Epidermal Growth Factor Homology Research Articles

Design, Synthesis, and Antiproliferative Activity of Quinazolin-4-One/Chalcone Hybrids via the EGFR Inhibition Pathway.

Quinazolinone scaffolds have drawn international attention due to their potent anticancer activity and therapeutic applications. Furthermore, Chalcone and Oxime are special chemical templates with a wide range of biological activities, including anti-cancer activity. As a result, the purpose of this research is to synthesize and develop a new series of 2-thioxo-3-substituted quinazolin-4-one/chalcone analogues and 2-thioxo-3-substituted quinazolin-4-one/oximes analogues in order to obtain a new cytotoxic agent that can target epidermal growth factor (EGFR) and/or V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAFV600E) oncogene. All synthesised compounds were tested for anticancer activity against four human cancer cell lines. The new hybrids' potential anti-cancer mechanism was evaluated using EGFR and BRAF enzymatic tests. The most active molecules within the target enzyme's active site were studied using molecular docking. Apoptosis and cell cycle analysis were also investigated. The target compounds 7a-j (series I) are obtained in high yields by alkylation of 2-mercapto-3-ethyl-(3H)- quinazolin-4-one 3a with acylated chalcones 6a-j. Alkylation of compounds 3b-c with N-(4-acetylphenyl)-2- bromoacetamide 8, the corresponding ketones intermediates 9b-c was produced in high yields. Compounds 7a-j, 9b-c, and 10b-c were tested for their antiproliferative activity against four human cancer cell lines using the MTT assay and doxorubicin as a control drug. The EGFR and BRAF assay tests were used to assess the inhibitory potency against EGFR and BRAF. Compounds 7c, 7d, 7f and 10c exhibited high proliferative activity and inhibited EGFR, which could serve as a potential target for antiproliferative activity. The most active hybrid, 7c, primarily caused cell cycle arrest in G0/G1 phase and S phase as well as cell apoptosis. Finally, the most active hybrids were docked well to the EGFR active site. 2-thioxo-3-substituted quinazolin-4-one/chalcone derivatives have significant apoptotic and antiproliferative properties.

TIE2-high cervical cancer cells promote tumor angiogenesis by upregulating TIE2 and VEGFR2 in endothelial cells

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2), the receptor for angiopoietins, has been found highly expressed in cervical cancer and associated with poor prognosis. However, the potential role of tumoral TIE2 in cervical cancer angiogenesis and the underlying mechanisms remain unexplored. Here, based on multicolor immunofluorescence of 64 cervical cancer tissues, we found that TIE2 level in cervical cancer cells was positively related to shorter survival and higher microvessel density in tumor. In vitro and in vivo experiments verified that TIE2-high cervical cancer cells could promote tumor angiogenesis. TIE2-high tumor cells induced an amplified expression of TIE2 and vascular endothelial growth factor receptor 2(VEGFR2) in HUVECs to promote angiogenesis via TIE2 -AKT/MAPK signals, which could be reversed or partially reversed by TIE2, AKT or MAPK inhibitors and activated by angiopoietin-1 and angiopoietin-2. In conclusion, TIE2-high cervical cancer cells promote tumor angiogenesis by upregulating TIE2 and VEGFR2 in endothelial cells via TIE2-AKT/MAPK axis inside tumor cells.

Effect of Novel Biotherapeutic Elevating Angiopoietin 1 on Progression of Diabetic Nephropathy in Diabetic/Obese Mice.

Diabetic nephropathy is a leading cause of end-stage renal disease, characterized by endothelial dysfunction and a compromised glomerular permeability barrier. Dysregulation of the angiopoietin 1 (ANGPT1)/angiopoietin 2 (ANGPT2) signaling axis is implicated in disease progression. We recently described the discovery of an IgG1 antibody, O010, with therapeutic potential to elevate circulating endogenous ANGPT1, a tyrosine kinase with Ig and epidermal growth factor (EGF) homology domains-2 (TIE2) agonist. Studies are described that detail the effect of various ANGPT1-elevating strategies to limit progression of renal dysfunction in diabetic-obese (db/db) mice. Results demonstrate that adeno-associated virus- or DNA minicircle-directed overexpression of ANGPT1 elicits a reduction in albuminuria (56%-73%) and an improvement in histopathology score (18% reduction in glomerulosclerosis). An improved acetylcholine response in isolated aortic rings was also observed indicative of a benefit on vascular function. In separate pharmacokinetic studies, an efficacious dose of the ANGPT1 DNA minicircle increased circulating levels of the protein by >80%, resulting in a concomitant suppression of ANGPT2. At a dose of O010-producing maximal elevation of circulating ANGPT1 achievable with the molecule (60% increase), no suppression of ANGPT2 was observed in db/db mice, suggesting insufficient pathway engagement; no reduction in albuminuria or improvement in histopathological outcomes were observed. To pinpoint the mechanism resulting in lack of efficacy, we demonstrate, using confocal microscopy, an interference with TIE2 translocation to adherens junctions, resulting in a loss of protection against vascular permeability normally conferred by ANGPT1. Results demonstrated the essential importance of ANGPT1 to maintain the glomerular permeability barrier, and, due to interference of O010 with this process, led to the discontinuation of the molecule for clinical development. SIGNIFICANCE STATEMENT: This body of original research demonstrates that elevation of systemic angiopoietin 1 (ANGPT1) is protective against diabetic nephropathy. However, using a novel biotherapeutic approach to elevate systemic ANGPT1 renoprotection was not observed; we demonstrate that protection was lost due to interference of the therapeutic with ANGPT1/ tyrosine kinase with Ig and EGF homology domains-2 translocation to adherens junctions. Thus, the clinical development of the antibody was terminated.

Tumor cell-derived exosomes deliver TIE2 protein to macrophages to promote angiogenesis in cervical cancer.

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2)-expressing macrophages (TEMs) are an angiogenesis-promoting subset of tumor-associated macrophages that have been demonstrated to be increased in solid tumors and associated with the progression of cervical cancer. However, the induction mechanism of TEMs remains unclear. Here, based on multicolor immunofluorescence of 58 cervical cancer tissues and the GEPIA database, we found that TEMs were increased in TIE2-high cervical cancer and related to shorter survival. In vitro and in vivo experiments verified that exosomes derived from TIE2-high cervical cancer cells transferred TIE2 protein directly to macrophages, thereby inducing TEMs. Similar to primary TEMs, TEMs induced by tumor-derived exosomes promoted angiogenesis, could be induced by angiopoietin-2, and possessed an M2-like phenotype. In conclusion, exosomes derived from TIE2-high cervical cancer cells induce TEMs by directly transporting TIE2 to promote tumor angiogenesis.

An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1

Activation of the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor by angiopoietin-1 (Ang1) is critical for vascular stabilization: it promotes survival signal transduction via auto-phosphorylation and reduces vascular permeability by strengthening tight junctions between endothelial cells. Thus, Tie2/Ang1 signaling is a promising therapeutic target for vascular diseases. However, in vivo use of existing Tie2 signaling modulators, such as recombinant Ang1, is restricted by limitations in manufacturability and stability. Here, we present a novel engineered tetra-valent agonistic antibody, ASP4021, which can specifically and fully activate the Tie2 receptor in an equivalent manner to Ang1. ASP4021 induced Tie2 self-phosphorylation and inhibited apoptosis in a human primary endothelial cell line. Additionally, single administration of ASP4021 significantly suppressed mustard-oil-induced vascular permeability in rats. ASP4021 may thus be a potential therapeutic candidate for diseases associated with vascular weakness such as diabetic retinopathy, diabetic macular edema and critical limb ischemia.

Circulating Tie2-Expressing Monocytes: A Potential Biomarker for Cervical Cancer.

BackgroundTyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2)-expressing monocytes (TEMs) are a highly proangiogenic subset of myeloid cells, which are characterized by expressing the angiopoietin receptor Tie2 with pro-tumor activity.PurposeThe present study aimed to determine the clinical value of circulating TEMs (cTEMs) for cervical cancer.Patients and MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from 7 healthy volunteers, 17 uterine fibroid patients, 24 cervical intraepithelial neoplasia (CIN) II patients, 31 CIN III patients and 99 patients with cervical cancer. The cTEMs were evaluated by the ratio of Tie2+ CD14+ cells to all CD14+ monocytes in the PBMCs through flow cytometry. The diagnostic value of cTEM was assessed by receiver operating characteristic (ROC) curves and the correlation between cTEM and clinicopathological characters in cervical cancer patients was analyzed.ResultsThe proportion of cTEMs was gradually increasing from healthy volunteers to patients with non-invasive lesions, then to cervical cancer patients. The area under the ROC curve was 0.913 when the level of cTEMs was used to distinguish cervical cancer from all the other women ranging from healthy volunteers to CIN III patients. In cervical cancer, an increased cTEM fraction was significantly correlated with advanced tumor stage, larger tumor size, lymph node metastasis (LNM), deep stromal infiltration, parametrial involvement and lymph-vascular space invasion and was an independent risk factor for LNM.ConclusionThe cTEM proportion might be a promising biomarker for the malignant transformation of cervical lesions and the progression of cervical cancer.

Systemic Endothelial Activation Is Associated With Early Acute Respiratory Distress Syndrome in Children With Extrapulmonary Sepsis.

Systemic endothelial activation may contribute to sepsis-associated organ injury, including acute respiratory distress syndrome. We hypothesized that children with extrapulmonary sepsis with versus without acute respiratory distress syndrome would have plasma biomarkers indicative of increased endothelial activation and that persistent biomarker changes would be associated with poor outcome. Observational cohort. Academic PICU. Patients less than 18 years old with sepsis from extrapulmonary infection with (n = 46) or without (n = 54) acute respiratory distress syndrome and noninfected controls (n = 19). None. Endothelial (angiopoietin-1, angiopoietin-2, tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2, vascular endothelial growth factor, soluble fms-like tyrosine kinase, von Willebrand factor, E-selectin, intercellular adhesion molecule, vascular cell adhesion molecule, thrombomodulin) and inflammatory biomarkers (C-reactive protein, interleukin-6, and interleukin-8) were measured from peripheral plasma collected within 3 days (time 1) of sepsis recognition and at 3-6 days (time 2) and 7-14 days (time 3). Time 1 biomarkers and longitudinal measurements were compared for sepsis patients with versus without acute respiratory distress syndrome and in relation to complicated course, defined as greater than or equal to two organ dysfunctions at day 7 or death by day 28. Angiopoietin-2, angiopoietin-2/angiopoietin-1 ratio, tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2, vascular endothelial growth factor, von Willebrand factor, E-selectin, intercellular adhesion molecule, vascular cell adhesion molecule, thrombomodulin, endocan, C-reactive protein, interleukin-6, and interleukin-8 were different between sepsis and noninfected control patients at time 1. Among patients with sepsis, those with acute respiratory distress syndrome had higher angiopoietin-2/angiopoietin-1 ratio, vascular endothelial growth factor, vascular cell adhesion molecule, thrombomodulin, endocan, interleukin-6, and interleukin-8 than those without acute respiratory distress syndrome (all p < 0.003). Angiopoietin-2 and angiopoietin-2/angiopoietin-1 ratio remained higher in sepsis with versus without acute respiratory distress syndrome after multivariable analyses. Time 1 measures of angiopoietin-2, angiopoietin-2/-1 ratio, von Willebrand factor, and endocan were indicative of complicated course in all sepsis patients (all area under the receiver operating curve ≥ 0.80). In sepsis without acute respiratory distress syndrome, soluble fms-like tyrosine kinase decreased more quickly and von Willebrand factor and thrombomodulin decreased more slowly in those with complicated course. Children with extrapulmonary sepsis with acute respiratory distress syndrome had plasma biomarkers indicative of greater systemic endothelial activation than those without acute respiratory distress syndrome. Several endothelial biomarkers measured near sepsis recognition were associated with complicated course, whereas longitudinal biomarker changes yielded prognostic information only in those without sepsis-associated acute respiratory distress syndrome.

Characterization of a Porcine Model for Von Willebrand Disease Type 1 and 3 Regarding Expression of Angiogenic Mediators in the Nonpregnant Female Reproductive Tract.

Von Willebrand disease (VWD), a blood coagulation disorder, is also known to cause angiodysplasia. Hitherto, no animal model has been found with angiodysplasia that can be studied in vivo. In addition, VWD patients tend to have a higher incidence of miscarriages for reasons unknown. Thus, we aimed to examine the influence of von Willebrand factor (VWF) on the female reproductive tract histology and the expression and distribution of angiogenic factors in a porcine model for VWD types 1 and 3. The disease-causing tandem duplication within the VWF gene occurred naturally in these pigs, making them a rare and valuable model. Reproductive organs of 6 animals (2 of each mutant genotype and 2 wildtype (WT) animals) were harvested. Genotype plus phenotype were confirmed. Several angiogenic factors were chosen for possible connections to VWF and analyzed alongside VWF by immunohistochemistry and quantitative gene expression studies. VWD type 3 animals showed angiodysplasia in the uterus and shifting of integrin αVβ₃ from the apical membrane of uterine epithelium to the cytoplasm accompanied by increased vascular endothelial growth factor (VEGF) expression. Varying staining patterns for angiopoietin (Ang)-2 were observed among the genotypes. As compared with WT, the ovaries of the VWD type 3 animals showed decreased gene expression of ANG2 and increased gene expression of TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) 2, with some differences in the ANG/TIE-system among the mutant genotypes. In conclusion, severely reduced VWF seems to evoke angiodysplasia in the porcine uterus. Varying distribution and expression of angiogenic factors suggest that this large animal model is promising for investigation of influence of VWF on angiogenesis in larger groups.

Prognostic importance of PTEN, EGFR, HER-2, and IGF-1R in gastric cancer patients treated with postoperative chemoradiation

Background/aimThis study aimed to describe the prognostic importance of epidermal growth factor (EGFR), phosphatase and tensin homolog (PTEN), human EGF receptor-2 (HER-2), and insulin-like growth factor 1 receptor (IGF-1R) in gastric cancer patients treated with postoperative chemoradiation therapy.Materials and methodsSixty-nine patients treated with adjuvant chemoradiation therapy were retrospectively evaluated. Tumor samples were stained immunohistochemically. ResultsAll patients were treated with 3D conformal radiation therapy with concomitant and adjuvant chemotherapy. Perineural invasion (PNI) (P = 0.042), prechemoradiation therapy albumin levels below 3.5 mg/dL (P = 0.011), and EGFR positivity (P = 0.008) had negative effects on overall survival (OS). The median OS was 26 months for patients with PNI (+), 34.9 months for those with PNI (–), 19.5 months for those with albumin levels below 3.5 mg/dL, and 33.2 months for those with albumin levels above 3.5 mg/dL. IGF-1R (+) (P = 0.035) and history of cigarette smoking (P = 0.033) were observed to have a statistically significantly negative effect on disease-free survival (DFS). The median DFS was 29.2 months for IGF-1R (+) patients, 37.9 months for those with IGF-1R (-), and 26.3 and 40.59 months for smokers and nonsmokers, respectively. ConclusionIGF-1R and EGFR may be used for patient selection in future prospective studies that evaluate the prognostic importance of these receptors.

Do Third Molars Contribute to Systemic Inflammation? Results From a Population-Based Study From Northeast Germany

Erupted and impacted third molars have been reported to contribute to systemic inflammation. This study investigated the systemic effect of third molars on serum levels of inflammatory parameters and on inflammatory messenger peptide hormones in a general population sample. Data of 2,151 participants from the Study of Health in Pomerania were included in this study. Erupted or impacted third molars were assessed with whole-body magnetic resonance imaging at 1.5T and associated with biomarkers of inflammation, lipid metabolism, glucose metabolism, and peptide hormones by linear regression. Models were adjusted for age, gender, smoking status, education, and type 2 diabetes mellitus. Neither erupted nor impacted third molars were associated with high-sensitivity C-reactive protein, white blood cell count, or fibrinogen as markers for systemic inflammation. Participants with erupted third molars had markedly lower serum levels of leptin (β coefficient, -2.47; 95% confidence interval [CI], -4.47 to -0.48), angiopoietin-2 (β coefficient, -135.1; 95% CI, 248.6 to -21.5), and ratio of angiopoietin-2 to tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2 (β coefficient, -6.57; 95% CI, -13.06 to -00.7) than participants without third molars. No such associations were observed for impacted third molars. The present results did not substantiate a relation between third molars and an increase in systemic inflammatory markers. Therefore, dental practitioners should be careful when considering this as the only indication for removal of third molars, especially in medically compromised patients. The results of this study showed that participants with erupted third molars had lower levels of messenger peptide hormones, such as leptin and angiopoetin-2.

Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility.

Introductory paragraphCell motility is essential for viral dissemination1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction2. However, F11L alone is not sufficient for VACV induced cell motility, indicating that additional viral factors must be involved. Here we show that the VACV epidermal growth factor homolog, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10 whereupon it acts largely in a paracrine fashion to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR/MAPK/FAK/ADAM10 inhibitors, or with VGF and F11 deleted viruses, revealed defects in radial velocity and directional migration efficiency leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish tie1 mRNA by Long Noncoding RNA During Brain Vascular Assembly.

Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus-tie1 antisense (tie1AS), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS. Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS).

Angiogenesis in adenosquamous cancer of pancreas.

Adenosquamous carcinoma of the pancreas (ASCP) is an uncommon variant of exocrine pancreatic malignancies, characterized by a histological admixture of adenomatous and squamous cell elements. This cancer is characterized by a poorly differentiated histology and a poorer clinical outcome compared to pancreatic ductal adenocarcinoma (PDAC). Unlike PDAC, that is characterized by a low microvascular density (MVD) and collapsed vasculature, no data are available about angiogenesis in ASPC. Immunohistochemical evaluation of MVD and trypatse-positive mast cells (MCs) were performed on a single case of ASCP compared to PDAC. Moreover, the levels of angiopoietin-1 and -2 (Ang-1, Ang-2), receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie-2), vascular endothelial growth factor A (VEGFA), hypoxia-inducible factor 1 alpha (HIF1A), miR-21-5p, miR-181a-5p, miR-122-5p, and miR-27a-3p were evaluated by real-time PCR. Higher number of tryptase-positive MCs and MVD are observed in the ASCP case compared to PDAC one. Lower levels of miR-122-5p and higher expression of VEGFA, HIF1A and Ang-2 genes were observed in ASCP. Furthermore, lower Ang-1 and Tie-2 transcript levels and higher increases of miR-21-5p, miR27a-3p and miR-181a-5p levels were found in the rarest form of pancreatic carcinoma. Our data demonstrate an important angiogenic activity in ASCP with a putative role of miR-21-5p, miR-181a-5p, miR-122-5p and miR-27a-3p in the regulation of this process.

Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells.

Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration.

Therapeutic hypothermia attenuates brain edema in a pig model of cardiac arrest: Possible role of the angiopoietin-Tie-2 system

ObjectiveThis study aimed to clarify whether therapeutic hypothermia protects against cerebral edema following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in a porcine model via regulating the angiopoietin-Tie-2 ligand-receptor system. MethodsMale pigs were randomized into the therapeutic hypothermia group, the normothermia group or the sham control group. CA was induced in pigs by untreated ventricular fibrillation for 8min. Brain edema was determined by measuring the cerebral cortical water content at 24h after the return of spontaneous circulation (ROSC). The serum levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2 (Tie-2), and S100B were measured using enzyme immunoassay kits at 0.5, 6, 12 and 24h after ROSC. The levels of the Ang-1, Ang-2, phosphorylated Tie-2 and Tie-2 proteins in the cerebral cortex at 24h after ROSC were determined by Western blotting. ResultsTherapeutic hypothermia lessened brain cortex edema, alleviated histopathology injury, and improved neurologic outcomes at 24h after ROSC. Therapeutic hypothermia inhibited the CA- and CPR-induced increases in serum Ang-2 protein expression and the Ang-2/Ang-1 ratio and attenuated the decrease in serum Ang-1 expression. Therapeutic hypothermia also increased the protein expression of Ang-1 and the phosphorylated Tie-2/Tie-2 ratio and inhibited the expression of Ang-2 in the cerebral cortex at 24h after ROSC. ConclusionsBased on our experiment, therapeutic hypothermia decreased cerebral edema after CA, which may be, at least in part, related to its ability to modulate the expression of components of the Ang-Tie-2 system.

Analyzing pERK Activation During Planarian Regeneration.

Planarians are an ideal model in which to study stem cell-based regeneration. After amputation, planarian pluripotent stem cells surrounding the wound proliferate to produce the regenerative blastema, in which they differentiate into the missing tissues and structures. Recent independent studies in planarians have shown that Smed-egfr-3, a gene encoding a homologue of epidermal growth factor (EGF) receptors, and DjerkA, which encodes an extracellular signal-regulated kinase (ERK), may control cell differentiation and blastema growth. However, because these studies were carried in two different planarian species, the relationship between these two genes remains unclear. We have optimized anti-pERK immunostaining in Schmidtea mediterranea using the original protocol developed in Dugesia japonica. Both protocols are reported here as most laboratories worldwide work with one of these two species. Using this protocol we have determined that Smed-egfr-3 appears to be necessary for pERK activation during planarian regeneration.

Current Literature Review.

Current Literature Review.

Tie2 Regulates Tumor Metastasis of Oral Squamous Cell Carcinomas.

The endothelial-specific receptor, tyrosine kinase with immunoglobulin-like loops and epidermal growth factor homology domains-2 (Tie2) is a member of the tyrosine kinase family and is ubiquitous in normal tissues; however, little is known about the mechanisms and roles of Tie2 in oral squamous cell carcinomas (OSCCs). In the current study, we investigated the expression status of Tie2 in OSCCs by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemistry and the functional mechanisms of Tie2 using its overexpressed OSCC (oeTie2) cells and Tie2 blocking by its antibody. We found that Tie2 expression was down-regulated significantly (p < 0.05) in OSCCs compared with normal counterparts in vitro and in vivo. Interestingly, oeTie2 cells showed higher cellular adhesion (p < 0.05) and lower cellular invasion (p < 0.05) compared with control cells; whereas there was similar cellular proliferation in both transfectants. Furthermore, cellular adhesion was inhibited and invasion was activated by Tie2 function-blocking antibody (p < 0.05), indicating that Tie2 directly regulates cellular adhesion and invasion. As expected, among the clinical variables analyzed, Tie2-positivity in patients with OSCC was correlated closely with negative lymph node metastasis. These results suggested for the first time that Tie2 plays an important role in tumor metastasis and may be a potential biomarker for OSCC metastasis.

Prognostic significance of the angiopoietin-2/angiopoietin-1 and angiopoietin-1/Tie-2 ratios for early sepsis in an emergency department.

IntroductionThis study was performed to assess the early diagnostic, risk stratification, and prognostic value of the angiopoietin-2/angiopoietin-1 ratio (Ang-2/Ang-1) and angiopoietin-1/tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2 ratio (Ang-1/Tie-2) and to compare these factors with procalcitonin (PCT) and the Mortality in Emergency Department Sepsis (MEDS) score in patients with early sepsis in the emergency department (ED).MethodsConsecutive patients with sepsis (n = 440) were enrolled in this study. They fulfilled the systemic inflammatory response syndrome (SIRS) criteria and were admitted to the ED of Beijing Chao-yang Hospital between August 2014 and February 2015. The control group consisted of 55 healthy blood donors. The patients were categorized into four groups: SIRS, sepsis, severe sepsis, and septic shock. Serum Ang-1, Ang-2, Tie-2, and PCT were measured, and the MEDS score was calculated upon ED arrival. The prognostic values of Ang-2/Ang-1, Ang-1/Tie-2, Ang-1, Ang-2, and Tie-2 were compared with the PCT and MEDS scores. All patients were followed for 28 days.ResultsUpon admission, the median levels of the serum Ang-2 level and Ang-2/Ang-1 ratio increased and the serum Ang-1 levels and Ang-1/Tie-2 ratios decreased with the severity of sepsis. The areas under the receiver operating characteristic curves of the Ang-2/Ang-1 and Ang-1/Tie-2 ratios were greater than those of the Ang-1, Ang-2, and PCT levels and MEDS scores in the diagnosis and prediction of 28-day mortality due to sepsis. Ang-2/Ang-1 was significantly higher and Ang-1/Tie-2 was significantly lower in nonsurvivors than in survivors at the 28-day follow-up examination. Ang-2/Ang-1, Ang-1/Tie-2, and MEDS score were found to be independent predictors of 28-day mortality in patients with sepsis. The levels of serum Ang-1, Ang-2, and Tie-2 were positively correlated with each other. The ratios of Ang-2/Ang-1 and Ang-1/Tie-2 were positively and negatively correlated, respectively, with the MEDS score in every septic group.ConclusionsThe Ang-2/Ang-1 and Ang-1/Tie-2 ratios are valuable for risk stratification in patients with sepsis and are associated with the poor clinical outcome of early sepsis in the ED.

Signal transduction by M3 muscarinic acetylcholine receptor in prostate cancer.

The present study aimed to investigate the potential mechanisms used during signal transduction by M3 muscarinic acetylcholine receptor (CHRM3) in prostate cancer. The microarray datasets of GSE3325, including 5 clinically localized primary prostate cancers and 4 benign prostate tissues, were downloaded from the Gene Expression Omnibus database. The differentially-expressed genes (DEGs) in primary prostate cancer tissues compared with benign controls were screened using the Limma package. Gene Ontology and pathway enrichment analyses were performed using the Database for Annotation Visualization and Integrated Discovery. Next, a protein-protein interaction (PPI) network was constructed. Additionally, microRNAs (miRNAs) associated with DEGs were predicted and miRNA-target DEG analysis was performed using a Web-based Gene Set Analysis Toolkit. Finally, the PPI network and the miRNA-target DEG network were integrated using Cytoscape. In total, 224 DEGs were screened in the prostate cancer tissues, including 113 upregulated and 111 downregulated genes. CHRM3 and epidermal growth factor (EGF) were enriched in the regulation of the actin cytoskeleton. EGF and v-myc avian myelocytomatosis viral oncogene homolog (Myc) were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway. EGF with the highest degree of connectivity was the hub node in the PPI network, and miR-34b could interact with Myc directly in the miRNA-target DEG network. EGF and Myc may exhibit significant roles in the progression of prostate cancer via regulation of the actin cytoskeleton and the MAPK signaling pathway. CHRM3 may activate these two pathways in prostate cancer progression. Thus, these two key factors and pathways may be crucial mechanisms during signal transduction by CHRM3 in prostate cancer.