Novel Peptides Activate GRP78 Heat Shock Protein Receptor as a New Target for Angiogenesis

Background: Impaired angiogenesis is one of the features of ischemic diseases. For the last decade the most established target for therapeutic angiogenesis has been VEGF and its receptors. However clinical trials to alleviate ischemia have been disappointing indicating the need for new molecules and molecular targets. We have identified a heat shock protein GRP78 on endothelial cell membranes as a novel target for angiogenesis that is independent of VEGF. We have demonstrated that binding to this target by a synthetic 12 amino acid peptide RoY induced therapeutic angiogenesis. A single local administration of the peptide significantly restored blood perfusion in mice post femoral artery excision. Recently, we have also demonstrated that intravenous administration of the peptide to mice with acute myocardial infarction resulted in increased capillary density in the ischemic tissue. Our aim was to study membrane GRP78 as a new target for angiogenesis and to identify its natural ligand.

Results: FACS analysis of endothelial cell cultures demonstrated that membrane GRP78 is increased under hypoxic conditions. In order to identify a natural ligand to GRP78, we used protein data base search. ADAM15, a disintegrin and metalloprotease glycoprotein was found to contain four out of twelve identical amino acids to the synthetic RoY peptide. We have therefore synthesized three peptides from the metalloprotease domain and studied endothelial cell binding, proliferation, migration and tube formation under hypoxia in the presence of these peptides. One of the peptides, termed ADoPep1, exhibited a significant angiogenic activity invitro and restored blood perfusion in a mouse hind limb ischemia model. Histological examinations showed an increase in capillary density, a decrease in GRP78 positive cells and apoptotic cells one week post treatment of ischemic leg with ADoPep1. We demonstrated that ADoPep1 inhibited endothelial cell hypoxia-induced apoptosis but not CoCl₂ induced apoptosis. We therefore suggest a possible mechanism of angiogenesis by inhibition of hypoxia-induced apoptosis is manifested through GRP78 receptor induced by ADAM15 derived peptide.

Conclusions: The peptide derived from ADAM15 induces angiogenesis by the activation of membrane GRP78 under hypoxia which may lead to the development of novel therapeutics for ischemic diseases. This study contributes to the search for new molecules that activates a new target for therapeutic angiogenesis in ischemic diseases.