Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor

Abstract

Focal adhesion kinase (FAK) is known to mediate endothelial cell adhesion and migration in response to vascular endothelial growth factor (VEGF). The aim of this study was to explore a potential role for FAK in VEGF regulation of microvascular endothelial barrier function. The apparent permeability coefficient of albumin (Pa) was measured in intact isolated porcine coronary venules. Treating the vessels with VEGF induced a time- and concentration-dependent increase in Pa. Inhibition of FAK through direct delivery of FAK-related non-kinase (FRNK) into venular endothelium did not alter basal barrier function but significantly attenuated VEGF-elicited hyperpermeability. Furthermore, cultured human umbilical vein endothelial monolayers displayed a similar hyperpermeability response to VEGF which was greatly attenuated by FRNK. Western blot analysis showed that VEGF promoted FAK phosphorylation in a time course correlating with that of venular hyperpermeability. The phosphorylation response was blocked by FRNK treatment. In addition, VEGF stimulation caused a significant morphological change of FAK from a punctate pattern to an elongated, dash-like staining that aligned with the longitudinal axis of the cells. Taken together, the results suggest that FAK contributes to VEGF-elicited vascular hyperpermeability. Phosphorylation of FAK may play an important role in the signal transduction of vascular barrier response to VEGF.

Figure 1. Topical application of vascular endothelial growth factor (VEGF; 10⁻¹⁰m) significantly increased albumin permeability in intact perfused coronary venules under control conditions

Inhibition of focal adhesion kinase (FAK) by direct delivery of FAK-related non-kinase (FRNK) peptide into endothelial cells of the venules attenuated VEGF-induced hyperpermeability in a dose-related manner. Numbers in parentheses represent the numbers of vessels studied. * Significant difference vs. basal; † significant difference vs. vehicle control (TranIt only).

Figure 2. VEGF (10⁻¹⁰m) induced an increase in albumin flux across cultured human umbilical vein endothelial cell monolayers in the same time course (3–5 min) as seen in venules

The hyperpermeability response was reduced in cells pre-treated with FRNK; n, number of dishes studied. * Significant difference vs. basal; † significant difference vs. vehicle control.

Figure 3. Western blots showing the effect of VEGF (10⁻¹⁰m) on tyrosine phosphorylation of FAK

A, human umbilical vein endothelial cells were stimulated with VEGF for different times and then lysed. The lysates were immunoprecipitated with an anti-phosphotyrosine antibody and followed by blotting with an anti-FAK antibody. B, cells were treated for 3–5 min with different concentrations of VEGF. Immunoprecipitation followed by immunoblotting was performed in cell lysates as described above. The second panel in B shows the protein content of FAK in each group. C, cells were pretreated with protein transfer vehicle alone or in combination with FRNK (1 or 3 μg) and then stimulated with VEGF (10⁻¹⁰m). Bar graphs show statistically evaluated optical densities of the protein bands. * Significant difference vs. basal.

Figure 4. Immunofluorescence staining of FAK in human umbilical vein endothelial cell monolayers

A, control cells under the basal condition. B, cells exposed to VEGF (10⁻¹⁰m) for 5 min. C, FRNK (3 μg ml⁻¹)-pretreated cells subjected to the same concentration of VEGF. Note that the upon VEGF stimulation FAK staining changed from a punctate pattern to an elongated, dash-like staining which seems to align with the longitudinal axis of the cells. The morphological change was diminished in cells transfected with FRNK.