Epidermal Growth Factor Gene Expression Research Articles

Acceleration of Wound Healing in Diabetic Rats through a Novel 3D Organo-Hydrogel Nanocomposite of Polydopamine/TiO2 Nanoparticles and Cu (PDA-TiO2@Cu).

Diabetic wound represents a serious issue with a substantial impact and an exceptionally complex pathology affecting patients' mental health and quality of life. So, we have developed a novel 3D organo-hydrogel nanocomposite of polydopamine/TiO2 nanoparticles and cu (PDA-TiO2@Cu) and examined its efficacy in diabetic wound healing. Forty-five adult male albino rats were divided into normal control rats (non-diabetic rats with non-treated skin wounds), diabetic control rats (diabetic rats with non-treated skin wounds), and organo-hydrogel-treated rats (diabetic wounds treated with topically applied organo- hydrogel once daily). Macroscopic changes of the wound were observed on days 0, 3, 5, 7, and 10 to measure wound diameters. Skin specimens from the wound tissue were taken on days 3, 7, and 10, respectively, and examined histologically and immunohistochemically. Also, the gene expressions of collagen I, Matrix Metalloproteinase-9 (MMP-9), and Epidermal Growth Factor (EGF), and levels of Interleukin 6 (IL-6) and Superoxide Dismutase (SOD) were assessed. Our observed results indicated that the developed patch significantly accelerated the healing time compared to the normal control and diabetic control groups. Moreover, the patchloaded group revealed complete re-epithelization and a highly significant increase in the mean area % of CD31 immunostaining on day 7. The organo-hydrogel-loaded group displayed a significant decrease in gene expression of MMP-9 and a significant increase in gene expression of EGF and collagen I. Additionally, the organo-hydrogel-loaded group exhibited a significant decrease in levels of IL-6 and a significant increase in levels of SOD, compared to the normal diabetic control groups. The organo-hydrogel can be used for treating and decreasing the healing period of diabetic wounds.

Endometrial Heparin-Binding Epidermal Growth Factor Gene Expression and Hormone Level Changes in Implantation Window of Obese Women with Polycystic Ovarian Syndrome

Polycystic ovarian syndrome (PCOS) is a common endocrine disorder amongst reproductive-age women, and 61% to 76% of women with PCOS are obese. Obese women with PCOS are usually burdened with infertility problems due to implantation failure. Thus, progesterone treatment is usually used to improve implantation rates. Although Hb-EGF expression is actively involved in endometrial receptivity and implantation, the data on heparin-binding epidermal growth factor (Hb-EGF) expression following progesterone therapy in obese women with PCOS are still lacking. To investigate the changes in serum follicle-stimulating hormone (FSH), luteinising hormone (LH), dehydroepiandrosterone sulphate (DHEA), progesterone and oestradiol levels and Hb-EGF expression in obese women with PCOS during the implantation window following progesterone therapy. A total of 40 participants aged 18-40 years old were recruited following the provision of written consent. The participants were divided into the obese PCOS, normal-weight PCOS, obese fertile and normal-weight fertile groups. First blood collection was done before ovulation. Then, daily oral micronised progesterone (Utrogestan 200 mg) was given to the PCOS group for 10 days. The treatment was followed by a second blood collection and endometrial tissue sampling by using a Pipelle de Cornier catheter. In the fertile group, ovulation was confirmed by using ultrasound, and a second blood sample was collected on days 7 to 9 postovulation. The serum levels of FSH, LH, DHEA, progesterone and oestradiol were measured in all participants. Wilcoxon signed-rank test was used to compare FSH, LH, DHEA, progesterone and oestradiol levels during pre- and postovulation. Mann-Whitney test was performed to compare FSH, LH, DHEA, progesterone and oestradiol levels between two groups: (1) the PCOS group and the fertile group, (2) the obese PCOS group and the non-obese PCOS group and (3) the obese group and the non-obese fertile group. Serum FSH levels were lower in obese women in their follicular phase than in women with normal weight regardless of their PCOS status, whereas serum LH/FSH ratios and DHEA levels were higher in women with PCOS than in women without PCOS. However, endometrial Hb-EGF expression was lower in the obese PCOS group than in the normal-weight PCOS group. Different patterns of hormonal levels and Hb-EGF expression levels were seen between the studied groups. However, further in vitro and in vivo studies are needed to investigate the mechanism underlying the changes in FSH, LH/FSH ratio, DHEA and Hb-EGF expression in PCOS after progesterone treatment.

Desquamated Epithelial Cells of Unstimulated Human Whole Saliva Express Both EGF Transcript and Protein.

The aim of this study was to investigate if desquamated oral epithelial cells (DOECs) express the epidermal growth factor (EGF) and if these cells thereby may contribute to salivary EGF contents. DOECs have recently been shown to harbor the antimicrobial peptide LL-37, proposing that they may also store other biologically important salivary peptides/proteins. The EGF peptide is a growth factor which plays a critical role to maintain epithelial integrity and promote epithelial healing. The EGF is produced by salivary glands, but it is not known whether DOECs contain the EGF and thereby contribute to salivary EGF levels. DOECs were isolated from unstimulated whole saliva collected from four healthy volunteers. EGF protein expression was determined in cell lysates by dot blot and ELISA. Cellular distribution of cytokeratin, the proliferation marker Ki67, and EGF immunoreactivity were assessed by immunocytochemistry. EGF gene expression was investigated by qPCR. Expression of EGF transcript and protein in DOECs was compared to that in the human cultured keratinocyte cell line (HaCaT) cells. EGF protein expression was detected in DOEC cell lysates by both dot blot and ELISA. Strong cytoplasmic EGF immunoreactivity was observed in DOECs, although some cells showed only a weak immunoreactive signal for EGF. Moreover, DOECs, besides containing EGF protein, also expressed transcript for EGF. Interestingly, ELISA analysis revealed that EGF protein contents were higher in DOECs than in HaCaT cells. ELISA analysis also disclosed that EGF concentration was about 10 times higher in whole saliva compared to DOECs. EGF transcript expression was about 50% lower in HaCaT cells stimulated with high (10%) compared to low (0.1%) concentration of fetal bovine serum, representing growth-stimulated and growth-restricted conditions, respectively, implying that growth-stimulus exerts negative feedback on EGF gene activity in HaCaT cells. Here, we show for the first time that DOECs express the EGF, arguing that these cells contribute to salivary EGF contents and hence may play a role in gingival epithelial repair and wound healing.

Lack of Association between Epidermal Growth Factor or Its Receptor and Reflux Esophagitis, Barrett's Esophagus, and Esophageal Adenocarcinoma: A Case-Control Study.

The epidermal growth factor (EGF) and its receptor (EGFR) gene-gene interactions were shown to increase the susceptibility to esophageal cancer. However, the role of the EGF/EGFR pathway in the development of gastroesophageal reflux disease (GERD) and its complications (reflux esophagitis (RE), Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC)) remains unclear. This association study is aimed at investigating functional EGF and EGFR gene polymorphisms, their mRNA expression in esophageal tissues, and EGF plasma levels in relation to RE, BE, and EAC development in the Central European population. 301 patients with RE/BE/EAC (cases) as well as 98 patients with nonerosive reflux disease (NERD) and 8 healthy individuals (controls) were genotyped for +61 A>G EGF (rs4444903) and +142285 G>A EGFR (rs2227983) polymorphisms using the TaqMan quantitative polymerase chain reaction (qPCR). In random subgroups, the EGF and EGFR mRNA expressions were analyzed by reverse transcription qPCR in esophageal tissue with and without endoscopically visible pathological changes; and the EGF plasma levels were determined by enzyme-linked immunosorbent assay. None of the genotyped SNPs nor EGF-EGFR genotype interactions were associated with RE, BE, or EAC development (p > 0.05). Moreover, mRNA expression of neither EGF nor EGFR differed between samples of the esophageal tissue with and without endoscopically visible pathology (p > 0.05) nor between samples from patients with different diagnoses, i.e., RE, BE, or EAC (p > 0.05). Nevertheless, the lower EGF mRNA expression in carriers of combined genotypes AA +61 EGF (rs4444903) and GG +142285 EGFR (rs2227983; p < 0.05) suggests a possible direct/indirect effect of EGF-EGFR gene interactions on EGF gene expression. In conclusion, EGF and EGFR gene variants and their mRNA/protein expression were not associated with RE, BE or EAC development in the Central European population.

Epidermal Growth Factor Is Associated with Loss of Mucosae Sealing and Peri-Implant Mucositis: A Pilot Study.

This study aimed to evaluate the correlation between epidermal growth factor (EGF) and receptor (EGFR) levels in different clinical stages of dental implant rehabilitation and trace mucositis development’s biological profile. Thirty-six participants from the Specialization in Implant Dentistry, Universidade Federal Fluminense, Brazil, were included in the study and underwent sample collection: inside the alveolar socket, immediately before implant placement (Group 1, n = 10); at the peri-implant crevicular fluid (PICF) during reopening (Group 2, n = 10); PICF from healthy peri-implant in function (Group 3, n = 8); and PICF from mucositis sites (Group 4, n = 18). Quantitative polymerase chain reaction (PCR) evaluated EGF/EGFR gene expression using the SYBR Green Master Mix detection system. The results showed that EGF expression in the peri-implant crevicular fluid was statistically different. There was a higher EGF expression for group C (peri-implant health) (p = 0.04) than for the other groups. Regarding EGFR, there was no statistical difference among the groups (p = 0.56). It was concluded that low levels of EGF gene expression in the peri-implant crevicular fluid are related to the development of peri-implant mucositis and the absence of mucosae sealing. There was no correlation between EGFR gene expression with health or mucositis.

Transcriptomal Evidence for Dysregulation of Angiogenic Factors and Inflammatory Markers after Allogeneic Hematopoietic Cell Transplantation

Introduction:Angiogenic factors (AF) are the prototypic growth factors required for wound healing, and are linked to the immune response after tissue injury. Angiogenesis and inflammation together influence recovery from organ damage after hematopoietic stem cell transplantation (HCT), as well as contribute to the risk for complications such as acute graft-versus-host disease (aGVHD). We have recently described alterations in circulating angiogenic factors occurring at the onset of aGVHD post-HCT (BBMT 2015 21(6):1029), with dysregulation in AF associated with tissue repair versus those reflective of tissue damage. We hypothesized that the AF-inflammatory signature identified in the plasma in severe aGVHD could be linked to the gene expression of circulating donor cells after HCT. We performed transcriptomal analysis of changes in the peripheral blood immune cells and correlated with plasma AF levels in order to understand if the circulating donor cells might contribute to the AF profile.Methods:Peripheral blood samples from 3 patients (uncomplicated without aGVHD, steroid responsive aGVHD, and steroid refractory aGVHD) were compared to a healthy stem cell donor. Total RNA was derived from whole blood samples, and samples were amplified and labeled using MessageAMP Premier RNA Amplification kit (Ambion). Microarray analyses were performed using Human PrimeView GeneChip arrays (Affymetrix). Plasma AF were quantified using a multiplex angiogenesis array (Millipore).Results:Patients were matched as able according to age, gender, conditioning intensity, donor characteristics, and disease (Table). All patients were 100% donor chimeras and without active infections at the time of blood draw. Compared to HCT donor, all HCT recipients had increased circulating levels of Angiopoietin-2 (Ang-2), endoglin, follistatin, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) (Figure). However, in steroid refractory aGVHD, levels of epidermal growth factor (EGF) were markedly lower compared to donor, steroid-responsive, and uncomplicated HCT recipients (1.4 pg/ml vs 32.4, 137.1, and 204.8 pg/ml respectively), while levels of VEGF-D, Ang-2, leptin, and PlGF were highest in steroid refractory aGVHD. In microarray analysis (Figure), steroid refractory aGVHD was again associated with a lower gene expression of EGF, but higher expression of only VEGF-D and leptin. Gene expression of Ang-2 and PlGF was not higher in hematopoietic cells, despite higher plasma levels, in steroid-refractory aGVHD, leading to the hypothesis that these growth factors may be predominantly host-derived. Interestingly, we found higher transcript expression of IFN-gamma, comparing steroid refractory versus steroid responsive aGVHD, along with a lower transcript expression of inflammatory cytokines TNF, IL-6, and IL-1B.Conclusions:The plasma profile of steroid-refractory aGVHD (low EGF, and high VEGF-D, Ang-2, leptin, and PlGF) appears to be only partially explained by donor hematopoietic cell gene expression. Gene expression of Ang-2 and PlGF are lower in steroid-refractory aGVHD, suggesting that the plasma elevations in these factors might be predominantly host-derived. Ang-2 likely reflects host endothelial tissue damage as previously described, while the PlGF response to aGVHD requires further investigation. The elevation in several damage-associated factors in the plasma and transcriptome in a healthy HCT recipient may reflect normal adaptation of donor cells to a new host environment. These results are hypothesis generating and mechanistic studies aimed at understanding and manipulating the dysregulated system of AF and downstream signaling pathways are indicated, with the goal of ameliorating severe aGVHD and facilitating tissue repair, without excess immunosuppression. [Display omitted] DisclosuresHoltan:Incyte: Other: One-time advisory board member.

Immunomodulatory effects of vitamin D3 on gene expression of MDGF, EGF and PDGFB in endometriosis

Research question: Endometriosis, an inflammatory disease, is assumed to be associated with an increased production of growth-related cytokines. Based on the emerging immunomodulatory role of vitamin D3 in different inflammatory conditions, this study aimed to examine its modulatory effect on the expression levels of the genes for platelet-derived growth factor-B (PDGFB), monocyte/macrophage-derived growth factor (MDGF, also known as PPBP) and epidermal growth factor (EGF) in peritoneal fluid mononuclear cells (PFMC) in women with and without endometriosis.Design: PFMC from 10 women with endometriosis and 10 control participants were treated with vitamin D3.The gene expression levels of PDGFB, MDGF and EGF were measured 6, 24 and 48 h following vitamin D3 administration using real-time PCR.Results: Gene expression levels of EGF and PDGFB were higher in the PFMC of women with endometriosis than the control group (P = 0.006, P < 0.001, respectively). Although MDGF expression showed an increase in the endometriosis group compared with non-endometriotic controls, no significant difference was found. Vitamin D3 significantly decreased EGF expression at 6, 24 and 48 h (P < 0.001, P < 0.001 and P = 0.007, respectively), MDGF at 24 and 48 h (P < 0.001 and P = 0.009, respectively) and PDGFB at 6 h (P = 0.047) in the endometriosis group. Vitamin D3 treatment had no significant effect on expression of the genes in the PFMC of non-endometriotic women.Conclusions: The study concluded that PDGFB and EGF gene expression increases in endometriosis, and vitamin D3 could markedly decrease this expression, suggesting its therapeutic potential in endometriosis.

Isoform-selective as opposed to complete depletion of fibroblast growth factor 2 (FGF-2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice.

We have previously shown that total knockout of fibroblast growth factor-2 (FGF-2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1G93A ) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1G93A mice, with distinct regulation patterns of FGF-2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF-2-isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1G93A FGF-2 high molecular weight- and SOD1G93A FGF-2 low molecular weight-knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform-dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1G93A FGF-2 low molecular weight knockout mice compared to single mutant SOD1G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1G93A FGF-2 low molecular weight knockout mice implies that FGF-2 low molecular weight knockout (or the presence of the FGF-2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.

276 - Transcriptomal Evidence for Dysregulation of Angiogenic Factors and Inflammatory Markers after Allogeneic Hematopoietic Cell Transplantation

Abstract Introduction: Angiogenic factors (AF) are the prototypic growth factors required for wound healing, and are linked to the immune response after tissue injury. Angiogenesis and inflammation together influence recovery from organ damage after hematopoietic stem cell transplantation (HCT), as well as contribute to the risk for complications such as acute graft-versus-host disease (aGVHD). We have recently described alterations in circulating angiogenic factors occurring at the onset of aGVHD post-HCT (BBMT 2015 21(6):1029), with dysregulation in AF associated with tissue repair versus those reflective of tissue damage. We hypothesized that the AF-inflammatory signature identified in the plasma in severe aGVHD could be linked to the gene expression of circulating donor cells after HCT. We performed transcriptomal analysis of changes in the peripheral blood immune cells and correlated with plasma AF levels in order to understand if the circulating donor cells might contribute to the AF profile. Methods: Peripheral blood samples from 3 patients (uncomplicated without aGVHD, steroid responsive aGVHD, and steroid refractory aGVHD) were compared to a healthy stem cell donor. Total RNA was derived from whole blood samples, and samples were amplified and labeled using MessageAMP Premier RNA Amplification kit (Ambion). Microarray analyses were performed using Human PrimeView GeneChip arrays (Affymetrix). Plasma AF were quantified using a multiplex angiogenesis array (Millipore). Results: Patients were matched as able according to age, gender, conditioning intensity, donor characteristics, and disease (Table). All patients were 100% donor chimeras and without active infections at the time of blood draw. Compared to HCT donor, all HCT recipients had increased circulating levels of Angiopoietin-2 (Ang-2), endoglin, follistatin, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) (Figure). However, in steroid refractory aGVHD, levels of epidermal growth factor (EGF) were markedly lower compared to donor, steroid-responsive, and uncomplicated HCT recipients (1.4 pg/ml vs 32.4, 137.1, and 204.8 pg/ml respectively), while levels of VEGF-D, Ang-2, leptin, and PlGF were highest in steroid refractory aGVHD. In microarray analysis (Figure), steroid refractory aGVHD was again associated with a lower gene expression of EGF, but higher expression of only VEGF-D and leptin. Gene expression of Ang-2 and PlGF was not higher in hematopoietic cells, despite higher plasma levels, in steroid-refractory aGVHD, leading to the hypothesis that these growth factors may be predominantly host-derived. Interestingly, we found higher transcript expression of IFN-gamma, comparing steroid refractory versus steroid responsive aGVHD, along with a lower transcript expression of inflammatory cytokines TNF, IL-6, and IL-1B. Conclusions: The plasma profile of steroid-refractory aGVHD (low EGF, and high VEGF-D, Ang-2, leptin, and PlGF) appears to be only partially explained by donor hematopoietic cell gene expression. Gene expression of Ang-2 and PlGF are lower in steroid-refractory aGVHD, suggesting that the plasma elevations in these factors might be predominantly host-derived. Ang-2 likely reflects host endothelial tissue damage as previously described, while the PlGF response to aGVHD requires further investigation. The elevation in several damage-associated factors in the plasma and transcriptome in a healthy HCT recipient may reflect normal adaptation of donor cells to a new host environment. These results are hypothesis generating and mechanistic studies aimed at understanding and manipulating the dysregulated system of AF and downstream signaling pathways are indicated, with the goal of ameliorating severe aGVHD and facilitating tissue repair, without excess immunosuppression. Download : Download high-res image (153KB) Download : Download full-size image Figure . Disclosures Holtan: Incyte: Other: One-time advisory board member.

Epidermal Growth Factor Represses Constitutive Androstane Receptor Expression in Primary Human Hepatocytes and Favors Regulation by Pregnane X Receptor.

Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.

Treatment of Oral Ulcers in Dogs Using Adipose Tissue-Derived Mesenchymal Stem Cells

Aim:Adipose tissue Derived Mesenchymal Stem cells (ADMSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. The goal of this study was to investigate the effects of ADMSCs transplantation on oral ulcer healing in dogs.Study design:Mesenchymal stem cells were isolated from adipose tissues of dogs obtained by suction-assisted lipectomy (liposuction), by dish adherence and were expanded in culture. Oral ulcers were induced by topical application of formocresol in the oral cavity of 18 dogs. The dogs were classified into 3 groups. Either autologousADMSCs, Corticosteriod (Dexamethasone) or vehicle (saline) was injected. The healing process of the ulcer was monitored histopathologically. Gene expression of vascular endothelial growth factor (VEGF),platelets derived growth factor (PDGF),epidermal growth factor (EGF) and collagen was assessed in biopsies obtained from all ulcers “as healing markers”, by real time polymerase chain reaction (PCR). Results:ADMSCs group showed significantly accelerated oral ulcer healing compared with the Dexamethasone and control groups. There was increased expression of VEGF, PDGF,EGF and collagen genes in ADMSCs-treated ulcers compared with Dexamethasone and controls. Conclusion:ADMSCs transplantation may help accelerate oral ulcer healing, possibly through the induction of angiogenesis by VEGF and PDGF, as well as epithelial and connective tissue proliferation as evidenced by increased EGF and collagen gene expression.

Placental gene expression patterns of epidermal growth factor in intrauterine growth restriction

ObjectiveIn this study, we compared human placental gene expression patterns of epidermal growth factor (EGF) in pregnancies with intrauterine growth restriction (IUGR) vs. normal pregnancies as control. Study designGene expression of EGF was determined from human placental samples collected from all pregnancies presenting with IUGR at our institution during the study period January 1, 2010–January 1, 2011. Multiple clinical variables were also assessed including maternal age, gestational weight gain, increase of BMI during pregnancy and fetal gender. ResultsA total of 241 samples were obtained (101 in the IUGR pregnancy group, 140 in the normal pregnancy group). EGF was found to be underexpressed in the IUGR group compared to normal pregnancy (Ln2α: −1.54; p<0.04). Within the IUGR group no fetal gender-dependent difference was seen in EGF gene expression (Ln2α: 0.44; p<0.06). Similarly, no significant difference in EGF expression was noted in cases with more vs. less severe forms of IUGR (Ln2α: −0.08; p=0.05). IUGR pregnancies were significantly more common in the maternal age group 35–44 years compared to other age groups. Gestational weight gain and gestational BMI increase were significantly lower in IUGR pregnancies compared to controls. ConclusionsPlacental expression of EGF was found to be reduced in IUGR pregnancies vs. normal pregnancies. This may partly explain the smaller placental size and placental dysfunction commonly seen with IUGR. An increased incidence of IUGR was observed with maternal age exceeding 35 years. The probability of IUGR correlated with lower gestational weight gain and lower BMI increase during pregnancy.

Gene expression of oncogenes, antimicrobial peptides, and cytokines in the development of oral leukoplakia

The aim of this study was to investigate the expression pattern of oncogenes, antimicrobial peptides, and genes involved in inflammation in leukoplakia of the oral cavity compared with healthy gingiva. Biopsies of healthy gingiva (n=20) and leukoplakia (n=20), were obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of alpha-defensin (DEFA) 1/3, DEFA-4, S100-A7, deleted-in-oral-cancer (Doc) 1, interleukin (IL) 1beta, IL-6, IL-8, IL-10, tumor necrosis factor (TNF) alpha, cyclooxygenase (Cox) 2, epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor (TGF) beta1, TGF-alpha, collagen-IA1 (Col-1), and tenascin-c were analyzed by real-time reverse-transcription polymerase chain reaction. The proteins encoded by the different genes were visualized by immunostaining. Compared with healthy gingiva (set as 1), there was an increased gene expression of DEFA-4 (179.2-fold), S100-A7 (25.4-fold), EGF (24.8-fold), TGF-beta1 (25.2-fold), and tenascin-c (34.3-fold) in oral leukoplakia. The expression of IL-1beta and Doc-1 was decreased (0.01-fold and 0.2-fold, respectively). The combination of an increased expression of the antimicrobial peptide DEFA-4, the oncogene S100-A7, EGF, and tenascin-c, and a decreased Doc-1 expression in oral leukoplakia might characterize its potency of malignant transformation. Chronic inflammation seems not to be involved in the development of this lesion.

Epidermal Growth Factor Variations in Amniotic Membrane Used forEx VivoTissue Constructs

The amniotic membrane (AM) is used for engineering ex vivo tissue constructs used in ocular surface reconstruction. Epidermal growth factor (EGF) content of the AM is believed to play a key role in supporting corneal epithelial cell expansion on AM. This study investigated EGF content in AM in relation to intra- and inter-donor variations and the effect of processing and preservation (handling). Fifteen human AM, both fresh and handled, were analyzed for EGF gene and protein expression by real-time polymerase chain reaction and ELISA, respectively. EGF gene expression was predominantly seen in the AM epithelium (p<0.01). Similarly, EGF protein too was predominantly seen in the epithelial layer (p<0.01) for fresh and handled samples. EGF protein content varied between membranes (inter-donor) and at different sites within the same membrane (intra-donor). The highest EGF protein concentration was noted in the AM apical and mid-region epithelium. Significant EGF protein loss (p<0.01) was observed after handling. There is a considerable variation in EGF content between and within donors. This is further affected by handling of the AM. Such variations could affect the clinical efficacy of tissue constructs. Current use of AM for ex vivo expansion of epithelial cells is not standardized and remains an area of concern.

Prostaglandin E1 and misoprostol increase epidermal growth factor production in 3D-cultured human annulus cells

Background context Epidermal growth factor (EGF) is a peptide known to modulate a number of cellular responses including embryogenesis, cell proliferation, and cell survival. Little is known about EGF and its regulation in human annulus cells. Previous work has identified EGF and its receptor in control outer annulus disc tissue, but not in herniated tissue. Purpose To determine if human annulus cells express EGF in vitro, to determine if the epidermal growth factor-receptor (EGF-r) was expressed in vivo and in vitro in disc cells, to test the effect of EGF on annulus cell proliferation and proteoglycan production in vitro, and to test the effect of prostaglandin E1 (PGE1) and misoprostol on disc cell production of EGF in vitro. Study design/setting Studies were approved by the authors' Human Subjects Institutional review Board. Human disc tissue was used for immunocytochemistry, and human annulus cells were tested in vitro. Patient sample Thirty-four disc specimens were used for studies of proteoglycan production, cell proliferation, and EGF production in vitro. An additional nine discs were used for EGF-r immunolocalization. Methods Disc tissue was used for immunocytochemical studies for the localization of EGF-r and as a source for cultured annulus cells. Monolayer culture was used to test proliferation responses to 0, 25, 50, or 75 ng/mL EGF over a 2-day culture period. Three-dimensional (3D) culture in a collagen sponge was used to test 100,000 cells cultured in a paired experimental design over 14 days for production of EGF and proteoglycans. Cells were exposed to control conditions, or to either misoprostol at 8 ng/mL or PGE1 at 10 −7 M. Conditioned media was harvested and assayed using an enzyme-linked immunosorbent assay (ELISA) assay with the Human Protein Cytokine Antibody Array I kit. Replicate EGF relative intensity values were averaged and normalized to controls assayed on the same membrane. 3D-cultured cells were also used to confirm EGF gene expression using microarray analysis. Standard statistical methods were used to analyze results. Results Microarray analysis of mRNA from annulus cells in 3D culture confirmed expression of EGF, and immunocytochemistry verified the presence of EGF-r in vitro and in vivo. PGE1, at a dose of 10 −7 M, and misoprostol (a synthetic PGE1 analog) at a dose of 8 ng/mL, both significantly increased EGF levels in annulus cells cultured in 3D compared with control levels (p=.031 and .034, respectively). No significant difference, however, was seen in cell proliferation or in total sulfated proteoglycan production in EGF-exposed annulus cells. Conclusions Data showed that EGF is expressed and produced by annulus cells in vivo and in 3D culture, with significantly greater in vitro EGF produced in the presence of PGE1 or the PGE1 analog misoprostol. Misoprostol, developed for prevention/treatment of nonsteroidal anti-inflammatory-induced gastropathy, has now been reported to have some interesting anabolic effects stimulating osteoblasts during fracture healing and during ovariectomy in animal models. Exogenous EGF did not increase cell proliferation in monolayer, or total production of proteoglycans in 3D culture. Additional work is needed to further delineate the role of EGF in the human disc.

Epidermal growth factor (EGF) A61G polymorphism and EGF gene expression in normal colon tissue from patients with colorectal cancer

Introduction. EGF/EGFR interactions are important mechanisms behind colorectal tumour development and growth. Recently a single nucleotide polymorphism in the EGF gene has been identified (EGF A61G). It may be a potential predictor for survival of patients receiving EGFR-inhibitor cetuximab treatment, but the clinical importance and the functional influence on EGF gene expression levels in colorectal cancer (CRC) patients have not yet been further assessed. The aim of the present study was to investigate the relationship between EGF A61G genotype and EGF gene expression levels in colorectal adenocarcinomas and normal colon tissue. Material and methods. Eighty-one CRC patients were included in the study. Tissue samples from normal colon, adenocacinomas and corresponding blood samples were analysed by real-time PCR for EGF gene expression and EGF A61G genotype, respectively. Results. Thirty-three percent were AA, 48% and 19% A/G and G/G respectively. We found a significantly lower median age in the A/A group compared to the G/G group, suggesting a later time of diagnosis in the G/G patients. There was a significant difference between the median EGF gene expression among the three genotypes in normal colon (p < 0.001) but not in adenocarcinomas. Furthermore, the median EGF gene expression was lower in CRC tissue than in normal colon samples, (0.13 (range 0.01–6.4) vs. 0.76, (range 0.013–5.55)). Conclusion. We suggest that EGF A61G genotype has a functional influence on EGF gene expression in normal colon in CRC patients. The clinical implications warrant further investigations in prospective trials.

Upregulation of the Expression of Epidermal Growth Factor and Its Receptor in Gingiva Upon Cyclosporin A Treatment

To understand the roles of epidermal growth factor (EGF) and EGF receptor (EGF-R) in cyclosporin A (CsA)-induced gingival overgrowth, expression of EGF and EGF-R upon CsA treatment was examined in an oral epidermoid carcinoma cell line of humans (OECM-1) and in edentulous gingiva of rats. In vitro study: after CsA treatment, OECM-1 cells were harvested to evaluate their mRNA and protein expression of EGF and EGF-R with reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry (ICC). In vivo study: 3 weeks after extraction of all maxillary molars, 20 male Sprague-Dawley rats were assigned to a CsA group (30 mg/kg, fed daily) and a control group. Five rats per group were sacrificed at weeks 1 and 4. Edentulous ridge specimens were obtained for evaluating their mRNAs and protein expression with RT-PCR, real-time RT-PCR, and immunohistochemistry (IHC). In both in vitro and in vivo experiments, the proliferating potential of epithelial cells was examined by the presence of proliferating cell nuclear antigen (PCNA). In vitro: dose-dependently increased mRNA expression of EGF and EGF-R in OECM-1 cells was noted after CsA treatment. Protein expressions of EGF and EGF-R were higher in OECM-1 with CsA treatment than without CsA. In vivo: higher mRNA and protein expressions of EGF and EGF-R were also observed in the gingival tissues of CsA-treated rats. In both in vitro and in vivo experiments, greater PCNA expression after CsA treatment was demonstrated. Higher expression of EGF and EGF-R upon CsA therapy was observed in OECM-1 epithelial cells of humans and in edentulous gingiva of rats. We suggest that CsA could upregulate gene and protein expression of EGF and EGF-R, and the upregulation may play a role in gingival overgrowth.

Gene expression of epidermal growth factor and epidermal growth factor receptor in oral lichen planus

To explore the expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), c-fos and c-jun in oral lichen planus (OLP). The levels of gene expression of EGF, EGFR, c-fos and c-jun were detected with reverse transcription-polymerase chain reaction (RT-PCR) in 10 cases of erosive OLP, 12 cases of reticular OLP and 9 cases of normal oral mucosa. Protein contents and distribution of EGF, EGFR, C-fos and C-jun were examined with immunohistochemical technique in different tissues. The endogenous levels of EGFR mRNA and protein in erosive OLP were (55.9 +/- 23.1)% and (71.1 +/- 10.1)%, respectively, and significantly higher than those in reticular OLP and normal oral mucosa. The mRNA contents of c-fos and c-jun were significantly higher in erosive OLP than in normal oral mucosa. The positive cell rates of c-fos and c-jun protein in erosive OLP were 1.3 and 1.5 times of those in normal oral mucosa, respectively (P < 0.05). The probability of carcinomatous change of erosive OLP might be higher than that of reticular OLP, in which EGF and EGFR might play an important role.

Activation of the MEK5/ERK5 Cascade Is Responsible for Biliary Dysgenesis in a Rat Model of Caroli's Disease

Polycystic kidney (PCK) rats exhibit a multiorgan cyst pathology similar to human autosomal recessive polycystic kidney disease, and are proposed as an animal model of Caroli's disease with congenital hepatic fibrosis (CHF). This study investigated the expression and function of selected components of the mitogen activated protein kinase (MAPK) pathway in cultured intrahepatic biliary epithelial cells (BECs) of PCK rats. Compared to the proliferative activity of cultured BECs of control rats, those of the PCK rats were hyperresponsive to epidermal growth factor (EGF). The increase in BEC proliferation was accompanied by overexpression of MAPK/extracellular signal-regulated protein kinase (ERK) kinase 5 (MEK5), and subsequent phosphorylation of ERK5 in vitro. The increased proliferative activity was significantly inhibited by the transfection of short interfering RNA against MEK5 mRNA. An EGF receptor tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839), also significantly inhibited the abnormal growth of cultured BECs of PCK rats. By contrast, treatment with PD98059 and U0126, inhibitors for MEK1/2, was less effective. These results suggest that the activation of the MEK5-ERK5 cascade plays a pivotal role in the biliary dysgenesis of PCK rats, and also provide insights into the pathogenesis of Caroli's disease with CHF. As the MEK5-ERK5 interaction is highly specific, it may represent a potential target of therapy.

Upregulated expression of EGF and TGF-? in the proximal respiratory epithelium in the human hypoplastic lung in congenital diaphragmatic hernia

Newborn infants with congenital diaphragmatic hernia (CDH) still have a high mortality rate. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are peptide growth factors involved in the fetal lung growth and development. The EGF and TGF-alpha have been reported to promote pulmonary branching activity and alveolar type-II pneumocyte proliferation. Epidermal growth factor and TGF-alpha immunoreactivity and mRNA expression in the bronchial and bronchiolar epithelium is maximal during early fetal life and barely detectable in the proximal airways of neonatal lung. The purpose of this study was to determine protein and gene expression of EGF and TGF-alpha in CDH lung in order to elucidate the potential role of these growth factors in the pathogenesis of pulmonary hypoplasia in CDH. Lung tissue specimens were obtained from archival lung tissue from 11 patients with CDH and 5 controls. Indirect immunohistochemistry was performed using ABC method with anti-EGF and anti-TGF-alpha antibodies. In situ hybridization was performed using EGF and TGF-alpha specific digoxigenin-labeled oligonucleotide probes. The most striking difference between hypoplastic CDH lung and control lung was the strong EGF and TGF-alpha mRNA expression and immunoreactivity in the bronchial and bronchiolar epithelium in CDH lung. The upregulated protein and gene expression of EGF and TGF-alpha in the proximal airways in the CDH hypoplastic lung suggests persistence of fetal stage of pulmonary airway development in CDH.