10 expression was analyzed by in situ hybridization and immunohistochemistry in prostate tissues obtained from 23 patients with prostate disease. ADAM-10 regulation was assessed using quantitative reverse transcription-PCR and Western blot analysis in the PCa cell line LNCaP.

Results: ADAM-10 expression was localized to the secretory cells of prostate glands, with additional basal cell expression in benign glands. ADAM-10 protein was predominantly membrane bound in benign glands but showed marked nuclear localization in cancer glands. By Western blot, the 100-kDa proform and the 60-kDa active form of ADAM-10 were synergistically up-regulated in LNCaP cells treated with insulin-like growth factor I plus 5α-dihydrotestosterone. Epidermal growth factor also up-regulated both ADAM-10 mRNA and protein.

Conclusions: This study describes for the first time the expression, regulation, and cellular localization of ADAM-10 protein in PCa. The regulation and membrane localization of ADAM-10 support our hypothesis that ADAM-10 has a role in extracellular matrix maintenance and cell invasion, although the potential role of nuclear ADAM-10 is not yet known.

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10-9 M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit C AT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat . Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease ( p 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven C AT expression, while TNF- alpha -enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat /TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

Background:  The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.

Methods:  Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age-matched wild-type littermates. Transcription of selected genes was validated by quantitative real-time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme-linked immunosorbent assay.

Results:  Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in Müller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to Müller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing Müller cell somata.

Conclusions:  An early spike in vascular endothelial growth factor induced significant long-term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and Müller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.

Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.

Epidermal growth factor receptor-tyrosine kinase inhibitors in advanced squamous cell carcinoma of the lung: a meta-analysis.

Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) are well established in treating metastatic pulmonary adenocarcinoma, especially patients with activating EGFR mutations. EGFR mutations are rare in pulmonary squamous cell carcinomas (SCCs). There are conflicting data supporting the efficacy of EGFR-TKIs in advanced lung SCC. We analyzed the impact of EGFR-TKIs on progression-free survival (PFS) and overall survival (OS) in unselected patients with lung SCC.

Coiled polymeric growth factor gradients for multi-luminal neural chemotaxis

In the injured adult nervous system, re-establishment of growth-promoting molecular gradients is known to entice and guide nerve repair. However, incorporation of three-dimensional chemotactic gradients in nerve repair scaffolds, particularly in those with multi-luminal architectures, remains extremely challenging. We developed a method that establishes highly tunable three-dimensional molecular gradients in collagen-filled multi-luminal nerve guides by anchoring growth-factor releasing coiled polymeric fibers onto the walls of collagen-filled hydrogel microchannels. Differential pitch in the coiling of neurotrophin-eluting fibers generated sustained three-dimensional chemotactic gradients that appropriately induced the differentiation of Pheochromocytoma (PC12) cells into neural-like cells along an increasing concentration of nerve growth factor (NGF). Computer modeling estimated the stability of the molecular gradient within the luminal collagen, which we confirmed by observing the significant effects of neurotrophin gradients on axonal growth from dorsal root ganglia (DRG). Neurons growing in microchannels exposed to a NGF gradient showed a 60% increase in axonal length compared to those treated with a linear growth factor concentration. In addition, a two-fold increment in the linearity of axonal growth within the microchannels was observed and confirmed by a significant reduction in the turning angle ratios of individual axons. These data demonstrate the ability of growth factor-loaded polymeric coiled fibers to establish three-dimensional chemotactic gradients to promote and direct nerve regeneration in the nervous system and provides a unique platform for molecularly guided tissue repair.

The control of epidermal growth factor grafted on mesoporous silica nanoparticles for targeted delivery

The performance of biomaterials in a biological environment is largely influenced by the surface properties of the biomaterials. In particular, grafted targeting ligands significantly impact the subsequent cellular interactions. The utilisation of a grafted epidermal growth factor (EGF) is effective for targeted delivery of drugs to tumours, but the amount of these biological attachments cannot be easily quantified as most characterization methods could not detect the extremely low amount of EGF ligands grafted on the surface of nanoparticles. In this study, hollow mesoporous silica nanoparticles (HMSNs) were functionalized with amine groups to conjugate with EGFs via carbodiimide chemistry. Time of flight secondary ion mass spectrometry (ToF-SIMS), a very surface specific technique (penetration depth <1.5 nm), was employed to study the binding efficiency of the EGF to the nanoparticles. Principal component analysis (PCA) was implemented to track the relative surface concentrations of EGFs on HMSNs. It was found that ToF-SIMS combined with the PCA technique is an effective method to evaluate the immobilization efficiency of EGFs. Based on this useful technique, the quantity and density of the EGF attachments that grafted on nanoparticles can be effectively controlled by varying the EGF concentration at grafting stages. Cell experiments demonstrated that the targeting performance of EGFR positive cells was affected by the number of EGFs attached on HMSNs.

Peripheral vascular endothelial growth factor as a novel depression biomarker: a meta-analysis

BACKGROUND: The neurotrophic hypothesis of major depressive disorder (MDD) postulates that the pathology of this illness incorporates a down-regulation of neurotrophin signaling. Brain-derived neurotrophic factor (BDNF) is the most studied neurotrophic mediator regarding the neurobiology of MDD. Nevertheless, emerging evidence has implicated the multi-competent angiogenic and neurogenic molecule - vascular endothelial growth factor (VEGF) - in hippocampal neurogenesis and depression pathophysiology. OBJECTIVE: To compare peripheral levels of VEGF between individuals with MDD and healthy controls. METHODS: We performed a systematic review and meta-analysis of original studies measuring peripheral levels of VEGF in participants with MDD compared to healthy controls. We searched the Pubmed/MEDLINE, EMBASE and PsycInfo databases for studies published in any language through December 16th, 2014. RESULTS: Fourteen studies met eligibility criteria (N=1633). VEGF levels were significantly elevated in individuals with MDD when compared to healthy controls (Hedges's g=0.343; 95% CI: 0.146-0.540; P<0.01). Funnel plot inspection and the Egger's test did not provide evidence of publication bias. A significant degree of heterogeneity was observed (Q=38.355, df=13, P<0.001; I(2)=66.1%), which was explored through meta-regression and subgroup analyses. Overall methodological quality, sample for assay (plasma versus serum), as well as the matching of MDD and control samples for age and gender emerged as significant sources of heterogeneity. CONCLUSIONS: Taken together, extant data indicate that VEGF shows promise as a biomarker for MDD, and supports that this mediator may be involved in neuroplasticity mechanisms underlying the pathophysiology of MDD.