Human Primary Cells
Pre-screened Human Endothelial Cells
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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.

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.

endothelial cell angiogenesis VEGF signaling phospho VEGFR2 Tyrosine 1175 Phospho-VEGFR2 (Tyr1175) Antibody
endothelial cell angiogenesis VEGF signaling VEGFR2 VEGFR2 Antibody

B.

endothelial cell angiogenesis VEGF signaling phospho Akt Serine 473 Phospho-Akt (Ser473) Antibody
endothelial cell angiogenesis VEGF signaling Akt Akt Antibody

C.

endothelial cell angiogenesis VEGF signaling phospho p44 p42 Phospho-p44/42 MAPK Antibody
endothelial cell angiogenesis VEGF signaling p44 p42 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.

HMVEC

HUVEC

A.

endothelial cell angiogenesis VEGF signaling Tie2 Tie2 Antibody

B.

endothelial cell angiogenesis VEGF signaling eNOS eNOS Antibody

C.

endothelial cell angiogenesis VEGF Axl Axl Antibody

D.

endothelial cell angiogenesis VEGF signaling Etk BMX Etk/BMX Antibody
VEGF (100ng/ml)
References: Each lot tested negative for HIV, Hepatitis B and Hepatitis C and negative for mycoplasma, bacteria, yeast, fungi.
Product Name Product Description Size (vol)

Cat. No.

Pre-Screened HUVEC Total Kit neonatal

Each kit contains an ampoule of cryopreserved HUVEC (S200-05n), 500 ml of Endothelial Cell Growth Medium (211-500), and a Subculture Reagent Kit (090K).

1 Kit 095S200K-05n
Cryopreserved Pre-Screened HUVEC neonatal (>500,000 cells in an ampoule) Cryopreserved in ampoules at the end of primary culture in Endothelial Cell Basal Medium that contains 10% FBS and 10% DMSO. 1 Ampoule 095S200-05n
Endothelial Cell Growth Medium

Optimized and ready for use

500 ml 095211-500
Endothelial Cell Growth Medium Kit Basal Medium & Growth Supplement packaged separately Yields 500 ml 095211K-500
Endothelial Cell Basal Medium Add Growth Supplement before use 500 ml 095210-500
Endothelial Cell Growth Supplement 15 ml 095211-GS
Subculture Reagent Kit 100ml each of HBSS, Trypsin/EDTA & Trypsin Neutralizing Solution 100 ml 095090K
Pre-Screened CADMECµ Total Kit adult neonatal

Each kit contains an ampoule of cryopreserved Pre-Screened CADMECµ (S100-05), 500 ml of CADMECµ Cell Growth Medium (112-500), a Subculture Reagent Kit (090K), two Extracellular Matrix Attachment Factor Coated T-25 Flasks (121-25-02), and 100 ml of Attachment Factor Solution (123-100).

1 Kit 095S100K-05a

095S100K-05n
Cryopreserved Pre-Screened CADMECµ adult neonatal (>500,000 cells in an ampoule) Cryopreserved at second passage in CADMECµ Cell Basal Medium that contains 10% FBS and 10% DMSO. 1 Ampoule 095S100-05a

095S100-05n
CADMECµ Growth Medium

Optimized and ready for use

500 ml 095112-500
CADMECµ Growth Medium Kit Basal Medium & Growth Supplement packaged separately Yields 500 ml 095112K-500
CADMECµ Basal Medium Add Growth Supplement before use 500 ml 095110-500
CADMECµ Growth Supplement 30 ml 095112-GS
Subculture Reagent Kit 100 ml each of HBSS, Trypsin/EDTA & Trypsin Neutralizing Solution 100 ml 095090K
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  3. Ortega, N. et al, Front. Biosci. 4:D141 (1999).
  4. Peters, K. G. et al, Recent Prog. Horm. Res. 59:51 (2004).
  5. Jones, N. et al, J. Cancer Metastasis Rev. 19:13 (2000).
  6. Ellis, A. et al, Can. J. Physiol. Pharmacol. 81:1013 (2003).
  7. Kondo, T. et al, Mol. Cell. Biochem. 264:25 (2004).
  8. Melaragno, M.G. et al, Trends Cardiovasc. Med. 9:250 (1999).
  9. OµDonnell, K. et al, Am. J. Pathol. 154:1171 (1999).
  10. Hasanbasic, I. et al, Am. J. Physiol. Heart Circ. Physiol. 287:H1207 (2004).
  11. Gallicchio, M. et al, Blood (2004).
  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).
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