JBiSE  Vol.3 No.6 , June 2010
Gene silencing of E-selectin block recruitment of endothelial progenitor cell to vascular endothelium under flow
ABSTRACT
Short interfering RNA (siRNA) is a powerful technique that can suppress gene expression in a variety of cells including mammalian cells. Endothelial progenitor cells (EPCs) are bone marrow—derived haematopoietic progenitor cells that have been implicated in vasculogenesis. We demonstrated for the first time that gene silencing of endothelial E-selectin using siRNA transfection in human umbilical vein endothelial cells (HUVECs) causes inhibition of EPC adhesion under flow conditions. Fluorescence immunobinding assay analysis showed that significant reduction of E-selectin surface expression in HUVECs (activated with IL-1β (10 U/mL) for 4 h) transfected with siRNA against E-selectin, but not in HUVECs transfected with LacZ siRNA (control). An EPC adhesion assay under flow conditions (shear stress = 1.0 dyne/cm2) then demonstrated that HUVECs transfected with E-selectin siRNA supported significantly less adhesion of EPCs than those HUVECs treated with control siRNA and no siRNA after activation by IL-1β (p < 0.05). Our experiments have shown the importance of E-selectin in EPC adhesion to HUVECs and the potential utility of gene silencing of E-selectin in EPC recruitment.

Cite this paper
nullSharma, S. and Yoshida, M. (2010) Gene silencing of E-selectin block recruitment of endothelial progenitor cell to vascular endothelium under flow. Journal of Biomedical Science and Engineering, 3, 350-355. doi: 10.4236/jbise.2010.36077.
References
[1]   Cines, D.B., Pollak, E.S., Buck, C.A., Loscalzo, J, Zimmerman, G.A., McEver, R.P., Pober, J.S., Wick, T.M., Konkle, B.A., Schwartz, B.S., Barnathan, E.S., McCrae, K.R., Hug, B.A., Schmidt, A.M. and Stern, D.M. (1998) Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood, 91(10), 3527-3561.

[2]   Shi, Q., Rafii, S., Wu, M.H., Wijelath, E.S., Yu, C., Ishida, A., Fujita, Y., Kothari, S., Mohle, R., Sauvage, L.R., Moore, M.A., Storb, R.F. and Hammond, W.P. (1998) Evidence for circulating bone marrow-derived endothelial cells. Blood, 92(2), 362-367.

[3]   Kakkar, A.K. and Lefer, D.J. (2004) Leukocyte and endothelial adhesion molecule studies in knockout mice. Current Opinion in Pharmacology, 4(2), 154-158.

[4]   Muller, W.A. (2003) Leukocyte—endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends in Immunology, 24(6), 326-333.

[5]   Chavakis, E., Aicher, A., Heeschen, C., Sasaki, K., Kaiser, R., El Makhfi, N., Urbich, C., Peters, T., Scharffetter- Kochanek, K., Zeiher, A.M., Chavakis, T. and Dimmeler, S. (2005) Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells. Journal of Experimental Medicine, 201(1), 63-72.

[6]   Vajkoczy, P., Blum, S., Lamparter, M., Mailhammer, R., Erber, R., Engelhardt, B., Vestweber, D. and Hatzopoulos, A.K. (2003) Multistep nature of microvascular recruitment of ex vivo-expanded embryonic endothelial progenitor cells during tumor angiogenesis. Journal of Experimental Medicine, 197(12), 1755-1765.

[7]   Nishiwaki, Y., Yoshida, M., Masuda, H. and Isobe, M. (2004) Recruitment of bone marrow-derived endothelial progenitor cells to vascular endothelium involves E-selectin dependent mechanism. Cardiovascular Pathology, 13(3), 171.

[8]   Nishiwaki, Y., Yokota, T., Hiraoka, M., Miyagishi, M., Taira, K., Isobe, M., Mizusawa, H. and Yoshida, M. (2003) Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion. Biochemical and Biophysical Research Communications, 310(4), 1062-1066.

[9]   Yoshida, M. and Gimbrone, M.A. Jr. (1997) Novel roles for E-selectin in endothelial-leukocyte adhesion. Annals of the New York Academic Sciences, 811, 493-497.

[10]   Yoshida, M., Szente, B.E., Kiely, J.M., Rosenzweig, A. and Gimbrone, M. Jr. (1998) Phosphorylation of the cytoplasmic domain of E-selectin is regulated during leukocyte-endothelial adhesion. The Journal of Immunology, 161(2), 933-941.

[11]   Bevilacqua, M.P., Pober, J.S., Mendrick, D.L., Cotran, R.S. and Gimbrone, M.A. (1987) Identification of an inducible endothelial-leukocyte adhesion molecule. Proceedings of the National Academy Sciences of USA, 84(24), 9238-9242.

[12]   Luscinskas, F.W., Cybulsky, M.I., Kiely, J.M., Peckins, C.S., Davis, V.M. and Gimbrone, M.A. (1991) Cytokine- activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. The Journal of Immunology, 146(5), 1617-1625.

[13]   Gerszten, R.E., Garcia-Zepeda, E.A., Lim, Y.C., Yoshida, M., Ding, H.A., Gimbrone, M. Jr., Luster, A.D., Luscinskas, F.W. and Rosenzweig, A. (1999) MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature, 398(6729), 718-723.

[14]   Kiely, J.M., Luscinkas, F.W. and Gimbrone, M. Jr. (1999) Leukocyte-endothelial monolayer adhesion assay (static conditions). Methods in Molecular Biology, 96, 131-136.

[15]   Gerszten, R.E., Luscinskas, F.W., Ding, H.T., Dichek, D.A., Stoolman, L.M., Gimbrone, M.A. Jr. and Rosenz- weig, A. (1996) Adhesion of memory lymphocytes to vascular cell adhesion molecule-1—transduced human vascular endothelial cells under simulated physiological flow conditions in vitro. Circulation Research, 79(6), 1205-1215.

[16]   Luscinskas, F.W., Kansas, G.S., Ding, H., Pizcueta, P., Schleiffenbaum, B.E., Tedder, T.F. and Gimbrone, M. Jr. (1994) Monocyte rolling, arrest and spreading on IL-4- activated vascular endothelium under flow is mediated via sequential action of L-selectin, β1-integrins, and β2-integrins. Journal of Cell Biology, 125(6), 1417-1427.

[17]   Bischoff, J. (1997) Cell adhesion and angiogenesis. Journal of Clinical Investigation, 99(3), 373-376.

[18]   Nguyen, M., Strubel, N.A. and Bischoff, J. (1993) A role for sialyl Lewis-X/A glycoconjugates in capillary morphogenesis. Nature, 365(6443), 267-269.

[19]   Kraling, B.M., Razon, M.J., Boon, L.M., Zurakowski, D., Seachord, C., Darveau, R.P., Mulliken, J.B., Corless, C.L. and Bischoff, J. (1996) E-selectin is present in proliferating endothelial cells in human hemangiomas. American Journal of Pathology, 148(4), 1181-1191.

[20]   Yu, Y., Flint, A.F., Mulliken, J.B., Wu, J.K. and Bischoff, J. (2004) Endothelial progenitor cells in infantile hemangioma. Blood, 103(4), 1373-1375.

[21]   Mazo, I.B., Gutierrez-Ramos, J.C., Frenette, P.S., Hynes, R.O., Wagner, D.D. and von Andrian, U.H. (1998) Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1. Journal of Experimental Medicine, 188(3), 465-474.

 
 
Top