Epidermal Growth Factor Group Research Articles

Effect of Epidermal Growth Factor on the Network Spiking Activity in Cultured Hippocampal Neurons

Effect of Epidermal Growth Factor on the Network Spiking Activity in Cultured Hippocampal Neurons

Comparative study on the effects of EGF and bFGF on the healing of human large traumatic perforations of the tympanic membrane.

We evaluated the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the healing of large traumatic tympanic membrane perforations (TMPs). Prospective clinical study. Tertiary university hospital. A randomized, prospective analysis was performed between June 2013 and August 2014 on the treatment of traumatic TMPs larger than 25% of the TM. Closure rate, closure time, hearing gain, and rate of otorrhea were compared between EGF and bFGF groups, as well as to an observation-only group. Final analysis was performed on 86 patients at 3 months. The closure rates of perforation in the EGF, bFGF, and observation groups were 86.2%, 89.3%, and 72.4%, respectively. The closure rates in the EGF and bFGF groups were 14% to 17% higher than in the observation group, although the difference was not statistically significant for the total closure rate among the three groups (P = 0.200). The average closure time was significantly longer (P < 0.01) in the observation group than in the EGF and bFGF groups. However, the closure times in the EGF and bFGF groups were not significantly different (P = 0.92). In addition, differences in purulent otorrhea rates among the groups were not statistically significant (P = 0.82). Both EGF and bFGF can accelerate the closure of human large traumatic TMPs. The healing outcomes among the two growth factors were not significantly different.

Enhanced osteoinductivity of recombinant human bone morphogenetic protein-2 in combination with epidermal growth factor in a rabbit tibial defect model

This study aims to explore the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone formation when treated with epidermal growth factor (EGF) using human mesenchymal stem cells (hMSCs) and a rabbit tibial defect model. The rhBMP-2 (250 ng/ml)+EGF (10 ng/ml) group showed higher alkaline phosphatase (ALP) activity, ALP expression, increased calcium amount than rhBMP-2 group. In micro-CT and histology results of animal experiments, the rhBMP-2+EGF group showed more amount of bone bridging compared to the rhBMP-2 group. Among the 8-week groups, the rhBMP-2+EGF group showed significantly higher percent bone volume and trabecular number compared to the rhBMP-2 group. The combined treatment with EGF and rhBMP-2 induced significantly higher bone formation compared to that of rhBMP-2 only in both hMSCs and a rabbit tibial defect model. Therefore, EGF is expected to facilitate bone formation effect of rhBMP-2 when both factors are treated in combination.

Epidermal growth factor improved alcohol-induced inflammation in rats

The purpose of this study was to investigate the effects of an epidermal growth factor (EGF) intervention on improving the inflammatory response of rats fed an ethanol-containing diet. Eight-week-old male Wistar rats were divided into ethanol (E) and control (C) groups. Rats in the E group were fed an ethanol liquid diet, while rats in the C group were pair-fed an isoenergetic diet without ethanol. After a 4-week ethanol-induction period, both the C and E group were respectively subdivided into 2 groups: a normal liquid diet without (C group, n = 8) or with EGF supplementation (C + EGF, n = 8), and the ethanol-containing diet without (E group, n = 8) or with EGF supplementation (E + EGF group, n = 8). The EGF (30 μg/kg body weight/day) intervention period was carried out for the following 8 weeks. At the end of the experiment, activity of aspartate transaminase (AST) and alanine transaminase (ALT) and hepatic levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in group E were significantly higher than those in group C. In addition, alterations in the gut microbiota profile were found in group E. In contrast, activity of AST and ALT and levels of TNF-α, IL-1β, and IL-6 in group E + EGF were significantly lower than those in group E. Significantly lower intestinal permeability and lower numbers of Escherichia coli in the fecal microbial culture were also found in group E + EGF. These results suggest that EGF improved the intestinal integrity by decreasing E. coli colonization and lowering intestinal permeability, which then ameliorated the inflammatory response under chronic ethanol exposure.

Comparison of skin effects of immediate treatment modalities in experimentally induced hydrofluoric acid skin burns

Hydrofluoric acid (HF) burns cause immediate damage and painful long-term sequellae. Traditionally, chelating agents have been used as the initial treatment for such burns. We have introduced epidermal growth factor (EGF) into an HF model to compare EGF with Ca(2+) and Mg(2+) treatments; 40 Sprague Dawley rats were divided into five groups. Each rat suffered a 6 × 4 cm(2) burn induced by 40% HF. Group 1 had no treatment, group 2 had saline injected beneath the burn, group 3 received magnesium sulphate injections, group 4 received calcium gluconate and group 5 received EGF. Specimens were evaluated via planimetry and biopsy at intervals of 4, 8, 24 and 72 hours. Fluid losses were significantly less in the Mg(2+) and EGF groups. The EGF group had the smallest burn area, least oedema, least polymorphonuclear granulocyte (PMN) infiltration, most angiogenesis and highest fibroblast proliferation of any group (P < 0·005). EGF limited HF damage morphologically and histologically more effectively than Ca(2+) or Mg(2+). This finding indicates that HF treatment via growth factors may be an improvement over chelation therapy.

118 EFFECTS OF EPIDERMAL GROWTH FACTOR ON IN VITRO SURVIVAL AND ANTRUM FORMATION OF ISOLATED OVINE PREANTRAL FOLLICLES

The aim of this study was to verify the beneficial effects of different concentrations of epidermal growth factor (EGF) on survival and antrum formation of isolated ovine preantral follicles cultured in vitro. Ovine ovaries (n = 50) were collected from a local slaughterhouse and secondary follicles (150–200 μm in diameter), without antral cavities, were mechanically isolated by microdissection using 26-gauge needles. After selection, the follicles were individually cultured in 100-μL droplets of culture medium at 39°C and 5% CO2 in air for 18 days. The basic control medium consisted of α-minimal essential medium (α-MEM) supplemented with BSA; insulin, transferring and selenium; glutamine; hypoxanthine; and ascorbic acid and then referred to as α-MEM+. For the experimental conditions, follicles were cultured in α-MEM+ alone (control) or in different concentrations of EGF (1, 10, or 50 ng mL–1). Every other day, 60 μL of the culture media was replaced with fresh media. The morphological aspects of all ovine follicles were assessed every 6 days using a precalibrated ocular micrometer in a stereomicroscope at 100× magnification. Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. The rupture of the basement membrane was also observed and characterised as follicle extrusion. In addition, antral cavity formation was defined as the emergence of a visible translucent cavity within the granulosa cell layers. Data from morphologically normal follicles, extruded follicles, and antrum formation rate during in vitro culture were expressed as percentages and compared by the chi-squared test, and differences were considered significant when P &lt; 0.05. The results showed that the percentage of morphologically normal follicles decreased significantly throughout the culture periods in all the treatments, except in the 50 ng mL–1 EGF group, which maintained the percentage of normal follicles from Day 0 to 6. Considering the same culture period, 50 ng mL–1 EGF treatment significantly increased the percentage of morphologically normal follicles at Day 18 compared with the control group. Moreover, the addition of EGF to the culture medium, at 50 ng mL–1, significantly reduced the precocious extrusion of oocytes and increased the percentage of antrum formation compared with the control and 1 ng mL–1 EGF after 18 days of culture. Notably, there were no significant differences between 10 ng mL–1 EGF, control medium, and 1 ng mL–1 EGF treatments. In conclusion, this study demonstrated that the addition of EGF to the in vitro culture medium, at 50 ng mL–1, increased the proportion of morphologically normal follicles and antrum formation rate of isolated ovine preantral follicles. This work was supported by FACEPE (Process APQ-0705–5.05/10). L. P. Santos is a recipient of a grant from FACEPE (Brazil).

Effects of qijingmingmu soup on the expression of matrix metalloproteinases in the conjuntival fibroblasts of conjunctivochalasis

Background Our previous study determined that expressions of matrix metalloproteinases (MMPs) and tissue matrix metalloproteinase inhibitors (TIMPs)change in the conjuntival fibroblasts of conjunctivochalasis in vitro.To seek a suitable drug is very important in the prevention and treatment of conjunctivochalasis.Objective This study was to explore the effect of qijingmingmu soup on the expressions of MMPs and TIMPs in human conjunctival fibroblasts of conjunctivochalasis.Methods Twenty-four SD rats were randomized into two groups.Qijingmingmu soup was administration gastrically for consecutive 3 days,and normal saline solution was given in the same way in the control group.The blood was collected from aortaventralis and drug serum was prepared.Human conjunctival samples were obtained during the surgery of conjunctivochalasis relaxation and cultured in the DMEM containing 10％ fetal bovine serum,20％,15％,10％,5％ of drug serum and 8 ml/L epidermal growth factor(EGF) was added into the medium respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of MMP1,MMP3,TIMP1 and TIMP3in conjunctival fibroblasts.Results Cultured cells grew well with the fusiform shape and showed the positive response for vimentin.The expression value of MMP1 (A value)in the cells was declined after administration of qijingmingmu soup.A significant difference was found in the expression of MMP1 among the control group,20％,15％,10％,5％ drug serum groups and EGF group(F=466.664,P＜0.05),and that in the 10％ ([9.92±0.14] mg/L) and 20％ ([11.87 ±0.11] mg/L) drug serum groups was significantly lowed in comparison with the control group([16.31±0.10] mg/L)(t=99.974,87.394,P＜0.05).The expression value of the MMP3in the cells in the various drug serum groups,EGF group and the control group was significantly different(F=158.168,P＜0.05),with a lower value in the 20％ drug serum group compared with the control group ([3.50±0.03] mg/L vs.[4.44 ± 0.11] mg/L) (t =21.991,P ＜ 0.05).Also,the significantly different expressing value of TIMP1 was seen among all the groups (F=183.508,P＜0.05),and expressing value of TIMP1 in the 15％ drug serum group was(1.88±0.06)mg/L,which was lower than(3.20±0.32) mg/L of the control group(t=10.353,P＜0.05).Furthermore,the expressing value of the TIMP3 in the cells was significantly different among the various groups(F=54.503,P＜0.05),and that of the 20％ drug serum group was (1.743±0.065)mg/L and it was significantly higher than (1.54 ± 0.05) mg/L of the control group (t =5.046,P =0.004).However,the expressing value of TIMP3of the 15％,10％ and 5％ drug serum groups was lower than that of the control group,respectively all at(P＜0.05).Conclusions Qijingmingmu soup drug serum at the concentration of 20％ can down-regulate the expressions of MMP1,MMP3,TIMP1 and up-regulate the expression of TIMP3 in human conjunctivochalasis bulbar conjunctival fibroblastsin vitro,which probably plays preventive and therapeutic effects on conjunctivochalasis. Key words: Conjunctivochalasis; Fibroblast; Chinese tranditional medicine/qijingmingmu soup; Matrix metalloproteinase

Regeneration of Chronic Tympanic Membrane Perforation Using an EGF-Releasing Chitosan Patch

Most chronic tympanic membrane (TM) perforations require surgical interventions such as tympanoplasty because, unlike with acute perforations, it is very difficult for the perforations to heal spontaneously. The purpose of this study was to develop novel therapeutic techniques and scaffolds that release growth factors to treat chronic TM perforations. We evaluated the cell proliferation effects of the epidermal growth factor (EGF) and fibroblast growth factor (FGF) on in vitro cultures of TM cells using an MTT assay. They both showed similar efficacy, so we used EGF because of its lower cost. We then constructed an EGF-releasing chitosan patch scaffold (EGF-CPS) based on previous studies. We analyzed its toxicity and strength, and we studied it using scanning electron microscopy. EGF was released from the EGF-CPS for 8 weeks in an in vitro system. In animal studies, the EGF group, which was treated with EGF-CPS, showed healing in 56.5% of the animals (13/23), while the control group, which did not receive any treatment, revealed 20.8% healing (4/24) (p=0.04). Transmission electron microscopic studies of regenerated eardrums in the EGF group showed much greater preservation of histological features, and TMs of the EGF group were thinner than spontaneously healed TMs. In conclusion, this novel EGF-CPS can be used as a nonsurgical intervention technique for treatment of chronic TM perforations.

Role of epidermal growth factor signaling system in the pathogenesis of endometriosis under estrogen deprivation conditions

To study the role of epidermal growth factor (EGF) , epidermal growth factor receptor(EGFR), extracellular signal-regulated kinase 1/2 (p-ERK1/2) in the pathogenesis of endometriosis under estrogen deprivation conditions. The estrogen was quickly-stripped in medium and the female nude mice were castrated by bilateral oophorectomy to build estrogen deprivation in vitro and in vivo experimental models, respectively. (1) In vitro experiments:according to different treatments the estrogen deprived ectopic endometrial cells were classified into 4 groups: a. EGF group:the ectopic endometrial cells were cultured for 72 hours with different concentrations of EGF (0.01, 0.1, 1, 10, 50, 100 ng/ml), the results of EGF group were represented by the result of cells treated by 10 ng/ml EGF cultured for 72 hours; b. EGF+PD98059 group:the ectopic endometrial cells were cultured for 72 hours with 5×10(-2) mol/L PD98059 (inhibitor of ERK), followed by a cultivation for 72 hours treated by 10 ng/ml EGF+5×10(-2) mol/L PD98059; c. EGF+ ICI182780 group: the ectopic endometrial cells were cultured for 72 hours with 10(-6) mol/L ICI182780 [inhibitor of estrogen receptor(ER)], followed by a cultivation for 72 hours treated by 10 ng/ml EGF+10(-6) mol/L ICI182780; d. Blank control group:the ectopic endometrial cells were cultured with no treatment. The proliferation activity of ectopic endometrial cells in all groups after treatment were examined by methyl thiazolyl tetrazolium (MTT) method represented by absorbance value (A). The expression of p-ERK1/2 protein were detected by western blot. (2) In vivo experiments: 64 female nude mice were randomly divided into control and castration groups (both n=32) using random number chart. The mice in castration group were castrated by bilateral oophorectomy on 3 weeks after the endometriosis model was established. The levels of EGF, EGFR, p-ERK1/2 protein in ectopic lesions of both groups were measured on 4, 6, 8 and 10 weeks after the endometriosis model was established by western blot. (1) The proliferation activity of ectopic endometrial cells:the proliferation activity of ectopic endometrial cells treated by different concentrations of EGF (0.01, 0.1, 1, 10, 50, 100 ng/ml) for 72 hours were 0.310±0.010, 0.340±0.020, 0.670±0.010, 0.980±0.030, 1.360±0.020, 1.670±0.020, respectively, the proliferation activity was increased along with of EGF concentrations.The proliferation activity was 0.680±0.030 at EGF+ PD98059 group, the differences exhibited significant difference when compared with that at EGF group with 100 ng/ml for 72 hours (P<0.01) .The proliferation activity of EGF+ ICI182780 and blank control groups were 0.330±0.030 and 0.310±0.030, respectively, which did not reached statistical differences (P>0.05). (2) The expression of EGF, EGFR, pERK1/2 protein: a. In vitro experiments:the levels of p-ERK1/2 protein in EGF and blank control groups were 0.670±0.020 and 0.600±0.010, respectively, which reached statistical differences (P<0.05). The level of p-ERK1/2 protein in EGF+ PD98059 group was 0.610±0.020, which exhibited significant differences with that at blank control group (P>0.05). b. In vivo experiments:at 4, 6 and 8 weeks after the endometriosis models were established, the expression of EGF protein in the ectopic lesions of castration group and control group were (0.530±0.015 versus 0.610±0.015), (0.400±0.029 versus 0.620±0.018), (0.560±0.026 versus 0.630±0.021), respectively, the levels of EGFR protein were (0.500±0.030 versus 0.640±0.030), (0.470±0.020 versus 0.630±0.020), (0.510±0.030 versus 0.610±0.020) respectively, and the level of p-ERK1/2 protein were (0.500±0.020 versus 0.580±0.020), (0.490±0.020 versus 0.580±0.020), (0.570±0.020 versus 0.590±0.020), respectively. The difference of EGF, EGFR, p-ERK1/2 protein expression levels between two groups did not exhibited significant difference (P<0.01, P<0.01, P<0.05). At 10 weeks after the endometriosis models were established, the levels of EGF protein in castration group and control group were both 0.620±0.020, the levels of EGFR protein were both 0.610±0.020, and the level of p-ERK1/2 protein were 0.590±0.010 and 0.600±0.020. No statistical difference (P>0.05) was found between those two groups (P>0.05). EGF could stimulate the proliferation of ectopic endometrial cells by activating the ERK pathway under estrogen deprivation conditions. The inhibition of EGF signaling system in ectopic lesions was alleviated along with the prolongation of the period of estrogen deprivation.

Effect of EGF and bFGF on Fibroblast Proliferation and Angiogenic Cytokine Production from Cultured Dermal Substitutes

Growth factors accelerate wound healing but the underlying mechanisms remain poorly understood. The aim of this study was to investigate the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on fibroblast proliferation and production of angiogenic factors from cultured dermal substitutes (CDS). In the first experiment, fibroblasts were seeded into a flask at a density of 1 × 104 cells/cm2.Cell proliferation was assessed after culturing in media containing EGF or bFGF at concentrations ranging from 2 to 50 μg. The number of fibroblasts increased significantly in the presence of EGF or bFGF, but fibroblasts detached from the flasks in the presence of 50 μg bFGF. In the second experiment, CDS were prepared by incorporating fibroblasts into collagen gels. To make a wound surface model, the CDS was elevated to the air–liquid interface, on which a spongy sheet of hyaluronic acid (HA) containing EGF or bFGF was placed. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS after 1 week of cultivation was measured by ELISA. When the CDS was covered with a HA sponge containing EGF (Group 1), fibroblasts released 3.5-times more VEGF compared with a HA-alone sponge (control group). When covered with a HA sponge containing bFGF (Group 2), 8.7-times more VEGF was released compared with the control group. Fibroblasts in Groups 1 and 2 released 9.6- and 9.3-times more HGF, respectively, compared with the control group. Thus, EGF stimulates fibroblasts to produce VEGF and HGF, in addition to its ability to enhance epidermal cell proliferation.

Effect of epidermal growth factor on expression of Survivin and signaling pathways involved in human small cell lung cancer cells

Objective To investigate the effects of epidermal growth factor (EGF) on the expression of Survivin and the signaling pathways involved in the human small cell lung cancer NIC-H446 cell line.Methods Methyl thiazol tetrazolium (MTT) assay was used to determine cell proliferation rate.Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the EGF mRNA and protein expression levels in NIC-H446 cells.Western blotting was used to detect p38MAPK,JNK and ERK expression levels as well as their phosphorylation levels in NIC-H446 cells.Results EGF could boost the multiplication of NIC-H446 cells in a concentration- and time-dependent manner.The expression levels of Survivin mRNA (0.69 ±0.12) and protein (0.89 ±0.19) in EGF group were significantly higher in EGF group than in control group (0.36 ±0.06,0.57 ±0.15),(P ＜0.01 ).The expression of p-p38MAPK (0.68 ±0.27) was significantly higher in EGF group than in control group (0.29 ±0.08,P ＜0.01 ).The expression of Surviving (0.56 ±0.17) and p-p38MAPK (0.41 ±0.11 ) was significantly higher in EGF + SB203580 group than that in EGF group (0.92 ±0.21,O.72 ±0.19,P ＜0.01 ).Conclusion EGF activates cell proliferation and up-regulates Survivin expression by activating pp38MAPK pathway in NIC-H446 cells,as maybe count for the progression of human small cell lung cancer. Key words: Lung carcinoma; Epidermal growth factor; Survivin; p38MAPK

67 SUPPLEMENTATION WITH EPIDERMAL GROWTH FACTOR AND GLIAL CELL-DERIVED NEUROTROPIC FACTOR DURING PORCINE OOCYTE MATURATION STIMULATES BLASTOCYST HATCHING RATE AND QUALITY

The growth factors glial cell-derived neurotropic factor (GDNF) and epidermal growth factor (EGF) have previously been shown to elicit a functional effect on oocyte competence and embryo development; however, their effects on embryo quality remain elusive. The objective of the present study was to investigate whether supplementation with EGF, GDNF, or a combination of the 2 growth factors during in vitro porcine oocyte maturation has a beneficial effect on the rate of embryonic development, cell number and relative expression of genes related to apoptosis (anti-apoptotic: BCLX; pro-apoptotic: BAK, CASPASE), cellular stress (HSP70), mitochondrial function (TFAM) and pluripotency (TERT). Cumulus-oocyte complexes (COC) aspirated from slaughterhouse ovaries were matured for 42 h in maturation medium supplemented with either 50 μg mL–1 of EGF (n = 3398), 50 μg mL–1 of GDNF (n = 1870), or a combination of both growth factors [EGF (50 μg mL–1) + GDNF (50 μg mL–1); EGF+GDNF-group; n = 1993). Subsequently, the oocytes were inseminated, the cleavage rate scored and the resultant embryos cultured in vitro for 7 days. The experiments were replicated a total of 7 times and zona-enclosed blastocysts were collected for Hoechst staining (cell counting) and for gene expression analyses. For gene expression analyses, total RNA was isolated from pools of embryos (n = 10), reverse transcribed into cDNA and subjected to comparative real-time PCR using the delta-delta Ct method (2–ΔΔCT) and statistical method with a significance level of P &lt; 0.05. The blastocyst rate was significantly lowest in the GDNF group (P &lt; 0.05), whereas the hatched blastocyst rate (P &lt; 0.05) was highest in the EGF+GDNF-group (Table 1). The GDNF-group displayed 2-fold up-regulation of BAK compared with the EGF group (P &lt; 0.05), 1.75-fold up-regulation of TFAM compared with the EGF+GDNF group (P &lt; 0.05) and 2.4-fold up-regulation of CASPASE compared with the EGF+GDNF group (P &lt; 0.05). In contrast, an 11-fold decrease of TERT compared with the EGF+GDNF and EGF group was observed (P &lt; 0.05). The EGF+GDNF-group displayed 1.16 and 1.56-fold up-regulation of BCLX compared with the EGF and GDNF (P &lt; 0.05) groups, respectively and a 1.1-fold up-regulation of TERT compared with the EGF group; in contrast, there was a 1.35 and 1.25 fold down-regulation of CASPASE and TFAM compared with the EGF group, respectively. The EGF-group displayed the lowest (P &lt; 0.05) expression level of BAK. In conclusion, compared among treatments, supplementation with a combination of EGF and GDNF during porcine in vitro oocyte maturation resulted in an increased rate of hatched blastocysts and increased expression of anti-apoptotic (BCLX) and pluripotency related (TERT) genes, whereas it resulted in decreased expression of the pro-apoptotic gene (CASPASE). The present study may help to improve the culture conditions, which are important for optimizing blastocyst rates and quality. Table 1.Effect of using growth factor in in vitro maturation on the cleavage rate and blastocyst yield (mean ± standard error of the means) in vitro

Effect of epidermal growth factor on the expression of matrix metalloproteinase-9 and the signalling pathways involved in the trophoblast cell line JEG-3

To investigate the effect of epidermal growth factor (EGF) on the expression of Matrix metalloproteinase-9 (MMP-9) and the signalling pathways involved in the trophoblast cell line JEG-3. The JEG-3 trophoblast cell line was used in this study. (1) JEG-3 cells were cultured with various concentrations of EGF (0, 1, 10, 20 ng/ml) for 24 hours and the expression of MMP-9 was tested by western blotting and reverse transcription PCR (RT-PCR). (2) Western blotting and RT-PCR were also used to investigate the expression of MMP-9 expression after incubation for 0, 4, 12 and 24 hours with EGF treatment (10 ng/ml) in JEG-3 cells. (3) According to the different added ingredients, JEG-3 cells were divided into some groups: control group (without EGF), EGF group (exposure to 10 ng/ml EGF), EGF+ inhibitors group (exposure to 10 ng/ml EGF+ 20 ng/ml SB203580 or exposure to 10 ng/ml EGF+ 10 ng/ml U0126), inhibitors group (exposure to 20 ng/ml SB203580 or exposure to 10 ng/ml U0126). Western blotting were used to investigate the expression levels of MMP-9, nuclear factor kappa B (NF-κB), p38MAPK, phospho-p38MAPK (p-p38MAPK), extracellular-signal regulated kinase (ERK) and phospho-ERK (p-ERK) protein in JEG-3 cells after incubation for 24 hours. (1) The profiles of MMP-9 mRNA were increased by various concentrations of EGF (0, 1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The expression of MMP-9 mRNA in JEG-3 cells exposure at 1 ng/ml of EGF (0.567±0.056), 10 ng/ml of EGF (1.392±0.133), 20 ng/ml of EGF (1.971±0.067) were significantly higher respectively (P<0.05), compared with 0 ng/ml of EGF treatment (0.166±0.015). Similarly, MMP-9 mRNAs were also increased with the increasing incubation time. Compared to EGF (10 ng/ml) stimulation for 0 h (0.253±0.044), the MMP-9 mRNA profiles were 0.470±0.026, 1.061±0.115, 1.453±0.180 for 4, 12 and 24 hours, respectively (P<0.05). (2) In accordance to the mRNA profiles, the expression of MMP-9 protein was also increased by different concentrations of EGF (0, 1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The abundance of MMP-9 protein in the three groups was 0.043±0.012, 0.085±0.008, 0.142±0.015, with a significantly higher expression, compared with 0 ng/ml of EGF treatment (0.004±0.001, P<0.05) respectively. Similarly, MMP-9 proteins were also increased with the increasing incubation time. Compared to EGF (10 ng/ml) stimulation for 0 h (0.030±0.009), the profiles of MMP-9 protein were 0.137±0.010, 0.240±0.010, 1.240±0.061 for 4, 12 and 24 hours, respectively (P<0.05). (3) Both p38MAPK and ERK signalling pathways were activated by EGF in JEG-3 cells. The expression of p-p38MAPK was significantly higher (without or with 10 ng/ml EGF, 234.1±4.1 vs. 260.9±2.5, P<0.05), however, the p38MAPK inhibitor SB203580 markedly suppressed the increase in p-p38MAPK content induced by EGF (227.9±2.4 vs. 260.9±2.5, P<0.05). Similarly, the expression of p-ERK was significantly higher with EGF treatment (812.2±3.5) vs. without EGF group (453.4±5.8) (P<0.05), while the ERK inhibitor U0126 significantly inhibited the increased p-ERK content in response to EGF treatment (71.0±1.2 vs. 812.2±3.5, P<0.05). (4) The p38MAPK inhibitor SB203580 significantly reduced the expression of EGF-induced MMP-9 (0.645±0.270 vs. 1.476±0.452, P<0.05) and NF-κB (0.530±0.026 vs. 0.959±0.017, P<0.05). (5) The ERK inhibitor U0126 also significantly reduced the expression of EGF-induced MMP-9 (0.623±0.030 vs. 2.112±0.056, P<0.05) and NF-κB (0.325±0.082 vs. 0.939±0.153, P<0.05). EGF induced the expression of MMP-9 in a time and dose-dependant manner in JEG-3 cells. EGF enhanced MMP-9 expression through the activation of p38MAPK and ERK signalling pathways in JEG-3 cells.

Abstract SY23-02: RTK endocytosis and other feedback mechanisms in cancer

Abstract The view of signal transduction pathways as a set of parallel pipelines connecting transmembrane receptor tyrosine kinases (RTKs) and the genome has given way, over the last decade, to the notion of layered networks, highly rich in internal connections. This paradigm is well exemplified by the RTK subfamily called ERBB, namely receptors for growth factors of the EGF (epidermal growth factor) and neuregulin groups: whereas ERBB-1 (also called EGFR) binds seven different growth factors and ERBB-4 binds several isoforms of neuregulins, ERBB-2/HER2 binds no known growth factor and the kinase activity of ErbB-3/HER3 is almost dead. Because both EGFR and HER2 frequently display aberrant expression and mutant forms in human tumors, their mode of action and regulation are of interest. Like other autonomous bi-stable systems, the ERBB network is regulated by feedback loops, both positive and negative. For example, one outcome of activation by growth factors is the transcriptional induction of EGF-like molecules (positive feedback). On the other hand, once activated the receptors are rapidly endocytosed and degraded in lysosomes (negative feedback). According to recent studies, negative feedback loops are often defective in tumors, whereas positive loops are frequently enhanced. In general, negative feedback regulation may be divided into two temporal phases: the immediate phase (approximately 20 minutes from stimulation) comprises posttranslational protein modifications, such as protein phosphorylation and ubiquitylation, as well as intracellular translocations of enzymes and adaptor proteins, including receptor endocytosis. The late phase of feedback regulation harnesses the transcriptional machinery, and it comprises newly synthesized proteins (e.g., phosphatases of the mitogen-activated protein kinases, MKPs), which restrain signaling. Interestingly, microRNAs complement the roles played by mRNA synthesis: multiple microRNAs undergo rapid turnover within minutes after growth factor stimulation, while another set is newly synthesized. In addition to the immediately down-regulated microRNAs (ID-miRs) and E3 ubiquitin ligases like CBL and Nedd4 family members, the first phase of negative RTK regulation entails tyrosine phosphatases like DEP-1, which dephosphorylates EGFR, and de-ubiquitinating enzymes (DUBs). E3 ligases, in concert with ubiquitin-binding proteins, which nucleate four different endosomal sorting complexes called ESCRTs, coordinate receptor sorting for either lysosomal degradation or recycling to the plasma membrane. In line with their negative regulatory functions, certain components of ESCRTs have been implicated in tumor suppression, and, on the other hand, inactive forms of the RTK-specific E3 ligase, namely CBL, are oncogenic. In analogy with regulators of the endocytic pathway, the group of immediate early genes (IEGs; e.g., FOS and JUN), which is induced by growth factors within the first 30 minutes, is endowed with an oncogenic potential. Likewise, tumors of epithelial and brain origins deviate from the physiological tight regulation of the IEGs: both ID-miRs and several RNA- and DNA-binding proteins, collectively termed delayed early genes (DEGs; e.g., the RNA-binding protein ZFP-36) shape the pulse like profile of IEG induction, but transformed cells escape this type of regulation. In summary, the emerging landscape of feedback regulation in malignant cells proposes multi-site manipulation of systems control: RTKs are often derailed to a recycling route by means of hyperactive RAB-coupling proteins (e.g., RAB11FIP1) and other aberrations, while both the ID-miRs and the DEGs are frequently down-regulated in tumors. Collectively, these aberrations enhance cytoplasmic signaling and augment expression of oncogenic transcription factors, which result in accelerated cell proliferation and higher invasiveness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY23-02. doi:10.1158/1538-7445.AM2011-SY23-02

Effects of epidermal growth factor dosage forms on mice full-thickness skin wound zinc levels and relation to wound strength.

Epidermal growth factor (EGF) and zinc promote re-epithelization and reparative tissue strength by enhancing deposition of collagen at the site of the wound. In this study two EGF dosage forms were chosen to assess the effect of zinc levels on wound healing and for comparison with wound tear strengths. A solution of EGF in 0.9% w/v NaCl and an EGF gel in 0.2% Carbopol 940 polymer (5 microL) were applied to full-thickness skin wounds of mice twice a day for 7 and 15 days. Wound zinc levels were higher on day 7 than on day 15, especially in wounds treated with EGF. The wound zinc levels of the gel + EGF group on day 15 were similar to those of normal control skin. These results imply that there is a close connection, but no direct relationship, between EGF application in both dosage forms and wound zinc levels during healing.

The Effect of Epidermal Growth Factor on Autogenous Fat Graft

Autogenous fat grafting is widely used for the correction of soft tissue contour deformity. However, the high absorption rate results in the need for overcorrection, and graft longevity is unpredictable. The authors hypothesized that epidermal growth factor (EGF), a potent stimulator of neovascularization, would improve fat graft survival. The experiment used two groups of New Zealand White Rabbit ear. Inguinal fat was harvested and injected with EGF or saline (n=24, each group). The 48 cases of fat grafting were managed for observation of volume and morphologic change. The fat was harvested 3months after the autogenous graft. The survival rate and the degree of neovascularization were measured. The grafts in the EGF group had a significantly higher survival rate than those in the control group. Histologic examination of the grafts demonstrated an increase in neovascularization and maintenance of fat cell morphology. These findings show that EGF can enhance fat graft survival and degree of neovascularization. Further well-controlled studies are required before EGF is used for clinical purposes.

Role of Wnt/β-catenin signaling pathway in impaired wound healing of diabetes mellitus

Objective To investigate the role of Wnt/β-catenin signaling pathway in impaired wound healing of diabetes mellitus.Methods The back skin defect was produced in rats with type1diabetes.All of these rats were divided into normal group, diabetes group, lithium chloride group, and epidermal growth factor (EGF) group.The back wound healing and β-catenin expression were observed.Results There were no signs of infection in the wound of rats after injury.Compared with diabetic group, the wound healing time was shorter,wound healing rate was higher, wound cavity volume was smaller, granulation tissue was more mature, and β-catenin positive cell rate was higher in normal group, lithium chloride group, and EGF group(P＜0.05 or P＜0.01).Conclusions Wnt/beta-catenin signaling pathway is involved in the process of wound healing in diabetic rats. Key words: Diabetes mellitus; Wound healing; Wnt/β-catenin

Effects of combined erythropoietin and epidermal growth factor on renal ischaemia/reperfusion injury: a randomized experimental controlled study

To investigate effects of combination of erythropoietin (EPO) and epidermal growth factor (EGF) on renal ischaemia and on reactive oxygen species in a rat model. In all, 90 male Sprague-Dawley rats were allocated into five groups of 18, designated: Sham; treated with right nephrectomy only; Control, subjected to left renal ischaemia for 45 min with no treatment; EPO-treated, as the control but with EPO pretreatment; EGF-treated, as the control but with EGF pretreatment; EPO + EGF-treated, as the control but with EPO and EGF pretreatment. Renal function, histopathology and malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) levels in kidneys were assessed at 1, 2 and 7 days after ischaemia. All rats except the controls had a significant improvement in serum creatinine, creatinine clearance and fractional excretion of Na(+) ; all three were significantly better in EPO + EGF group than in all other groups Histopathological examination showed marked structural damage in control rats. The tubular damage was least in the EPO + EGF group. The control group had a significant increase in MDA level and a significant decrease in SOD and GSH, while the EPO + EGF group had a marked significant reduction in MDA and increase in GSH and SOD. The protection against ischaemia/reperfusion injury might be maximal when EPO and EGF are administered concomitantly, and their protective effect might be partly due to their antioxidant effects.

Comparison of EGF with VEGF Non-Viral Gene Therapy for Cutaneous Wound Healing of Streptozotocin Diabetic Mice

BackgroundTo accelerate the healing of diabetic wounds, various kinds of growth factors have been employed. It is the short half-life of administered growth factors in hostile wound beds that have limited wide-spread clinical usage. To overcome this limitation, growth factor gene therapy could be an attractive alternative rather than direct application of factors onto the wound beds. We administered two growth factor DNAs, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) into a cutaneous wound on diabetic mice. We compared the different characteristics of the healing wounds.MethodsStreptozotocin was injected intraperitoneally to induce diabetes into C57BL/6J mice. The ultrasound micro-bubble destruction method with SonoVue as a bubbling agent was used for non-viral gene delivery of EGF828 and VEGF165 DNAs. Each gene was modified for increasing efficacy as FRM-EGF828 or minicircle VEGF165. The degree of neoangiogenesis was assessed using qualitative laser Doppler flowmetry. We compared wound size and histological findings of the skin wounds in each group.ResultsIn both groups, accelerated wound closure was observed in the mice receiving gene therapy compared with non treated diabetic control mice. Blood flow detected by laser doppler flowmetry was better in the VEGF group than in the EGF group. Wound healing rates and histological findings were more accelerated in the EGF gene therapy group than the VEGF group, but were not statistically significant.ConclusionBoth non-viral EGF and VEGF gene therapy administrations could improve the speed and quality of skin wound healing. However, the detailed histological characteristics of the healing wounds were different.

Epidermal growth factor effects on marmoset monkey (Callithrix jacchus) oocyte in vitro maturation, IVF and embryo development are altered by gonadotrophin concentration during oocyte maturation

This is the first study of the effect of epidermal growth factor (EGF) on marmoset monkey oocytes matured in vitro. We have evaluated the effects of 10 ng/ml EGF in combination with 1 or 10 IU/ml of gonadotrophins (FSH/hCG 1:1 ratio) during in vitro maturation (IVM) of marmoset oocytes. Immature cumulus-oocyte complexes (COCs) were retrieved from ovarian antral follicles of unprimed monkeys. COCs from six animals (n= 268) used in this study were randomly distributed among four experimental groups: (A) 1 FSH +1 hCG; (B) 10 FSH +10 hCG; (C) 1 FSH +1 hCG + EGF; and (D) 10 FSH +10 hCG + EGF (where 1 and 10 are concentrations, IU/ml). After IVM, oocytes were fertilized in vitro and embryos were allowed to progress up to 87-88 h. the highest rate of total and radial cumulus expansion was observed in Group A, with the lowest in Group B (P < 0.05). Neither maturation nor fertilization rate were affected by gonadotrophin concentration or presence of EGF. Addition of EGF increased degeneration and decreased first cleavage rate, which was significantly lower in Group C than Group A (P < 0.005). Interestingly, in the EGF groups some embryos cleaved faster than without EGF. The effects of EGF are highly dependent on concentration of gonadotrophins present in IVM medium. EGF has a negative effect on oocytes in the presence of low gonadotrophins, but contrastingly partially protects oocytes from the negative effects of high gonadotrophins. We propose that these observed negative effects of EGF may suggest use of an inappropriate dose of growth factor.