Angiopoietin-1 Reduces VEGF-Stimulated Leukocyte Adhesion to Endothelial Cells by Reducing ICAM-1, VCAM-1, and E-Selectin Expression
Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are potent vasculogenic and angiogenic factors that hold promise as a means to produce therapeutic vascularization and angiogenesis. However, VEGF also acts as a proinflammatory cytokine by inducing adhesion molecules that bind leukocytes to endothelial cells, an initial and essential step toward inflammation. In the present study, we used human umbilical vascular endothelial cells (HUVECs) to examine the effect of Ang1 on VEGF-induced expression of three adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Interestingly, Ang1 suppressed VEGF-induced expression of these adhesion molecules. Furthermore, Ang1 reduced VEGF-induced leukocyte adhesion to HUVECs. These results demonstrate that Ang1 counteracts VEGF-induced inflammation by reducing VEGF-induced endothelial adhesiveness.
Two endothelial cell–specific growth factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1), act cooperatively and interactively during prenatal and postnatal vascular development.1 In fact, co-overexpression of VEGF and Ang1 in mouse skin or rabbit ischemic hindlimb produces an additive increase in vessel formation.2,3 Although overexpression of VEGF alone in mouse skin produced profound angiogenesis, it also produced enhanced leukocyte rolling and adhesion, vascular leakage, and inflammation.2,4 Thus, VEGF is also a proinflammatory cytokine in addition to being an angiogenic factor. However, co-overexpression of VEGF and Ang1 in mouse skin showed less vascular leakage and inflammation compared with VEGF alone.2 These data clearly indicate that Ang1 counteracts some subset of activities of VEGF in endothelial cells.
We recently demonstrated that VEGF stimulates the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin mRNAs in endothelial cells through the Flk-1/KDR receptor.5 This stimulation is mediated through nuclear factor-κB (NF-κB) activation and is suppressed by phosphatidylinositol (PI) 3′-kinase.5 Other studies have shown that Ang1 strongly activates the PI 3′-kinase/Akt pathway in endothelial cells through its binding to the Tie2 receptor.6,7 Therefore, in this study, we assessed the specific role of Ang1 in VEGF-induced expression of adhesion molecules in endothelial cells. Interestingly, our results indicate that Ang1 counteracts VEGF-induced expression and activity of adhesion molecules.
Materials and Methods
Ang1*, rTie1-Fc, and rTie2-Fc were obtained from Regeneron Pharmaceuticals, Inc. Ang1* is a recombinant version of Ang1. Recombinant human VEGF165 was purchased from R&D Systems. Human umbilical vascular endothelial cells (HUVECs) were prepared and maintained as previously described.6
RNase protection assay (RPA), Western blot analyses, and flow cytometric analyses for human ICAM-1, VCAM-1, and E-selectin were performed as previously described.5 Leukocyte-endothelial adhesion was measured as previously described.5
All signals were visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film). Data are expressed as mean±SD. Statistical significance was tested using 1-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Results and Discussion
We developed a method of RPA by which we can simultaneously detect the mRNA levels of ICAM-1, VCAM-1, E-selectin, and cyclophilin. Because VEGF produces a maximum effect at 4 hours on expression of these adhesion molecules,5 we examined the effect of VEGF at this time point. VEGF stimulated expression of these adhesion molecules in a dose-dependent manner (Figure 1A). Ang1 (200 ng/mL) inhibited ≈23% to 29%, 46% to 48%, and 62% to 68% of the VEGF-induced ICAM-1, VCAM-1, and E-selectin mRNAs, respectively, at 4 hours and 6 hours, whereas Ang1 (200 ng/mL) by itself did not produce any significant effect on mRNA levels of these adhesion molecules (Figure 1B). A 5-fold molar excess of rTie2-Fc, but not rTie1-Fc, mostly blocked Ang1-induced suppression of VEGF-induced mRNA of adhesion molecules (Figure 1C). These results indicate that Ang1 exerts its effects on endothelial cells through mainly Tie2 receptor binding, but not through Tie1.
We previously demonstrated that VEGF stimulated the expression of ICAM-1, VCAM-1, and E-selectin mRNAs mainly through activation of phospholipase-Cγ and NF-κB.5 This induction was suppressed by activation of PI 3′-kinase.5 Because Ang1 is a strong activator of the intracellular PI 3′-kinase/Akt signaling system,6,7 Ang1-induced activation of PI 3′-kinase/Akt could be the main pathway for suppressing the VEGF-induced expression of adhesion molecules. As we expected, suppression of basal PI 3′-kinase by the pharmacological inhibitor wortmannin (30 nmol/L) induced expression of adhesion molecules (Figure 1D). Furthermore, addition of wortmannin (30 nmol/L) not only enhanced VEGF-induced expression of adhesion molecules but also reversed Ang1-induced suppressive effect on VEGF-induced expression of adhesion molecules (Figure 1D). Addition of another PI 3′-kinase inhibitor, LY294002 (100 nmol/L), produced the same results (data not shown). In fact, our preliminary results indicated that selective activation (or inactivation) of PI 3′-kinase/Akt using adenoviral transfer reduced (or enhanced) VEGF-induced expression of adhesion molecules (data not shown). Thus, Ang1 counteracts VEGF-induced expression of these adhesion molecules, possibly through activation of the PI 3′-kinase/Akt pathway.
We looked further at the protein levels of ICAM-1, VCAM-1, and E-selectin in HUVECs treated with Ang1 and VEGF. Consistent with the RPA data, Ang1 (200 ng/mL) by itself did not produce any significant effect, whereas VEGF (20 ng/mL) increased protein levels at 6 hours (Figure 2). Addition of Ang1 (200 ng/mL) inhibited ≈43%, 44%, and 62% of the VEGF-induced ICAM-1, VCAM-1, and E-selectin protein levels (Figure 2). Using flow cytometry, we also confirmed that Ang1 inhibited VEGF-induced expression of these adhesion molecules on the cell surface of HUVECs (data not shown).
Because the induction of adhesion molecules in endothelial cells induces leukocyte adhesion, we examined whether Ang1 reduces VEGF-induced leukocyte adhesion to HUVECs. Ang1 (200 ng/mL) by itself did not produce any effect, whereas VEGF (20 ng/mL) produced an increase of ≈2.8-fold in leukocyte adhesion after 8 hours compared with addition of control buffer (Figure 3). Ang1 (200 ng/mL) reduced ≈46% of the VEGF-induced leukocyte adhesion (Figure 3). A 5-fold molar excess of rTie2-Fc, but not rTie1-Fc, completely blocked Ang1-induced suppression of VEGF-induced leukocyte adhesion (Figure 3). These results indicate that Ang1 exerts its effects in endothelial cells through Tie2 receptor binding. Function-blocking antibodies to ICAM-1, VCAM-1, and E-selectin, either singly or as a triple combination, suppressed VEGF-induced leukocyte adhesion to varying extents (Figure 3). These data suggest that VEGF-induced adhesiveness requires combined activity of each of these adhesion molecules, because inhibition of the individual molecules could not completely impair the effects of VEGF.
To our knowledge, these results are the first to demonstrate that Ang1 can suppress the expression of adhesion molecules. Furthermore, a recent in vitro experiment demonstrated that Ang1 decreases basal and VEGF-induced endothelial permeability.8 Taken together, Ang1 counteracts VEGF-induced inflammation in endothelial cells while having an additive effect on vessel formation.2,3 Therefore, combined treatment with VEGF and Ang1 could be better than sole treatment with one for enhancing therapeutic vascularization and angiogenesis while avoiding inflammation.
This work was supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology. We thank Drs John S. Rudge and George D. Yancopoulos for enthusiastic support and for providing angiopoietin- and Tie-related reagents.
Original received March 13, 2001; resubmission received May 9, 2001; revised resubmission received July 31, 2001; accepted July 31, 2001.
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