Nanoscale obstacle arrays frustrate transport of EphA2-Ephrin-A1 clusters in cancer cell lines.

Juxtacrine signaling interactions between the EphA2 receptor tyrosine kinase and its ephrin-A1 ligand contribute to healthy tissue maintenance and misregulation of this system is observed in at least 40% of human breast cancer. Hybrid live cell-supported membrane experiments in which membrane-linked ephrin-A1 displayed in supported membranes interacts with EphA2 in living cells have revealed large scale clustering of EphA2/ephrin-A1 complexes as well as their lateral transport across the cell surface during triggering. Here, we utilize 100 nm spaced hexagonally ordered arrays of gold nanodots embedded within supported membranes to present defined obstacles to the movement and assembly of EphA2 clusters. By functionalizing both the supported membrane and the nanodots with ephrin-A1, we perform a type of affinity chromatography on EphA2 signaling clusters in live cell membranes. Analysis of 10 different breast cancer cell lines reveals that EphA2 transport is most frustrated by nanodot arrays in the most diseased cell lines. These observations suggest that strong physical association among EphA2 receptors, as well as their assembly into larger clusters, correlates with and may contribute to the pathological misregulation of the EphA2/ephrin-A1 pathway in breast cancer.

Intramyocardial administration of chimeric ephrinA1-Fc promotes tissue salvage following myocardial infarction in mice.

The purpose of this study was to investigate the role of intramyocardial administration of chimeric ephrinA1-Fc in modulating the extent of injury and inflammation in non reperfused myocardial infarction (MI). Our results show that intramyocardial injection of 6 µg ephrinA1-Fc into the border zone immediately after permanent coronary artery ligation in B6129s mice resulted in 50% reduction of infarct size, 64% less necrosis, 35% less chamber dilatation and 32% less left ventricular free wall thinning at 4 days post-MI. In the infarct zone, Ly6G+ neutrophil density was 57% reduced and CD45+ leukocyte density was 21% reduced. Myocyte damage was also reduced in ephrinA1-Fc-treated hearts, as evidenced by 54% reduced serum cardiac troponin I. Further, we observed decreased cleaved PARP, increased BAG-1 protein expression, increased phosphorylated AKT/total AKT protein, and reduced NF-κB protein with ephrinA1-Fc administration, indicating improved cellular survival. Of the eight EphA receptors known to be expressed in mice (A1­A8), RT-PCR revealed that A1­A4, A6 and A7 were expressed in the uninjured adult myocardium. Expression of EphA1­A3 and EphA7 were significantly increased following MI while EphA6 expression decreased. Treatment with ephrinA1-Fc further increased EphA1 and EphA2 gene expression and resulted in a 2-fold increase in EphA4. Upregulation and combinatorial activation of these receptors may promote tissue survival. We have identified a novel, beneficial role for ephrinA1-Fc administration at the time of MI, and propose this as a promising new target for infarct salvage in non reperfused MI. More experiments are in progress to identify receptor-expressing cell types as well as the functional implications of receptor activation.