Human Pre-Screened Endothelial Cells

Pre-Screened Endothelial Cells:

VEGF Signaling Pathway Tested Umbilical Vein Endothelial Cells, Microvascular Endothelial Cells, Aortic Endothelial Cells

Endothelial cells are important parts of blood and lymph vessels. They play a pivotal role in a diverse range of physiological and pathophysiological processes from working as selective barrier between the blood and the surrounding bodily tissue to regulation of angiogenesis. They are closely involved in arterial disease and cancer development.

The most commonly used sources of human endothelial cells to study specific characteristics are Human Umbilical Vein Endothelial Cells, HUVEC, isolated from the vein of the umbilical cord, Human Microvascular Endothelial Cells, HMVEC,  isolated from capillaries of the dermis and Human Aortic Endothelial Cells, HAOEC, isolated from capillaries of the aorta. At Cell Applications, primary cultures of HUVEC and HMVEC were pre-screened for angiogenesis and other endothelial cell signaling studies.

 

VEGF-Stimulated Signaling in Pre-Screened HUVEC, HMVEC and HAOEC

VEGF receptor-2 (VEGFR-2) is a major receptor transducing the signal of VEGF into the cells. Its expression is almost exclusively restricted to cells of endothelial lineage. VEGFR-2-mediated signaling plays a critical role in regulation of proliferation, differentiation, cell movement, and survival of endothelial cells and angiogenesis (1). VEGF-induced receptor dimerization triggers activation of VEGFR-2 tyrosine kinase and autophosphorylation at a specific set of tyrosine residues, which serve as docking sites for downstream signaling components containing SH2 or PTB domains. A regulated cascade of protein-protein interaction lead to activation of important signaling pathways within cells, such as Akt and MAP kinase activation (2, 3).

     

	Pre-Screened HUVEC	Pre-Screened HMVEC
A.			Phospho-VEGFR2 (Tyr1175) Antibody
			VEGFR2 Antibody
B.			Phospho-Akt (Ser473) Antibody
			Akt Antibody
C.			Phospho-p44/42 MAPK Antibody
			p44/42 MAPK Antibody
VEGF (100ng/ml)

      Fig. 1. Activation of VEGFR-2 signaling in Pre-Screened HUVEC and HMVEC by VEGF.  Pre-Screened HUVEC and HMVEC were cultured in CAI Endothelial Cell Growth Medium and CADMEC Growth Medium respectively. When cell density reaches 70-80% confluence, CAI HUVEC and HMVEC were maintained in CAI Endothelial Cell Serum-Free Defined Mediums for 16 hours and starved in CAI Endothelial Cell Starvation Medium for 4 hours prior to stimulation with VEGF (100 ng/ml) for 5 minutes or 15 minutes respectively. Cell lysates were prepared and subjected to Western blot analysis. (A) VEGFR-2 autophosphorylation was probed using a phospho-VEGFR-2 (Y1175) specific antibody. (B) Akt activation was detected using a phospho-Akt (S473) specific antibody. (C) MAP kinase activation was detected using a phospho-p44/42 MAP kinase (T202/Y204) antibody. The results showed that VEGF stimulated significant VEGFR-2 phosphorylation and downstream Akt and MAP kinase activation in Pre-Screened HUVEC and HMVEC.

     Detection of Expression of Important Endothelial Cell Signaling Molecules in Pre-Screened HUVEC and HMVEC

Tie2 is another important receptor tyrosine kinase critical for functional expression of endothelial cells and regulation of angiogenesis. Coordinated expression of Tie2 and its ligand angiopoietins is required for the angiogenic remodeling and vessel stabilization processes that occur subsequently to the initial vasculogenic action of VEGF receptors (4). It was demonstrated that Tie2-mediated signaling can effect changes in angiogenic sprout formation and survival of endothelial cells. Thus, Tie2 is also an important anti-angiogenesis drug target (5). The expression of Tie2 was detected in CAI pre-screened HUVEC and HMVEC cells as shown in Fig. 2, panel A.

 

Nitric oxide (NO) is an important signaling molecules that acts in many tissues to regulate a diverse range of physiological events and participates in various disease processes (6). NO is produced by the endothelial nitric oxide synthase (eNOS). Activation of eNOS leads to production of NO in endothelial cells, which regulates functional expression of endothelial cells in vascular remodeling and angiogenesis as well as cell survival (7). Abnormal control of NO synthesis has been implicated in a variety of diseases. Thus eNOS is an important endothelial cell signaling component target for drug development. The expression of eNOS was detected in CAI pre-screened HUVEC and HMVEC cells as shown in Fig. 2, panel B.

 

Axl is a new receptor tyrosine kinase family member. The survival factor growth arrest-specific gene 6 product (Gas6) is Axl ligand. Axl and Gas6 are both expressed in endothelial cells, where they are involved in many processes such as leukocyte transmigration through capillaries and neointima formation in injured vessels (8). Gas6 bound to HUVECs, protected HUVECs from apoptosis in response to growth factor withdrawal and from TNFalpha-mediated cytotoxicity. It was suggested Gas 6-Axl signaling may be relevant to endothelial cell survival in the quiescent environment of the vessel wall (9, 10). In addition, Axl stimulation by Gas6 results in inhibition of the ligand-dependent activation of vascular endothelial growth factor (VEGF) receptor 2 (11). There is a cross talk between Axl and VEGF receptor 2 in angiogenesis regulation. The expression of Axl receptor tyrosine kinase was detected in CAI pre-screened HUVEC and HMVEC cells as shown in Fig. 2, panel C.

 

The endothelial/epithelial tyrosine kinase (Etk/Bmx), a member of the Btk nonreceptor tyrosine kinase family, has been implicated in cell adhesion, migration, proliferation, and survival. Etk participates in signal transduction stimulated by growth factor receptors, cytokine receptors, G-protein-coupled receptors, antigen receptors, and integrins (12, 13). Etk has been implicated in the signaling of Tie-2 and VEGF receptors (14). Recently, it was demonstrated Etk is one of key players in regulation of TNF-induced inflammatory angiogenesis (15). The expression of Etk/Bmx tyrosine kinase was detected in CAI pre-screened HUVEC and HMVEC cells as shown in Fig. 2, panel D.

    

Fig. 2. Expression of various cell signaling proteins in CAI HUVEC and HMVEC.  Cell lysates of CAI HUVEC and HMVEC were prepared and directly subjected to Western blot analysis using  different specific antibodies.

 

References:

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8. Melaragno, M.G. et al, Trends Cardiovasc. Med. 9:250 (1999).

9. O’Donnell, K. et al, Am.  J. Pathol. 154:1171 (1999).

10. Hasanbasic, I. et al, Am. J. Physiol. Heart Circ. Physiol. 287:H1207 (2004).

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12. Chen, R. et al, Nat. Cell Biol. 3: 439 (2001).

13. Mao, J. et al, EMBO J . 17:5638 (1998).

14. Zhang, R. et al, J. Biol. Chem. 278: 51267 (2003).

15. Pan, S. et al, Mol. Cell. Biol. 22: 7512 (2002).