Epidermal Growth Factor Supplementation Research Articles

Protective effect of epidermal growth factor on cryopreservation of dromedary camel epididymal spermatozoa: Evidence from in vitro and in silico studies

Epidermal growth factor (EGF) plays a crucial role in maintaining male reproductive capacity in mammals, however, its protective effects on cryopreserved dromedary camel epididymal spermatozoa have not been thoroughly investigated. This study aims to investigate the potential protective role of EGF on cryopreserved camel epididymal spermatozoa, supported by evidence from a molecular docking study. We assessed sperm motility, kinematics parameters, oxidative stress, ultrastructural changes, apoptosis, and molecular docking markers in camel epididymal spermatozoa following cryopreservation. Camel epididymal spermatozoa (n=30 pairs of testes) were collected from local slaughterhouses. The epididymal spermatozoa were diluted with a freezing medium (SHOTOR extender) supplemented with different concentrations of EGF; 0 (EGF0), 50 (EGF50), 100 (EGF100), 200 (EGF200), and 400 (EGF400) ng/mL in SHOTOR extender and cryopreserved using a standard protocol. All EGF groups showed significant improvements in sperm progressive motility, viability, and sperm membrane function after equilibration at 5 °C for 24hours. Regarding frozen-thawed samples, sperm progressive motility and some kinematic parameters (DAP, VSL, VCL and AHL) were significantly higher in the EFG400 group compared to the other groups (P < 0.01). A significant increase in the percentage of live/acrosome-intact sperm was observed, accompanied by a significant decrease in malondialdehyde levels in all EGF groups (P < 0.05). Both the EGF200 and EGF400 groups showed significantly higher sperm viability and significantly lower percentages of apoptotic and necrotic sperm compared to the other groups (P < 0.05). EGF supplementation preserved the ultrastructural integrity and cryotolerance of epididymal camel spermatozoa. The docking analysis indicated that EGF exhibited higher binding affinity with apoptosis sperm markers, including caspase-3 and bcl-2-associated X (Bax) proteins, with binding energies of -502.0 and -621.0kcal/mol, respectively. In conclusion, the addition of EGF to SHOTOR extender was found to have beneficial effects on sperm motility, kinematics parameters, sperm viability, acrosome integrity, sperm ultrastructural features, and reduced oxidative stress and apoptosis-like changes in cryopreserved epididymal camel sperm.

Potential Benefits of Epidermal Growth Factor for Inhibiting Muscle Degrative Markers in Rats with Alcoholic Liver Damage.

This study investigated the beneficial effects of epidermal growth factor (EGF) on muscle loss in rats with chronic ethanol feeding. Six-week-old male Wistar rats were fed either a control liquid diet without EGF (C group, n = 12) or EGF (EGF-C group, n = 18) for two weeks. From the 3rd to 8th week, the C group was divided into two groups. One was continually fed with a control liquid diet (C group), and the other one was fed with an ethanol-containing liquid diet (E group); moreover, the EGF-C group was divided into three groups, such as the AEGF-C (continually fed with the same diet), PEGF-E (fed with the ethanol-containing liquid diet without EGF), and AEGF-E (fed with the ethanol-containing liquid diet with EGF). As a result, the E group had significantly higher plasma ALT and AST, endotoxin, ammonia, and interleukin 1b (IL-1b) levels, along with liver injuries, such as hepatic fatty changes and inflammatory cell infiltration. However, plasma endotoxin and IL-1b levels were significantly decreased in the PEGF-E and AEGF-E groups. In addition, the protein level of muscular myostatin and the mRNA levels of forkhead box transcription factors (FOXO), muscle RING-finger protein-1 (MURF-1) and atorgin-1 was increased considerably in the E group but inhibited in the PEGF-E and AEGF-E groups. According to the principal coordinate analysis findings, the gut microbiota composition differed between the control and ethanol liquid diet groups. In conclusion, although there was no noticeable improvement in muscle loss, EGF supplementation inhibited muscular protein degradation in rats fed with an ethanol-containing liquid diet for six weeks. The mechanisms might be related to endotoxin translocation inhibition, microbiota composition alteration as well as the amelioration of liver injury. However, the reproducibility of the results must be confirmed in future studies.

Effects of dietary epidermal growth factor supplementation on liver antioxidant capacity of piglets with intrauterine growth retardation.

The present experiment was conducted to study the effects of dietary epidermal growth factor (EGF) supplementation on the liver antioxidant capacity of piglets with intrauterine growth retardation (IUGR). The present study consists of two experiments. In experiment 1, six normal-birth-weight (NBW) and six IUGR newborn piglets were slaughtered within 2 to 4 h after birth to compare the effects of IUGR on the liver antioxidant capacity of newborn piglets. The results showed that compared with NBW piglets, IUGR piglets had a lower birth weight and liver relative weight; IUGR piglets had a higher serum malondialdehyde (MDA) level, liver MDA level and hydrogen peroxide (H2O2) level, and had a lower liver total antioxidant capacity (T-AOC) level and glutathione peroxidase (GSH-Px) activity; IUGR trended to increase serum alanine aminotransferase activity, aspartate aminotransferase activity, and H2O2 level, and trended to decrease liver total superoxide dismutase activity. In experiment 2, six NBW piglets, and 12 IUGR piglets weaned at 21 d of age were randomly divided into the NC group (NBW piglets fed with basal diet); IC group (IUGR piglets fed with basal diet), and IE group (IUGR piglets fed with basal diet plus 2 mg/kg EGF), and feeding for 14 d. Organ index, serum parameters, liver antioxidant capacity, and liver antioxidant-related genes expression were measured. The results showed that compared to the IC group, dietary EGF supplementation (IE group) significantly reduced serum malondialdehyde level and H2O2 level, and liver protein carbonyl (PC) level and 8-hydroxydeoxyguanosine level of piglets with IUGR; dietary EGF supplementation (IE group) significantly increased serum T-AOC level, liver T-AOC level and GSH-Px activity; dietary supplemented with EGF (IE group) enhanced liver Nrf2, NQO1, HO1, and GPX1 mRNA expression compared to IC group. Pearson's correlation analysis further showed that EGF can alleviate liver oxidative injury caused by IUGR and improve the performance of IUGR piglets. In conclusion, EGF exhibited potent protective effects on IUGR-induced liver oxidative injury, by activating the Nrf2 signaling pathway to mediate the expression of downstream antioxidant enzymes and phase II detoxification enzymes (NQO1 and HO1), thereby alleviating liver oxidative damage and promoting the growth performance of IUGR piglets.

Dietary Epidermal Growth Factor Supplementation Alleviates Intestinal Injury in Piglets with Intrauterine Growth Retardation via Reducing Oxidative Stress and Enhancing Intestinal Glucose Transport and Barrier Function.

Simple SummaryIt is well known that intrauterine growth retardation (IUGR) seriously affects the postnatal growth and intestinal health of piglets. Keeping intestinal health is of great importance to pig production. Numerous studies have shown that epidermal growth factor (EGF) has a positive effect on the repair of intestinal injury of animals. However, whether EGF can promote the intestinal health of IUGR piglets is unknown. Therefore, in our present study, a total of 12 IUGR piglets and 6 normal birth weight (NBW) piglets were selected to study the effects of EGF on the growth performance and intestinal health of IUGR piglets. Our study found that basal diet supplemented with 2 mg/kg EGF significantly improved the growth performance, jejunum morphology, jejunum glucose absorption and antioxidant capacity, and jejunum barrier function of piglets with IUGR. Therefore, this study confirmed that EGF has positive effects on the growth and intestinal development of piglets with IUGR.EGF plays an important role in the intestinal repair and nutrients transport of animals. However, the effect of EGF on the intestinal health of piglets with IUGR has not been reported. Thus, the present study was performed to investigate the effects of EGF on the intestinal morphology, glucose absorption, antioxidant capacity, and barrier function of piglets with IUGR. A total of 6 NBW piglets and 12 IUGR piglets were randomly divided into three treatments: NC group (NBW piglets fed with basal diet, n = 6), IC group (IUGR piglets fed with basal diet, n = 6), and IE group (IUGR piglets fed with basal diet supplemented with 2 mg/kg EGF, n = 6). Growth performance, serum biochemical profile, jejunum histomorphology, jejunum glucose absorption and antioxidant capacity, and jejunal barrier function were measured. The results showed that EGF supplementation significantly increased the final body weight (FBW), average daily gain (ADG), and average daily feed intake (ADFI) of piglets with IUGR; EGF supplementation significantly increased the total protein (TP), glucose (GLU), and immunoglobulin G (IgG) levels compared with the IUGR piglets in the IC group; EGF administration effectively exhibited an increased jejunum villus height (VH) and the villus-height-to-crypt-depth ratio (V/C) of IUGR piglets compared with the IC group; EGF supplementation significantly increased sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, intestinal alkaline phosphatase (AKP) activity, glucose transporter sodium/glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), and AMP-activated protein kinase α1 (AMPK-α1) mRNA expressions in the jejunum of IUGR piglets compared with the IC group; EGF supplementation exhibited increased superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) levels, tended to increase glutathione peroxidase (GSH-Px) and catalase (CAT) activities, and tended to decrease the malondialdehyde (MDA) level in the jejunum of IUGR piglets compared with the IC group; EGF supplementation significantly increased ZO-1, Claudin-1, Occludin, and MUC2 mRNA expressions and improved secreted immunoglobulin A (sIgA) secretion in the jejunum of IUGR piglets compared with the IC group and tended to decrease the interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) levels in the jejunum of IUGR piglets compared with the IC group. Pearson’s correlation analysis further showed that EGF can promote intestinal development and nutrient absorption by promoting intestinal barrier function, thus improving the growth performance of IUGR piglets.

Differentiation ability of rat‐mesenchymal stem cells from bone marrow and adipose tissue to neurons and glial cells

Mesenchymal stem cells (MSCs) are multipotent cells and can differentiate into neurons and glial cells. In vitro differentiation would be done by the addition of various factors. There remains no comparison for the differentiation of MSCs from rat bone marrow (rBMMSCs) and adipose tissue (rATMSCS) into neurons and glial cells with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and brain‐derived neurotrophic factor (BDNF). The aims of this study were to investigate the effect of bFGF, EGF, and BDNF supplementation on the differentiation ability of rBMMSCs and rATMSCs into neurons and glial cells. MSCs were cultured with bFGF and EGF for 4 days and then BDNF was added until day 8. Characterization of MSCs before and after induction was carried out by observing the cell morphology and several cell markers. Flowcytometry analysis was performed for MSCs markers (CD90, CD29) and neurons and glial cell markers (A2B5, Beta‐III‐tubulin, PSAN‐CAM); while MAP‐2, a neuron marker, was analyzed by immunocytochemistry. Induction of both types of MSCs showed MAP‐2‐positive cells, decreased MSCs markers, and in rBMMSCs showed increased neuron markers. The number of neuron marker positive cells in rBMMSCS was higher than rATMSCs. This study showed that the addition of bFGF, EGF, and BDNF to the medium induced rBMMSCs into neurons and glial cells, but the conditions were not optimal for rATMSC as judged by the expression of neural markers (A2B5, Beta‐III‐tubulin, PSAN‐CAM, and MAP‐2).

Modulation of the Systemic Immune Response in Suckling Rats by Breast Milk TGF-β2, EGF and FGF21 Supplementation.

Breast milk is a rich fluid containing bioactive compounds such as specific growth factors (GF) that contribute to maturation of the immune system in early life. The aim of this study was to determine whether transforming growth factor-β2 (TGF-β2), epidermal growth factor (EGF) and fibroblast growth factor 21 (FGF21), compounds present in breast milk, could promote systemic immune maturation. For this purpose, newborn Wistar rats were daily supplemented with these GF by oral gavage during the suckling period (21 days of life). At day 14 and 21 of life, plasma for immunoglobulin (Ig) quantification was obtained and spleen lymphocytes were isolated, immunophenotyped and cultured to evaluate their ability to proliferate and release cytokines. The main result was obtained at day 14, when supplementation with EGF increased B cell proportion to reach levels observed at day 21. At the end of the suckling period, all GF increased the plasma levels of IgG1 and IgG2a isotypes, FGF21 balanced the Th1/Th2 cytokine response and both EGF and FGF21 modified splenic lymphocyte composition. These results suggested that the studied milk bioactive factors, mainly EGF and FGF21, may have modulatory roles in the systemic immune responses in early life, although their physiological roles remain to be established.

Epidermal growth factor promotes intestinal secretory cell differentiation in weaning piglets via Wnt/β-catenin signalling

Small intestinal epithelium homeostasis involves four principal cell types: enterocytes, goblet, enteroendocrine and Paneth cells. Epidermal growth factor (EGF) has been shown to affect enterocyte differentiation. This study determined the effect of dietary EGF on goblet, enteroendocrine and Paneth cell differentiation in piglet small intestine and potential mechanisms. Forty-two weaned piglets were used in a 2 × 3 factorial design; the major factors were time post-weaning (days 7 and 14) and dietary treatment (0, 200 or 400 µg/kg EGF supplementation). The numbers of goblet and enteroendocrine cells were generally greater with the increase in time post-weaning. Moreover, the supplementation of 200 µg/kg EGF increased (P < 0.01) the number of goblet and enteroendocrine cells in villus and crypt of the piglet small intestine as compared with the control. Dietary supplementation with 200 µg/kg EGF enhanced (P < 0.05) abundances of differentiation-related genes atonal homologue 1, mucin 2 and intestinal trefoil factor 3 messenger RNA (mRNA) as compared with the control. Piglets fed 200 or 400 µg/kg EGF diet had increased (P < 0.05) abundances of growth factor-independent 1, SAM pointed domain containing ETS transcription factor and pancreatic and duodenal homeobox 1 mRNA, but decreased the abundance (P < 0.01) of E74 like ETS transcription factor 3 mRNA as compared with the control. Animals receiving 400 µg/kg EGF diets had enhanced (P < 0.05) abundances of neurogenin3 and SRY-box containing gene 9 mRNA as compared with the control. The mRNA abundance and protein expression of lysozyme, a marker of Paneth cell, were also increased (P < 0.05) in those animals. As compared with the control, dietary supplementation with 200 µg/kg EGF increased the abundance of EGF receptor mRNA and the ratio of non-phospho(p)-β-catenin/β-catenin (P < 0.05) in villus epithelial cells at days 7 and 14. This ratio in crypt epithelial cells was higher (P < 0.05) on the both 200 and 400 µg/kg EGF groups during the same period. Our results demonstrated that dietary EGF stimulated goblet, enteroendocrine and Paneth cell differentiation in piglets during the post-weaning period, partly through EGFR and Wnt/β-catenin signalling.

Epidermal growth factor attenuates lingual papillae lesions in a rat model of sialoadenectomy

Salivary epidermal growth factor (EGF) plays an important role in the maintenance of the oral and gastro-esophageal mucosa. Sialoadenectomy delays healing of oral wounds and affects lingual papillae. In this work, we aimed to determine the effect of EGF deficiency induced by sialoadenectomy and evaluate the effect of exogenous EGF administration on the lingual papillae and taste buds in rats. Thirty male adult Wistar albino rats were equally divided into 3 groups; sham-operated control group, sialoadenectomy group and group of sialoadenectomy + EGF. EGF was given 8 weeks after sialoadenectomy in a dose of 1 μg /ml/day in drinking water for 2 weeks. The anterior two-thirds of the tongue was dissected and cut longitudinally into two halves; one half for light microscope and the other for electron microscope examinations. Saliva and blood were collected to determine salivary and plasma EGF. Our results revealed that sialoadenectomy significantly reduced plasma and saliva levels of EGF which resulted in severe disruption of the architecture of lingual papillae. These changes were effectively improved by the exogenous EGF administration. In conclusion, EGF supplementation reversed the effects of sialoadenectomy and restored almost normal architecture of lingual papillae and taste buds.

Leptin and EGF Supplementation Enhance the Immune System Maturation in Preterm Suckling Rats.

In preterm newborns the immaturity of the immune system is remarkable, with reduced innate and adaptive immune responses. Many bioactive compounds in breast milk, such as growth factors and adipokines, contribute to the immune system’s maturation in early life. However, studies on the immunoregulatory activity in preterm neonates are practically nonexistent. The aim of the present study was to determine whether a nutritional supplementation in early life with leptin or epidermal growth factor (EGF) was able to promote the maturation of the systemic and intestinal immune system in preterm conditions. For this purpose, premature rats were daily supplemented by oral gavage with leptin or EGF. Term and Preterm groups receiving vehicle were used as controls. Preterm rats showed deficiencies compared to full-term ones, such as lower body weights, erythrocyte counts, plasma IgG and IgM concentrations and B cell percentages, and higher values of Th and Tc TCRαβ+ cells in mesenteric lymph nodes, and intestinal permeability, among others. However, leptin and EGF supplementation were able to revert some of these deficiencies and to improve the premature immune system’s development. These results suggest that leptin and EGF are involved in enhancing the maturation of the systemic and intestinal immune system in preterm conditions.

Enhance hepatic differentiation of human Wharton's jelly–derived mesenchymal stromal cells by using sodium butyrate pre-treated

Background & Aim Human Wharton's jelly–derived mesenchymal stromal cells (hWJ–MSCs) of umbilical cord tissue are a richest and more attractive source for stem cell–based therapy. Despite of their ability to undergo self–renewal, they also differentiate into all mesenchyme and some non–mesenchyme tissues, including hepatocytes. This study, we differentiated hWJ–MSCs into hepatocyte–like cells by using sodium butyrate (NaB) pre–treated with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) supplementation. Methods, Results & Conclusion Hepatic differentiation of hWJ–MSCs was induced with 3–step differentiation protocol. The differentiated cells were examined for the expression of hepatic–specific markers and hepatocyte function by using immunocytochemistry, real–time reverse transcriptase–polymerase chain reaction (RT–PCR) and Periodic acid–Schiff (PAS) staining. Before hepatic differentiation of hWJ–MSCs, we identified embryonic definitive endodermal cells by using RT–PCR, which increase of CXCR4 mRNA level, after NaB pre–treated along with EGF and bFGF supplementation. Hepatic differentiation of hWJ–MSCs by using NaB pre–treated along with EGF and bFGF supplementation, the differentiated hWJ–MSCs performed high hepatic–specific markers at gene and protein levels, and increased signal of glycogen storage accumulation. Our result indicate that hWJ–MSCs are able to differentiate into hepatocyte–like cells by inducing of hepatic differentiation medium and NaB pre–treated with EGF and bFGF supplementation. Therefore, the differentiated hWJ–MSCs to hepatocyte–like cells by inducing of NaB pre–treated with EGF and bFGF condition could represent an alternative source of end–stage of liver diseases.

Effects of dietary supplementation with epidermal growth factor on nutrient digestibility, intestinal development and expression of nutrient transporters in early‐weaned piglets

The abnormalities in intestinal morphology and digestive function during weaning are associated with the loss of milk-borne growth factors. Epidermal growth factor (EGF) has been shown to stimulate the growth of animals. This study was to determine the effect of dietary EGF on nutrient digestibility, intestinal development and the expression of genes encoding nutrient transporters in weaned piglets. Forty-two piglets were weaned at 21days and assigned to one of three treatment groups: (1) basal diet (control), (2) basal diet+200µg/kg EGF or (3) basal diet+400µg/kg EGF. Each treatment consisted of 14 replicates, and seven piglets from each treatment were sampled on day 7 and 14. The EGF supplementation significantly elevated (p<0.05) the coefficients of total tract apparent digestibility of crude protein, calcium and phosphorus, but tended to decrease sucrase activity (p<0.10) than the control group. At day 7 post-weaning, animals receiving EGF diets showed a tendency (p<0.10) towards greater ileal villus height (VH), jejunal crypt depth (CD) and duodenal VH:CD when compared with the control group. Moreover, the mRNA levels of glucose transporter 2 (Slc2a2), neutral amino acid transporter (Slc6a19) and calbindin D9k (S100G) tended to be higher (p<0.10) for EGF groups than the control group. By day 14, EGF supplementation markedly enhanced (p<0.05) the VH, CD and VH:CD in the jejunum compared to the control group. This addition also up-regulated (p<0.05) the mRNA level and the protein abundance of peptide transporter 1 than the control group. These findings demonstrated that dietary EGF beneficially enhanced nutrient digestibility, improved intestinal development and increased the mRNA expression of nutrient transporters in weaned piglets.

Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients

ObjectiveRegenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. MethodsThe secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. ResultsBoth BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. ConclusionsPL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

The impact of epidermal growth factor supernatant on pig performance and ileal microbiota.

Weaning of pigs can lead to low-feed intake resulting in a lag in growth performance, reduced gut health, and diarrheal diseases. Epidermal growth factor (EGF), the most abundant growth factor in milk, increased weaned pig BW gain and feed efficiency in our previous work. It is believed that intestinal microbiota plays an important role in gut health and pig growth, but limited data are available on the impact of feed additives, such as EGF, on the microbial communities of the intestines. The objective of the study was to investigate if the positive influence of EGF supplementation on weight gain and gut health was related to differences in intestinal microbiota. To examine the efficacy of EGF, a 21-d animal trial was performed using 72 pigs (two equal blocks of 36 pigs with three barrows and three gilts/pen). Pigs were assigned to one of two dietary treatments at weaning (20 ± 2 d of age; n = 6 pens/treatment) balancing across treatment for litter, gender, and initial BW. Recombinant yeast supernatant containing EGF at 120 μg/kg BW/d and without EGF (control) was added to the feed for 21 d, followed by a common diet for 7 d. Pig performance was measured weekly and ileal digesta was collected at day 21 from six pigs/treatment for microbiome analysis. Pigs fed diets containing EGF fermentation supernatant had greater (P = 0.01) daily gain in week 3 and overall resulting in heavier (P = 0.029) BW at day 28, which was consistent to our previous finding. No difference in alpha-diversity (Chao1, Shanon, and Simpson indices) and beta-diversity (weighted and unweighted UniFrac distances) of ileal digesta microbiota between EGF supplemented and control pigs were observed. The relative abundances of bacterial taxa did not differ among treatment groups at the phylum level. The relative abundances of Corynebacterium (0.0 vs. 0.9%), Blautia (0.003 vs. 0.26%), and Coprococcus (0.0 vs. 0.05%) genera, and Rumminococcaceae family (0.001 vs. 0.08%) were decreased (P < 0.05) in EGF group compared to control and were negatively correlated (P < 0.05, r > 0.60) with growth performance. Pathways related to detoxification and carbohydrate metabolism were differentially represented in the luminal bacterial populations. The improved growth of pigs supplemented with EGF supernatant produced by Pichia pastoris may be related to changes in functional capacity of the gut microbial populations. However, the impact on mucosa-associated or large intestinal communities is still unknown.

Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis

We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF- cells) or the presence (EGF+ cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100μM) decreased cell viability in EGF- but not in EGF+ cells. In EGF- cells, Tl(I) decreased mitochondrial potential, enhanced H2O2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF- cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF- and EGF+ cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF- cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF- and EGF+ cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

Requirement of Leukemia Inhibitory Factor or Epidermal Growth Factor for Pre-Implantation Embryogenesis via JAK/STAT3 Signaling Pathways.

Leukemia inhibitory factor (LIF) plays a key role in the survivability of mouse embryos during pre-implantation. In this study, we verified the role of LIF by detecting gene expression in morula stage embryos through DNA microarray. Our results showed that LIF knockdown affected expression of 369 genes. After LIF supplementation, the epidermal growth factor (EGF) is most affected by LIF expression. To observe the correlation between LIF and EGF, the LIF knockdown embryos were supplemented with various growth factors, including LIF, EGF, GM-CSF, TGF, and IGF II. Only LIF and EGF caused the rate of blastocyst development to recover significantly from 52% of control to 83% and 93%, respectively. All of the variables, including the diameter of blastocysts, the number of blastomeres, and cells in ICM and TE, were almost restored. Moreover, EGF knockdown also impaired blastocyst development, which was reversed by LIF or EGF supplementation. The treatment with various signaling suppressors revealed that both EGF and LIF promoted embryonic development through the JAK/STAT3 signaling pathway. These data suggest that the EGF and LIF can be compensatory to each other during early embryonic development, and at least one of them is necessary for sustaining the normal development of pre-implantation embryos.

Abstract PR14: Extracellular matrix control of metastasis and dormancy

Abstract Introduction: Paget's classical seed and soil hypothesis states that specific interactions between the metastatic cell (the seed) and the recipient tissue microenvironment (the soil) mediate the non-random patterns of spread observed in breast cancer (tropism). We hypothesize that the ability of metastatic cells to survive dormancy, exit quiescence, and colonize a specific tissue depends upon the ability of the soil to first sustain survival, and subsequently trigger outgrowth. To this end, we created a biomaterial platform that varies extracellular matrix (ECM) density and composition, and used integrin-mediated phenotyping to study how highly metastatic cells differentiate between secondary sites via integrin binding. With this relationship in hand, we have found that both the ECM and growth factors also mediate dormancy in cells that display metastatic latency in vivo. Methods: ECM microenvironments were created by linking combinations of ECM proteins to glass coverslips using silane chemistry. Human breast cancer cell lines were seeded onto the surfaces, and we quantified initial cell adhesion and long-term migration via microscopy. Using cell lines that only metastasize to the bone, brain, or lung (provided by J. Massagué), we created fingerprints of behavior characteristic of each tropism, and compared these with the phenotypes of other cell lines with known metastasis. Quiescence was induced on ECMs via culture in serum free medium for up to 14 days, and subsequent serum-stimulation for allowed for recovery and growth. Proliferation was assayed via immunofluorescent staining for Ki67, and cell number was determined via DAPI staining. Results: We identified unique phenotypes of bone, brain, and lung tropic subpopulations, and confirmed these in seven of nine other known, more heterogeneous, metastatic cell lines, establishing a connection between in vitro phenotype and in vivo fate. Function-affecting antibodies to integrins each shift a cell line's predicted tropism in vitro, and these in vitro phenotyping results mimic the clinical metastasis patterns dictated by both α2 and α6 integrin gene expression. Interestingly, however, our functional predictions regarding β1 integrin actually provide insights that are largely independent of clinical gene expression, potentially identifying new metastatic therapeutic targets, and emphasizing the need for this type of functional analysis. However, when we applied this approach to cell lines that display latency in vivo (e.g., ZR-75-1), our predictions were not correct. These cells were generally insensitive to ECM and growth factors in adhesion and motility, but instead, we found that these factors regulate entrance into dormancy. We uniformly observe that a laminin-coupled ECM is unable to permit survival of dormant cells, whereas binding to collagen I maintains cell survival for at least 14 days. Epidermal growth factor supplementation improves the entrance into dormancy on collagen alone. We have found that this collagen-specific, EGF-enhanced survival ability is mediated by β1 integrin binding and activation of ERK1/2, suggesting that β1 integrin function is critical both for aggressive metastasis and for permitting dormancy. Conclusions: We are the first to report that bone, brain, and lung tropism can be distinguished via simple integrin-mediated phenotyping, and that this approach can be used to predict in vivo tropism. We suggest that functional β1 integrin binding mediates both aggressive and latent metastasis. In sum, we propose that this functional screen of cell-matrix interactions can predict in vivo outcomes, and thus, is a valuable tool to provide insight toward integrins as druggable targets for metastatic disease. Citation Format: Lauren E. Barney, Ari Gilman, Arthur Mercurio, Shelly R. Peyton. Extracellular matrix control of metastasis and dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR14.

Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.

Milk-borne epidermal growth factor modulates bilirubin levels in neonatal rats

Summary Objectives Breast milk jaundice (BMJ) is commonly of undetermined etiology. Recently, it has been shown that the degree of BMJ was associated with the increased levels of milk-borne EGF (Epidermal Growth Factor). The aim of the present study was to test the effects of enteral administration of EGF on the development of hyperbilirubinemia in neonatal rats. Methods Pups were divided into three groups: Group 1; artificially fed with formula, Group 2; artificially fed with formula supplemented with 500 ng/ml rat EGF, Group 3; fed by their mothers. Hyperbilirubinemia was induced by an intraperitoneal injection of unconjugated bilirubin suspension once daily for 5 consecutive days. Bilirubin levels, length of ideal villi, and EGF-R expression in the intestine was determined at postnatal day 6. Results In the groups of 2 and 3, serum bilirubin levels were significantly higher when compared to animals in the group 1. There was no significant difference in terms of serum bilirubin levels between the Group 2 and Group 3. Length of ileal villi was significantly longer in the Group 2 and Group 3 than the Group 1 animals. The pattern of EGF-R expression in the Group 2 was similar to that seen in Group 3 animals. In contrast, animals given formula without EGF had markedly decreased expression of the EGF-R. Conclusion Results suggest that EGF supplementation in newborn rats leads to a significant increase in intestinal mucosal proliferation and a significant decrease in bilirubin elimination. These data suggest that EGF possibly increases intestinal bilirubin absorption and may have a role in development of breast milk jaundice. Further studies are needed to confirm this hypothesis.

The EGF signaling pathway influences cell migration and the secretion of metalloproteinases by myoepithelial cells in pleomorphic adenoma.

During tumor development, benign neoplastic cells are influenced by the expression of cytokines, growth factors, and proteases present in the tumor microenvironment. Epidermal growth factor (EGF) is the most studied growth factor and is considered important for cell proliferation and migration. Metalloproteinases (MMPs) are also involved in tumor progression. The present study aimed to analyze the proliferation, viability and migration index of pleomorphic adenoma myoepithelial cells, in addition to the secretion of MMPs with EGF supplementation. Benign myoepithelial cells were cultured with two different EGF doses (5 and 10ng/ml), and the influence of EGF on cell proliferation and viability, using trypan blue and MTT assays, respectively, after 24, 48, and 72h, was evaluated. To analyze cellular morphology, hematoxylin-eosin staining and indirect immunofluorescence using the anti-vimentin antibody, was performed. In vitro migration assays were performed in Transwell chambers with an 8-μm pore covered with Matrigel and supplemented with 5 or 10ng/ml of EGF, after 96h. After 4days of cell culture, ELISA was performed to determine the MMP-2 and MMP-13 levels. One-way analysis of variance (ANOVA) with post hoc Tukey test was applied, with a significance level of 0.05. The results revealed that EGF influences myoepithelial cell morphology, without alteration of proliferation and viability. The migration assay showed that EGF increased the mean index from 16% in the control group to 40 and 76% for 5 and 10ng/ml of EGF, respectively. ELISA revealed that when the cells were supplemented with eitherof the EGF doses, an increase in MMP-2 levels was observed when compared with the control group (C). This study concludes that EGF aids in the production of MMP-2, which favors the dissolution of the basement membrane, contributing to cell migration and tumor progression, hence permitting contact between the myoepithelial cells and stroma.

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.