Angiogenesis experiments using HUVEC cells and transfection of anti-VEGFR2 siRNA with LONZA nuclear transfection

Srinivasan Kokatam1, Kanchan Tiwari1, Jenny Schroeder2, Andrea Toell2, Lubna Hussain3, Preeti Kapoor1.

1. Lonza India Pvt Ltd, Hyderabad, India; 2. Lonza Cologne GmbH, Cologne, Germany; 3. Lonza Walkersville, Inc., Walkersville, MD, USA

Experimental Materials:

Medium formula:

Introduction to the experimental process:

1. Pre-experiment using pmaxGFP plasmid to optimize the nuclear transfer conditions of HUVEC cells;

2. Detecting whether nuclear turnover conditions have an effect on the tube formation of HUVEC cells ;

3. Transfect siRNA to determine the optimal concentration that has the least effect on cell viability, and determine the optimal time point for detecting decreased protein expression;

4. To evaluate the effect of siRNA on Tube Formation of HUVEC cells .

Experimental results:

Figure 1. HUVEC cells transfected with GFP plasmid, the transfection efficiency was about 82% , and had no significant effect on cell morphology:

Figure 2. After nuclear transduction of HUVEC cells, there is no significant effect on Tube formation :

Figure 3. Cells transfected with Anti-VEGFR2 siRNA did not respond to VEGF , demonstrating that siRNAs function.

references:

1. Timar, J. et. al. (2001) Angiogenesis-dependent diseases and angiogenesis therapy. Pathol. Oncol. Res.7 (2): 85-94

2. Murga M1, Fernandez-Capetillo O, Tosato G. (2005) Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2. Blood 105 (5): 1992-9

3. Yang, S, Xin, X, Zlot, C et. al. (2001) Vascular Endothelial Cell Growth Factor-Driven Endothelial Tube Formation Is Mediated by Vascular Endothelial Cell Growth Factor Receptor-2, a Kinase Insert Domain-Containing Receptor. Arterioscler. Thromb. Vasc. Biol. 21: 1934-1940

4. Zumbansen M, Altrogge L, Toell A, Leake D, Muller-Hartmann H (2009) First siRNA Library Screening in Difficult-to-Transfect HUVEC and Jurkat Cells. White paper.

5. Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M, Dahm R (2007) High-efficiency transfection of mammalian neurons via Nucleofection. Nat. Protocols 2: 1692 – 1704

6. Kapoor, R., Reddy, V. and Kapoor, P. (2014) Tube Formation Assay with Primary Human Umbilical Vein Endothelial Cells. Resource Notes, 2014: 9-12.