Angiopoietin-1 Reduces VEGF-Stimulated Leukocyte Adhesion to Endothelial Cells by Reducing ICAM-1, VCAM-1, and E-Selectin Expression

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.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1. Ang1 suppresses VEGF-induced mRNA levels of ICAM-1, VCAM-1, and E-selectin in HUVECs. A, HUVECs were incubated for 4 hours with the indicated dose of VEGF₁₆₅. Yeast total RNA (10 μg) was similarly assayed in the presence of RNase (T). To clarify the identity of the bands, ICAM-1 (I), VCAM-1 (V), and E-selectin (E) probes were applied individually to the total RNA from HUVECs treated with VEGF for 4 hours to reveal protected bands of 367, 279, and 187 bp, respectively. B, HUVECs were incubated for 4 hours and 6 hours with control buffer (CB), Ang1 (A1, 200 ng/mL), VEGF₁₆₅ (VE, 20 ng/mL), or Ang1 plus VEGF₁₆₅ (AV). C, HUVECs were incubated for 4 hours with control buffer (CB), Ang1 (A1, 200 ng/mL), VEGF₁₆₅ (VE, 20 ng/mL), or Ang1 plus VEGF₁₆₅ (AV) with or without 5-fold molar excess of rTie2-Fc (T2) or rTie1-Fc (T1). D, HUVECs were incubated for 4 hours with control buffer (CB), wortmannin (WT, 30 nmol/L), VEGF₁₆₅ (VE, 20 ng/mL), wortmannin plus VEGF₁₆₅ (WV), Ang1 plus VEGF₁₆₅ (AV), or Ang1 plus VEGF₁₆₅ plus wortmannin (AVW). Total RNAs (10 μg) were subjected to multiplex RPA probed with 4 antisense RNAs: ICAM-1, VCAM-1, E-selectin, and cyclophilin. Bottom panels, Densitometric analyses are presented as the relative ratio of ICAM-1, VCAM-1, or E-selectin mRNA to cyclophilin mRNA. The relative ratio measured in the CB is arbitrarily presented as 1. Bars represent the mean±SD from 4 experiments. *P<0.05 vs 0 ng/mL of VEGF₁₆₅ or CB; +P<0.05 vs VE; and #P<0.05 vs AV.

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).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2. Ang1 suppresses VEGF-induced protein levels of ICAM-1, VCAM-1, and E-selectin in HUVECs. HUVECs were incubated for 6 hours with control buffer (CB), Ang1 (A1, 200 ng/mL), VEGF₁₆₅ (VE, 20 ng/mL), or Ang1 plus VEGF₁₆₅ (AV). Each lane contains 50 μg of total protein from the cells. A through C, Western blots were probed with appropriate antibody (top panels). The blots were reprobed with an anti–actin antibody (bottom panels). D, Densitometric analyses are presented as the relative ratio of ICAM-1:actin, VCAM-1:actin, and E-selectin:actin. The relative ratio in the CB is arbitrarily presented as 1. Bars represent the mean±SD from 5 experiments. *P<0.05 vs CB; +P<0.05 vs VE.

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.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3. Ang1 suppresses VEGF-induced HUVEC adhesiveness. A, Representative phase-contrast photographs of leukocyte adhesion to HUVECs. Note that there are more leukocytes in HUVECs treated with VEGF than in those treated with control buffer (CB), Ang1, or Ang1 plus VEGF (A+V). Bar=50 μm. B, Quantification of the leukocyte adhesion to HUVECs. Leukocytes were labeled with Vybrant DiD (Molecular Probes) and added to confluent monolayers of HUVECs, which were treated with and without VEGF₁₆₅ (20 ng/mL) for 8 hours and were also treated with CB, Ang1 (A1, 200 ng/mL) with or without 5-fold molar excess of rTie2-Fc (Tie2) or rTie1-Fc (Tie1), and anti–ICAM-1 antibody (IA, 10 μg/mL), anti–VCAM-1 antibody (VA, 10 μg/mL), anti–E-selectin antibody (SA, 10 μg/mL), or a triple combination of these antibodies (TA). Then, leukocyte adhesion was measured as described in Materials and Methods. Bars represent the mean±SD from 5 experiments. *P<0.05 vs CB; +P<0.05 vs CB plus VEGF₁₆₅ (20 ng/mL).

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.