The “Angiogenic Switch” and Functional Resources in Cyclic Sports Athletes

Regular physical activity in cyclic sports can influence the so-called “angiogenic switch”, which is considered as an imbalance between proangiogenic and anti-angiogenic molecules. Disruption of the synthesis of angiogenic molecules can be caused by local changes in tissues under the influence of excessive physical exertion and its consequences, such as chronic oxidative stress and associated hypoxia, metabolic acidosis, sports injuries, etc. A review of publications on signaling pathways that activate and inhibit angiogenesis in skeletal muscles, myocardium, lung, and nervous tissue under the influence of intense physical activity in cyclic sports. Materials: We searched PubMed, SCOPUS, Web of Science, Google Scholar, Clinical keys, and e-LIBRARY databases for full-text articles published from 2000 to 2020, using keywords and their combinations. Results: An important aspect of adaptation to training loads in cyclic sports is an increase in the number of capillaries in muscle fibers, which improves the metabolism of skeletal muscles and myocardium, as well as nervous and lung tissue. Recent studies have shown that myocardial endothelial cells not only respond to hemodynamic forces and paracrine signals from neighboring cells, but also take an active part in heart remodeling processes, stimulating the growth and contractility of cardiomyocytes or the production of extracellular matrix proteins in myofibroblasts. As myocardial vascularization plays a central role in the transition from adaptive heart hypertrophy to heart failure, further study of the signaling mechanisms involved in the regulation of angiogenesis in the myocardium is important in sports practice. The study of the “angiogenic switch” problem in the cerebrovascular and cardiovascular systems allows us to claim that the formation of new vessels is mediated by a complex interaction of all growth factors. Although the lungs are one of the limiting systems of the body in cyclic sports, their response to high-intensity loads and other environmental stresses is often overlooked. Airway epithelial cells are the predominant source of several growth factors throughout lung organogenesis and appear to be critical for normal alveolarization, rapid alveolar proliferation, and normal vascular development. There are many controversial questions about the role of growth factors in the physiology and pathology of the lungs. The presented review has demonstrated that when doing sports, it is necessary to give a careful consideration to the possible positive and negative effects of growth factors on muscles, myocardium, lung tissue, and brain. Primarily, the “angiogenic switch” is important in aerobic sports (long distance running). Conclusions: Angiogenesis is a physiological process of the formation of new blood capillaries, which play an important role in the functioning of skeletal muscles, myocardium, lung, and nervous tissue in athletes. Violation of the “angiogenic switch” as a balance between proangiogenic and anti-angiogenic molecules can lead to a decrease in the functional resources of the nervous, musculoskeletal, cardiovascular, and respiratory systems in athletes and, as a consequence, to a decrease in sports performance.     

Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics

Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein-protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N-terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling.     

δ-Catenin Participates in EGF/AKT/p21Waf Signaling and Induces Prostate Cancer Cell Proliferation and Invasion

Prostate cancer (PCa) is the second most leading cause of death in males. Our previous studies have demonstrated that δ-catenin plays an important role in prostate cancer progression. However, the molecular mechanism underlying the regulation of δ-catenin has not been fully explored yet. In the present study, we found that δ-catenin could induce phosphorylation of p21^Waf and stabilize p21 in the cytoplasm, thus blocking its nuclear accumulation for the first time. We also found that δ-catenin could regulate the interaction between AKT and p21, leading to phosphorylation of p21 at Thr-145 residue. Finally, EGF was found to be a key factor upstream of AKT/δ-catenin/p21 for promoting proliferation and metastasis in prostate cancer. Our findings provide new insights into molecular controls of EGF and the development of potential therapeutics targeting δ-catenin to control prostate cancer progression.     

Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

EGF +61基因多态性与乙型肝炎肝细胞癌的相关性

目的探讨表皮生长因子(EGF)G61A位点(EGF +61)多态性与慢性乙型肝炎肝细胞癌的相关性。方法运用多聚酶链式反应-限制性片段长度多态性(PCR-RFLP)技术,检测中国人群中215例慢性乙型肝炎肝细胞癌患者和104例正常对照者EGF+61位点的基因型,分析基因型及等位基因的分布频率。结果EGF +61位点基因型及等位基因在乙型肝炎肝细胞癌患者及对照者的分布频率差异均无统计学意义。TNMⅠ、Ⅱ及TNMⅢ、Ⅳ两组的基因型及等位基因的分布频率差异也无统计学意义。结论在中国人群中,EGF +61位点的基因多态性与乙型肝炎肝细胞癌的发生和进展程度无密切关系。     

Cyanidin suppresses neoplastic cell transformation by directly targeting phosphatidylinositol 3-kinase

Cyanidin, an anthocyanidin found in fruits and vegetables, has been reported to possess anti-cancer effects. However, there is no study on the chemopreventive effect of cyanidin against neoplastic cell transformation and its molecular mechanisms. In the present study, we compare the anti-carcinogenic effects of cyanidin and cyanidin-3-glucoside (C3G) and investigate their underlying mechanisms. The inhibitory effect of cyanidin on EGF-induced cell transformation was higher than those of C3G in JB6 P+ mouse epidermal (JB6 P+) cells. Both cyanidin and C3G showed dose-dependent radical scavenging activities. It is indicated that the divergent inhibitory effects of cyanidin and C3G are not due to their antioxidant activities. We found that cyanidin, but not C3G, inhibited the EGF-induced Akt/p70S6K phosphorylation. Moreover, cyanidin directly suppressed the activity of PI3K by binding to PI3K directly in an ATP-competitive manner, which indicates that PI3K is one of the molecular targets of cyanidin.     

血栓调节蛋白核心片段的基因克隆、表达及鉴定

目的 获得血栓调节蛋白及其核心片段,以便研制临床诊断治疗制品。方法 应用ＰＣＲ获得血栓 调节蛋白ＥＧＦ４～６的基因片段,构建克隆载体ＥＧＦ４－６／ＰＹ和表达载体ＥＧＦ４－６／ＰＢＶ２２０）,表达血栓调节蛋白ＥＧＦ４-６。结果 原核细胞表达量约占菌体可溶性蛋白的１２％,经ＫＰＴＴ试剂盘检测具有生物学活性。结论 为进一步 研究临床诊断治疗制剂创造了条件。     

Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy,surface replication,freeze-etching and label fracture

In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno-gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO₄ fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors.     

Role of growth factors and their receptors in gastric ulcer healing

The repair of gastric ulcers requires the reconstitution of epithelial structures and the underlying connective tissue, including vessels and muscle layers. Several growth factors have been implicated in this process, since they are able to regulate important cell functions, such as cell proliferation, migration, differentiation, secretion, and degradation of extracellular matrix, all of which are essential during tissue healing. Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), hepatocyte growth factor (HGF), and trefoil factors (TFFs) are mainly involved in the reconstitution of the epithelial structures. Platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-beta) play a major role in the reconstitution of connective tissue, including vessels and smooth muscle cells, and provide the extracellular matrix substrate for cell migration and differentiation. The expression of these growth factors and their receptors is increased during ulcer healing and, in some cases, intracellular signaling related to receptor binding and transduction has been demonstrated. EGF, TGF-alpha and TFFs are normally present either in the gastric juice or in the mucosa, and may exert their effects immediately after damage, before newly synthesized EGF and TFFs are released from the ulcer margin. The inhibition of their effects by neutralizing antibodies may result in delayed ulcer healing, while the administration of recombinant or natural analogues may improve ulcer repair. In this review, we will summarize the basic molecular characteristics of some of these growth factors, and will discuss available evidence supporting their role in the ulcer repair process.