The role of gravin in endothelial wound healing

During vascular wound healing, endothelial cells undergo several changes, including increased proliferation, increased migration and altered gene expression Gravin, a 300 kDa, intracellular protein, is upregulated in endothelial cells under conditions that promote cell spreading and migration and is upregulated in cells at the wound edge in wounded endothelial cell monolayers. To test the hypothesis that this protein may play a role in cell migration during wound healing, we examined the effect on wound heating of inhibiting gravin expression using an antisense oligonucleotide (20 mer) complementary to the 3'UTR of the gravin mRNA. In all experiments, confluent cultures of human umbilical vein endothelial cells (HUVEC) were treated with either the antisense oligonucleotide or a missense control oligonucleotide (0 5 and 0.1 u,M) in the presence of 10 ng/ml Lipofectin in OptiMEM for 4 hr. Uptake of the oligonucleotides by endothelial cells under the treatment conditions was confirmed using a FITC-labelled 20 mer. Western blotting revealed that the antisense oligonucleotide inhibited gravin protein expression to approximately 50% ofthat in missense treated cells. The effect of the antisense oligonucleotide on wound healing was determined by wounding confluent HUVEC monolayers with a pasteur pipette after treatment with the antisense and control oligonucleotides as described above and measuring wound width immediately after wounding and at 1718 hr after wounding. Wound closure was significantly inhibited in cultures treated with the antisense oligonucleotide when compared to those treated with the control oligonucleotide. We conclude from these data that gravin expression is important in wound healing and may play role in cell adhesion and migration (Supported by an American Heart Association Grant-in-Aid).