To decipher the mechanism of action, we found that ALKBH5 is required to maintain eNOS phosphorylation and SPHK1 protein levels. In this study, we report that ALKBH5 levels are upregulated following ischemia and are associated with maintaining ischemia-induced ECs angiogenesis. siRNA mediated ALKBH5 gene silencing was used for examining the loss of function. Methods and Results: ECs were treated with ischemic insults (lipopolysaccharide and 1% hypoxia) to determine the role of ALKBH5 in ECs angiogenesis. We aimed to study the role of m 6A RNA demethylase alkB homolog 5 (ALKBH5) in ECs angiogenesis during ischemic injury. The molecular mechanism for endothelial dysfunction in the heart is still not clearly understood. 3Department of Pathology, Molecular and Cellular Pathology, The University of Alabama at Birmingham, Birmingham, AL, United Statesīackground: Endothelial cells dysfunction has been reported in many heart diseases including acute myocardial infarction, and atherosclerosis.2Department of Biomedical Engineering, The University of Alabama at Birmingham, Birmingham, AL, United States.1Division of Cardiovascular Disease, Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL, United States.Error bars correspond to the number of analysed images (n = 15 acquired from 3 wells of 190 mm 2 for ETFA, and n = 32 acquired from 8 wells of 32 mm 2 for FBA) ± SEM providing from one experiment performed with each method used.Rajesh Kumari 1 †, Roshan Dutta 1, Prabhat Ranjan 1, Zainab Gbongbo Suleiman 2, Sumanta Kumar Goswami 1, Jing Li 1, Harish Chandra Pal 3 and Suresh Kumar Verma 1,2 * Each result in the presence of VEGF-A or Sunitinib was compared to the corresponding Ctrl experiment. Statistical analyses were performed by One-way ANOVA followed by Dunett’s multiple comparison test. Common parameters are Number of Junction (JN), Total Length (TL) and Total Segment Length (TSL). ( A) MMS: Mean Mesh Size ( B) TL: Total Length ( C) TMA: Total Mesh Area ( D) TSL: Total Segment Length ( E) JN: Number of Junction ( F) AJN/S: Number of Anchorage Junction per sphere ( G) TL/S: sum of branches and segments length per Sphere ( H) AJN/S + JN/S: sum of number of Anchorage Junction and Junction per sphere ( I) TSL/S: sum of segment length per Sphere ( J) JN/S: Number of Junction per sphere. Crtl, medium only (black bars) increasing concentrations of VEGF-A (5, 10, 25 and 50 ng/ml) and sunitinib (5 and 25 nM) (white bars). ETFA: HUVEC were cultured for 1 day in Matrigel FBA: HUVEC were cultured for 4 days on the surface of Cytodex 3 microbeads. Although the two methods do not assess the same biological step, our data suggest that they display specific and complementary information on the angiogenesis processes analysis.Ĭomparative measurement of parameters obtained from image analysis in ETFA and FBA. ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC). We detailed these two algorithms and used the new AA version to compare both methods (i.e. Such a method is presented for the first time in fully automated mode and using non-destructive image acquisition.
In this work, we developed and implemented a new algorithm for AA able to recognize microspheres and to analyze the attached capillary-like structures from the FBA model. We previously developed the "Angiogenesis Analyzer" for ImageJ (AA), a tool allowing analysis of ETFA-derived images, according to characteristics of the pseudo-capillary networks. The analytical evaluation of these two widely used assays still remains challenging in terms of observation method and image analysis. An alternative approach to ETFA is the "Fibrin Bead Assay" (FBA), based on the use of Cytodex 3 microspheres, which promote the growth of 3D capillary-like patterns from coated EC, suitable for high throughput in vitro angiogenesis studies. In suitable culture conditions, EC form two-dimensional (2D) branched structures that can lead to a meshed pseudo-capillary network. Among the different methods applied to study angiogenesis, the most commonly used is the "Endothelial Tube Formation Assay" (ETFA). Angiogenesis assays based on in vitro capillary-like growth of endothelial cells (EC) are widely used, either to evaluate the effect of anti- and pro-angiogenesis drugs of interest, or to test and compare the functional capacities of various types of EC and progenitor cells.
0 kommentar(er)
