Effects Of EGF Research Articles

MiR-191-5p suppresses PRRSV replication by targeting porcine EGFR to enhance interferon signaling.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major thread to the global swine industry, lack of effective control strategies. This study explores the regulatory role of a small non-coding RNA, miR-191-5p, in PRRSV infection. We observed that miR-191-5p significantly inhibits PRRSV in porcine alveolar macrophages (PAMs), contrasting with negligible effects in MARC-145 and HEK293-CD163 cells, suggesting a cell-specific antiviral effect. Further investigation unveiled that miR-191-5p directly targets the porcine epidermal growth factor receptor (EGFR), whose overexpression or EGF-induced activation suppresses type I interferon (IFN-I) signaling, promoting PRRSV replication. In contrast, siRNA-or miR-191-5p-induced EGFR downregulation or EGFR inhibitor boosts IFN-I signaling, reducing viral replication. Notably, this miRNA alleviates the suppressive effect of EGF on IFN-I signaling, underscoring its regulatory function. Further investigation revealed interconnections among miR-191-5p, EGFR and signal transducer and activator of transcription 3 (STAT3). Modulation of STAT3 activity influenced IFN-I signaling and PRRSV replication, with STAT3 knockdown countering EGFR activation-induced virus replication. Combination inhibition of STAT3 and miR-191-5p suggests that STAT3 acts downstream in EGFR's antiviral response. Furthermore, miR-191-5p's broad efficacy in restricting various PRRSV strains in PAMs was identified. Collectively, these findings elucidate a novel mechanism of miR-191-5p in activating host IFN-I signaling to inhibit PRRSV replication, highlighting its potential in therapeutic applications against PRRSV.

Retraction: Comparison of the effects of EGF, FGF-b, and NGF on the proliferation, migration, and reprogramming of primary rat Müller cells.

[This retracts the article DOI: 10.3389/fncel.2024.1338129.].

FRI375 Validation Of A Novel Mouse Placenta Explant Protocol For Screening Of Drugs Modulating Circadian Rhythms

Abstract Disclosure: J.S. Lee: None. H.M. Hoffmann: None. Introduction Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia. Methods We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&E we validated the explants to be >80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells. Results Through H&E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for <6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ∼4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects. Conclusion We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta. Presentation: Friday, June 16, 2023

Influence of EGF and pro-NGF on EGFR/SORTILIN interaction and clinical impact in head and neck squamous cell carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) remains a cancer with a poor prognosis, with a 5-year survival rate of less than 50%. Although epidermal growth factor receptor (EGFR) is almost always overexpressed, targeted anti-EGFR therapies have modest efficacy and are mainly used in palliative care. Growth factors such as Nerve Growth Factor (NGF) and its precursor proNGF have been shown in our laboratory to play a role in tumor growth and aggressiveness. Interestingly, an interaction between Sortilin, a proNGF receptor, and EGFR has been observed. This interaction appears to interfere with the pro-oncogenic signaling of EGF and modulate the membrane expression of EGFR. The aim of this study was to characterize this interaction biologically, to assess its impact on clinical prognosis and to analyze its role in the cellular trafficking of EGFR. Using immunohistochemical staining on tumor sections from patients treated at our university center and PLA (Proximity Ligation Assay) labeling, we showed that Sortilin expression is significantly associated with reduced 5-year survival. However, when Sortilin was associated with EGFR, this association was not found. Using the Cal-27 and Cal-33 cancer cell lines, we observed that proNGF reduces the effects of EGF on cell growth by inducing the internalization of its receptor. These results therefore suggest a regulatory role for Sortilin in the degradation or renewal of EGFR on the membrane. It would be interesting in future work to show the intracellular fate of EGFR and the role of (pro)neurotrophins in these mechanisms.

NSCLC presents metabolic heterogeneity, and there is still some leeway for EGF stimuli in EGFR-mutated NSCLC

BackgroundMetabolic remodeling is crucial in carcinogenesis and cancer progression. Oncogenic mutations may promote metabolic reprogramming in cancer cells to support their energy and biomass requirements. EGFR mutations are commonly found in non-small cell lung cancer (NSCLC) and may induce NSCLC metabolic rewiring. Whether EGFR-driven metabolic reprogramming triggers cell vulnerabilities with therapeutic potential remains unknown. MethodsThe role of EGFR signaling activation by EGF was investigated using NSCLC cell lines with different EGFR and KRAS status: A549 (EGFR WT and KRAS c.34G > A), H292 (EGFR WT and KRAS WT) and PC-9 (EGFR exon 19 E746-A750 deletion and KRAS WT). The effect of EGF on NSCLC cell death and cell cycle was evaluated using flow cytometry, and cell migration was assessed through wound healing. EGFR, HER2, MCT1, and MCT4 expression was analyzed through immunofluorescence or western blotting. We explored the impact of glucose and lactate bioavailability on NSCLC cells' metabolic profile using nuclear magnetic resonance (NMR) spectroscopy. Moreover, the expression of several relevant metabolic genes in NSCLC cells or patient samples was determined by RT-qPCR. ResultsWe showed that cell lines presented different metabolic profiles, and PC-9 cells were the most responsive to EGF stimulus, as they showed higher rates of cell proliferation and migration, together with altered metabolic behavior. By inhibiting EGFR with gefitinib, a decrease in glucose consumption was observed, which may be related to the fact that despite PC-9 harbor EGFR mutation, they still express the EGFR WT allele. The analysis of NSCLC patients' RNA showed a correlation between MCT1/MCT4 and GLUT1 expression in most cases, indicating that the metabolic information can serve as a reference in patients' follow-up. ConclusionTogether, this study shows that NSCLC cell lines have heterogeneous metabolic profiles, which may be underlaid by different genetic profiles, revealing an opportunity to identify and stratify patients who can benefit from metabolism-targeted therapies.

Epithelial to mesenchymal transition in mammary gland tissue fibrosis and insights into drug therapeutics.

The epithelial-mesenchymal transition (EMT) is a multi-step morphogenetic process in which epithelial cells lose their epithelial properties and gain mesenchymal characteristics. The process of EMT has been shown to mediate mammary gland fibrosis. Understanding how mesenchymal cells emerge from an epithelial default state will aid in unravelling the mechanisms that control fibrosis and, ultimately, in identifying therapeutic targets to alleviate fibrosis. The effects of EGF and high glucose (HG) on EMT in mammary epithelial cells, MCF10A and GMECs, as well as their pathogenic role, were studied. In-silico analysis was used to find interacting partners and protein-chemical/drug molecule interactions. On treatment with EGF and/or HG, qPCR analysis showed a significant increase in the gene expression of EMT markers and downstream signalling genes. The expression of these genes was reduced on treatment with EGF+HG combination in both cell lines. The protein expression of COL1A1 increased as compared to the control in cells treated with EGF or HG alone, but when the cells were treated with EGF and HG together, the protein expression of COL1A1 decreased. ROS levels and cell death increased in cells treated with EGF and HG alone, whereas cells treated with EGF and HG together showed a decrease in ROS production and apoptosis. In-silico analysis of protein-protein interactions suggest the possible role of MAPK1, actin alpha 2 (ACTA2), COL1A1, and NFκB1 in regulating TGFβ1, ubiquitin C (UBC), specificity protein 1 (SP1) and E1A binding protein P300 (EP300). Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment suggests advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE) signalling pathway, relaxin signalling pathway and extra cellular matrix (ECM) receptor interactions underlying fibrosis mechanism. This study demonstrates that EGF and HG induce EMT in mammary epithelial cells and may also have a role in fibrosis.

Hemostatic Effect and Mechanism of Epidermal Growth Factor-Hemostatic Powder after Endoscopic Submucosal Dissection in a Porcine Model.

Among several methods used to prevent endoscopic submucosal dissection (ESD) bleeding, the recently developed hemostatic powder (HP) has few technical limitations and is relatively easy-to-use. This study aimed to analyze the hemostatic effects and mechanisms of two HPs using a porcine upper gastrointestinal hemorrhage model. We evaluated HPs (Endospray and epidermal growth factor [EGF]-endospray) for adhesion, waterproofing ability, permeability, and absorption in vitro. ESD was performed to induce bleeding ulcers in the porcine stomachs. In a total of three pigs, three bleeding ulcers per animal were generated. Hemostasis and rebleeding were evaluated endoscopically. After 72 hours, the animals were sacrificed, and histologically analyzed. The water absorption of HPs was over 20 times the initial value within 30 minutes. The gelated HPs completely blocked water penetration into the applied site within 5 minutes and strongly adhered to the Petri-dish surface for up to 6 hours. The initial hemostasis rates within 5 minutes were 33.3%, 100.0%, and 66.7%, and the rebleeding rates at 6 to 72 hours after HP application were 33.3%, 16.7%, and 33.3% (control, Endospray, and EGF-endospray groups, respectively). Histological analysis revealed the thickness of the regenerated mucosa (522.1, 514.5, and 680.3 μm) and the submucosal layer (1,510.3, 2,848.2, and 3,062.3 μm) and the number of newly formed blood vessels (15.3, 17.9, and 20.5) in the control, Endospray, and EGF-endospray groups, respectively. The endoscopic HPs demonstrated the ability to elicit effective initial hemostasis and the histological ulcer-healing effect of EGF in an animal model of hemorrhagic gastric ulcers.

Epithelial cell cluster size affects force distribution in response to EGF-induced collective contractility

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin β1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.

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.

A Rat Experimental Model for Investigation of the Effect of Diabetes on Submandibular Salivary Glands Treated with Epidermal Growth Factor

Background: Despite the plethora of research around the negative effects of diabetes on different body organs, this topic still attracts a lot of attention in order to find potential remedies that could counteract or reverse the damaging effect of diabetes.
 Aim: In this study, we developed a reliable experimental rat model that can be used for investigation of the ability of epidermal growth factor (EFG) in restoring the normal architecture of oral tissues after being damaged by diabetes.
 Methods: Eighty adult male albino rats (average weight ±220 gm) were used in the current study. Twenty rats served as control and received no treatment. Diabetes was induced in forty rats using a single injection of 65mg/kg of Streptozotocin (STZ). Out of the forty diabetic rats, twenty rats received a single daily intraperitoneal injection of EGF (10 µg/Kg) for 8 weeks. Furthermore, twenty healthy rats received the same dose of EGF and served as positive controls. The submandibular salivary glands of all rats were examined for Immunohistochemical detection of myosin in the glandular structure.
 Results: The EGF treated group showed comparable myosin expression to the control group. The diabetic group revealed deterioration of all components of the submandibular salivary glands. Finally, the diabetic + EGF group has demonstrated restoration of the myosin expression levels in the submandibular salivary glands to a level that is not significantly different from healthy (non-diabetic) rats in the control group (p>0.05) and significantly higher than the diabetic group (p<0.0001).
 Conclusion: The findings of the present study confirm previous studies and validates the use of our animal model as predictable experimental tool to investigate the effects of diabetes and EGF on different oral tissues. It also highlights the importance of further research investigating EGF as a promising treatment modality for restoration of the condition and functions of tissues damaged by diabetes not only in the oral cavity but also around the whole body.

FIP-nha, a fungal immunomodulatory protein from Nectria haematococca, induces apoptosis and autophagy in human gastric cancer cells via blocking the EGFR-mediated STAT3/Akt signaling pathway

FIP-nha, a fungal immunomodulatory protein from Nectria haematococca, has been demonstrated a broad spectrum of antitumor activity and cell selectivity against human cancers in our previous study. However, the effect and mechanism of FIP-nha on gastric cancer remains unclear. In this study, we systematically observed the cytotoxicity, biological effect, regulatory mechanism and interaction target of FIP-nha on human gastric cancer cell lines, AGS and SGC7901. Our results demonstrated that FIP-nha inhibited the growth of AGS and SGC7901 cells in a dose-dependent manner and exerted proapoptotic effects on both cells as confirmed by flow cytometry, DAPI staining and western blot analysis. Additionally, the exposure of AGS and SGC7901 to FIP-nha induced autophagy as indicated by western blot analysis, GFP-LC3 and mCherry-GFP-LC3 transfection and acridine orange staining. Furthermore, we found that FIP-nha decreased the phosphorylation of EGFR, STAT3 and Akt and inhibited activation effect of ligand factor EGF to EGFR and its downstream signal molecule STAT3 and Akt. Finally, we proved that FIP-nha located on the surface of gastric cancer cells and bound directly to the transmembrane protein of EGFR by immunoprecipitation, cellular localization, molecular docking, microscale thermophoresis assay. The above findings indicated that FIP-nha inhibited the growth of gastric cancer and induced apoptosis and autophagy through competitively binding to EGFR with EGF to blocking the EGFR-mediated STAT3/Akt pathway. In summary, our study provided novel insights regarding the activity of FIP-nha against gastric cancer and contributed to the clinical application of FIP-nha as a potential chemotherapy drugs that targeted EGFR for human gastric cancer.

Anti-epidermal growth factor vaccine antibodies increase the antitumor activity of kinase inhibitors in ALK and RET rearranged lung cancer cells

Advanced NSCLC patients harboring EML4-ALK and CCDC6-RET rearrangements derive benefit from treatment with ALK and RET TKIs but not immune checkpoint inhibitors. New immunotherapeutic approaches, such as immunization against growth factors, can be of particular interest for combination treatment in these patients. Here, we investigated the effects of anti-EGF antibodies generated by vaccination (anti-EGF VacAbs), TKIs and combinations in EML4-ALK and CCDC6-RET NSCLC cell lines. We found that EGF and tumor growth factor alpha (TGFα) significantly decreased the antiproliferative activity of the RET inhibitor BLU-667 in CCDC6-RET cells and brigatinib, alectinib and crizotinib in EML4-ALK translocated cells. The addition of anti-EGF VacAbs reversed the effects of EGF and TGFα, potentiated the antitumor effects of the kinase inhibitors and delayed the appearance in vitro of resistant clones. Western blotting demonstrated that the combination of anti-EGF VacAbs with ALK or RET TKIs effectively suppressed EGFR downstream pathways in EML4-ALK translocated and CCDC6-RET cells, respectively. In conclusion, anti-EGF VacAbs significantly increased the antitumor activity of TKIs in ALK and RET-positive cell lines. Clinical trials of an EGF vaccine in combination with ALK and RET TKIs are warranted.

Potentiation of calcium-activated chloride secretion and barrier dysfunction may underlie EGF receptor tyrosine kinase inhibitor-induced diarrhea.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFr TKIs) are first‐line therapies for various cancers, and cause dose‐limiting severe diarrhea in many patients. We hypothesized that diarrhea caused by EGFr TKIs might reflect actions on epithelial transport, barrier function, or both, which we tested using cell cultures including murine and human enteroid‐derived monolayers (EDMs), analyzed using electrophysiological and other relevant methods. EGFr TKIs (such as afatinib, erlotinib, and osimertinib) reversed the acute inhibitory effect of EGF on chloride secretion induced by carbachol (CCh) across T84 human colonic epithelial cells, which correlated with the diarrhea‐inducing effect of each agent clinically. EGFr TKIs also reduced transepithelial electrical resistance (TEER), whereas co‐treatment with CCh delayed the decrease in TEER compared with that of cells co‐treated with EGF. Furthermore, afatinib and erlotinib prevented EGF‐ or CCh‐induced EGFr phosphorylation. EGFr TKIs also suppressed phosphorylation of extracellular signal‐regulated kinase (Erk)1/2 in response to EGF, whereas they had weaker effects on CCh‐induced Erk1/2 phosphorylation. In human EDMs, EGF potentiated ion transport induced by CCh, whereas afatinib reversed this effect. The ability of EGFr TKIs to reverse the effects of EGF on calcium‐dependent chloride secretion could contribute to the diarrheal side effects of these agents, and their disruption of epithelial barrier dysfunction is likely also pathophysiologically significant. CCh‐activated Erk1/2 phosphorylation was relatively insensitive to EGFr TKIs and delayed the deleterious effects of EGFr TKIs on barrier function. These findings confirm and extend those of other authors, and may be relevant to designing strategies to overcome the diarrheal side effects of EGFr TKIs.

Praeruptorin B Mitigates the Metastatic Ability of Human Renal Carcinoma Cells through Targeting CTSC and CTSV Expression.

Renal cell carcinoma (RCC) is the most common adult kidney cancer, and accounts for 85% of all cases of kidney cancers worldwide. Praeruptorin B (Pra-B) is a bioactive constituent of Peucedanum praeruptorum Dunn and exhibits several pharmacological activities, including potent antitumor effects. However, the anti-RCC effects of Pra-B and their underlying mechanisms are unclear; therefore, we explored the effects of Pra-B on RCC cells in this study. We found that Pra-B nonsignificantly influenced the cell viability of human RCC cell lines 786-O and ACHN at a dose of less than 30 μM for 24 h treatment. Further study revealed that Pra-B potently inhibited the migration and invasion of 786-O and ACHN cells, as well as downregulated the mRNA and protein expression of cathepsin C (CTSC) and cathepsin V (CTSV) of 786-O and ACHN cells. Mechanistically, Pra-B also reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), p-mitogen-activated protein kinase kinase (MEK), and p-extracellular signal-regulated kinases (ERK) in RCC cells. In addition, Pra-B treatment inhibited the effect of EGF on the upregulation of EGFR–MEK–ERK, CTSC and CTSV expression, cellular migration, and invasion of 786-O cells. Our findings are the first to demonstrate that Pra-B can reduce the migration and invasion ability of human RCC cells through suppressing the EGFR-MEK-ERK signaling pathway and subsequently downregulating CTSC and CTSV. This evidence suggests that Pra-B can be developed as an effective antimetastatic agent for the treatment of RCC.

ENHANCEMENT OF WOUND HEALING BY TOPICAL APPLICATION OF EPIDERMAL GROWTH FACTOR IN ANIMAL MODEL

Objective: Wound healing is a complex process of biological events involving re-epithelialization and granulation that are mainly mediated by several endogenously released growth factors such as epidermal growth factor. This work was undertaken to study the effects of various doses of locally applied recombinant human epidermal growth factor (rhEGF) on wound healing in rats.
 Methods: Recombinant human EGF consists of 53 amino acids. In vitro, rhEGF promoted it’s obvious cell growth and proliferation when added to cultured 3T3 cells using MTT assay. In the test groups, in vivo, wound sites were given daily with a solution containing 2, 5, 10, 50ug of EGF spray and 40ug of EGF ointment, respectively. We presented evidence that a significant decreased healing time in wound was observed in all rhEGF groups when compared with the control, and reach to its maximal efficacy at 10ug/ml of rhEGF spray. Results: The rate of wound closure was over 50 per cent at initial 3 days of treatment. Treatment with rhEGF significantly decreased the length of time to over 50 per cent healing by approximately 4-5 days, and that to 70 per cent and 90 per cent healing by approximately 3-4 days and 3 days, respectively. A stimulatory, dose-dependent effect of EGF on wound healing was observed with increased hEGF concentration. In toxicological group, higher doses of 100ug/ml of rhEGF spray was applied by local dorsal incision in rats. Moreover, a dose of single 200ug, single 300ug or 300ug within 24 hours of subcutaneous and intramuscular rhEGF injection was given respectively. There were no significant adverse side effects.Conclusion: Current study recommended a proposal of clinical drug doses in wound at 2µg, 5 µg and 10 µg /ml of rhEGF spray, and 10 µg and even higher 40 µg rhEGF/g of ointment. The results indicated that prepared rhEGF by our genetic engineering is safe, and is emerging in clinical effective use in assisting wound healing time.
 Peer Review History: 
 Received 8 January 2020; Revised 12 February; Accepted 2 March, Available online 15 March 2020
 UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.
 Received file 
 
 Average Peer review marks at initial stage: 6.5/10
 Average Peer review marks at publication stage: 8.5/10
 Reviewer(s) detail:
 Name: Asmaa Ahmed Mohamed Ahmed Khalifa 
 Affiliation: Pharos University, Alexandria, Egypt
 E-mail: asmaa.khalifa@pua.edu.eg
 
 Name: Dr. Sabah Hussien El-Ghaiesh 
 Affiliation: Tanta University, Egypt
 E-mail: s.ghaiesh@gmail.com
 Comments of reviewer(s): 
 
 Similar Articles:
 POTENTIAL OF SNAKEHEAD FISH (OPHIOCEPHALUS STRIATUS) IN ACCELERATING WOUND HEALING

Proteomic and Phosphoproteomic Analysis Reveals that Neurokinin-1 Receptor (NK1R) Blockade with Aprepitant in Human Keratinocytes Activates a Distinct Subdomain of EGFR Signaling: Implications for the Anti-Pruritic Activity of NK1R Antagonists.

Background: Epidermal growth factor receptor (EGFR) inhibitors can cause serious cutaneous toxicities, including pruritus and papulopustular acneiform skin eruptions. Increasingly, the neurokinin-1 receptor (NK1R) antagonist aprepitant is being utilized as an anti-pruritic agent in the treatment of EGFR-inhibitor induced pruritus. Aprepitant is believed to reduce itching by blocking NK1R on the surface of dermal mast cells. However, the effects of aprepitant on human keratinocytes remains unexplored. Methods: Herein, we examine the effects of aprepitant on EGFR stimulation in HaCaT cells using a phosphoproteomic approach including reverse phase protein arrays and Ingenuity Pathway Analysis. Changes in EGFR phosphorylation were visualized using Western blotting and the effect of EGF and aprepitant on the growth of HaCaT cells was determined using the WST-1 Cell Proliferation Assay System. Results: We found that aprepitant increased the phosphorylation of EGFR, as well as 10 of the 23 intracellular proteins phosphorylated by EGF. Analysis of phosphoproteomic data using Ingenuity Pathway Analysis software revealed that 5 of the top 10 pathways activated by EGF and aprepitant are shared. Conclusions: We propose that aprepitant produces its antipruritic effects by partially activating EGFR. Activation of EGFR by aprepitant was also seen in primary human keratinocytes. In addition to itch reduction through partial activation of shared EGFR pathways, aprepitant exerts a dose-dependent cytotoxicity to epithelial cells, which may contribute to its antitumor effects.

PDGFB-expressing mesenchymal stem cells improve human hematopoietic stem cell engraftment in immunodeficient mice

The bone marrow (BM) niche regulates multiple hematopoietic stem cell (HSC) processes. Clinical treatment for hematological malignancies by HSC transplantation often requires preconditioning via total body irradiation, which severely and irreversibly impairs the BM niche and HSC regeneration. Novel strategies are needed to enhance HSC regeneration in irradiated BM. We compared the effects of EGF, FGF2, and PDGFB on HSC regeneration using human mesenchymal stem cells (MSCs) that were transduced with these factors via lentiviral vectors. Among the above niche factors tested, MSCs transduced with PDGFB (PDGFB-MSCs) most significantly improved human HSC engraftment in immunodeficient mice. PDGFB-MSC-treated BM enhanced transplanted human HSC self-renewal in secondary transplantations more efficiently than GFP-transduced MSCs (GFP-MSCs). Gene set enrichment analysis showed increased antiapoptotic signaling in PDGFB-MSCs compared with GFP-MSCs. PDGFB-MSCs exhibited enhanced survival and expansion after transplantation, resulting in an enlarged humanized niche cell pool that provide a better humanized microenvironment to facilitate superior engraftment and proliferation of human hematopoietic cells. Our studies demonstrate the efficacy of PDGFB-MSCs in supporting human HSC engraftment.

Abstract P197: TRPM7 is Involved in the Effects of VEGF and EGF in Vascular Cells

TRPM6 and 7 are channels important in Mg 2+ and Ca 2+ homeostasis. We demonstrated that TRPM7 is influenced by angiotensin II. TRPM6 is influenced by EGF and cancer patients treated with EGFR inhibitors exhibit hypomagnesemia and hypertension by unclear mechanisms. Whether growth factors influence vascular TRPM7 is unknown. Here we questioned if VEGF/EGF mediate vascular effects through TRPM7. Studies were performed in human VSMC, wild type (WT) and TRPM7-deficient (M7+/Δ) mice. VSMC were stimulated with VEGF or EGF (50ng/ml) in the absence/presence of vatalanib, gefitinib (1μM), 2APB (30μM) and NS8593 (40μM), inhibitors of VEGFR, EGFR and TRPM7 respectively. Ca 2+ and Mg 2+ levels were assessed by Cal-520 and Mg-green. VEGF/EGF signaling was assessed by immunoblotting and vascular function by myography in mesenteric arteries from WT and M7+/Δ mice and treated with EGF or VEGF (50ng/ml). TRPM7 expression in aortas and kidneys from WT treated with vatalanib or gefitinib (100mg/Kg/day, 2 weeks) was assessed by immunoblotting. VEGF and EGF increased TRPM7 expression (50% and 67% respectively) and phosphorylation (2-fold), promoted influx of Ca 2+ (8% and 10%) and Mg 2+ (8%), effects that were reduced by vatalanib, gefitinib, NS8593, and 2-APB. EGF but not VEGF increased phosphorylation of PKC (43%), p38MAPK (47%), and ERK1/2 (120%). These responses were reduced by gefitinib, however only ERK1/2 phosphorylation was inhibited by NS8593, and 2-APB. Mice treated with vatalanib or gefitinib showed reduced expression of TRPM7 in aortas (50% and 74% respectively) and kidneys (36% and 66% respectively). Vessels exposed to EGF were less responsive to acetylcholine (ACh)-induced relaxation, [Emax %: WT (veh 97±3 vs EGF 63±10, p<0.05), M7+/Δ (veh 89%±5% vs EGF 69%±5%, p<0.05)]. Vessels from M7+/Δ treated with VEGF were less sensitive to sodium nitroprusside (SNP)-induced relaxation [pD2: WT (veh 7±0.12 vs VEGF 7.4±0.12, p<0.05), M7+/Δ (veh 6.7±0.15 vs VEGF 6.9±0.09)]. EGF and VEGF regulate VSMCs through TRPM7-dependent pathways. These processes involve MAP kinases and influence vascular function. Our findings identify novel mechanisms whereby growth factors influence vascular contraction/relaxation and suggest that TRPM7-regulated Mg 2+ and Ca 2+ are important.

Active PKG II inhibited the growth and migration of ovarian cancer cells through blocking Raf/MEK and PI3K/Akt signaling pathways.

Despite advances in chemotherapy, ovarian cancer (OC) is still the most lethal gynecologic malignancy. So, it is imperative to explore its mechanism and find novel targets to improve the outcome. Type II cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG II) has been recently reported to inhibit proliferation and metastasis in several tumors. The present study is to clarify the effect of PKG II combined with l-arginine (l-Arg) on OC cells. SKOV3 and A2780 cells were infected with adenovirus coding cDNA of PKG II to increase PKG II expression and l-Arg was applied to activate this kinase. CCK8 assay, Transwell migration and TUNEL assay were applied to detect the proliferation, migration and apoptosis of the OC cells, respectively. Western blotting was used to detect the level of total and phosphorylated proteins. Our results showed that co-treatment with PKG II and l-Arg inhibited EGF-induced proliferation and the expression of Proliferating Cell Nuclear Antigen (PCNA), Cyclin E and N-Cadherin, whereas up-regulated the expression of E-Cadherin, abolished the anti-apoptotic effect of EGF, prevented the process of epithelial-to-mesenchymal transition (EMT) as well as blocked EGF-triggered Raf-MEK and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. Our results suggested that PKG II activated by l-Arg could inhibit proliferation and migration and promote the apoptosis of OC cells. Based on the above results and our previous data, it is speculated that PKG II is an inhibitor of cancer with extensive effects.

Revisiting a controversy: The effect of EGF on EGFR dimer stability

EGFR is a receptor tyrosine kinase that plays a critical role in cell proliferation, differentiation, survival and migration. Its activating ligand, EGF, has long been believed to stabilize the EGFR dimer. Two research studies aimed at quantitative measurements of EGFR dimerization, however, have led to contradicting conclusions and have questioned this view. Given the controversy, here we sought to measure the dimerization of EGFR in the absence and in the presence of saturating EGF concentrations, and to tease out the effect of ligand on dimer stability, using a FRET-based quantitative method. Our measurements show that the dissociation constant is decreased ~150 times due to ligand binding, indicative of significant dimer stabilization. In addition, our measurements demonstrate that EGF binding induces a conformational change in the EGFR dimer.