Low-dose Endothelial Monocyte-activating Polypeptide-II Increases Permeability of Blood-tumor Barrier by Caveolae-mediated Transcellular Pathway

Low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase blood-tumor barrier (BTB) permeability via the paracellular pathway. The role of the transcellular pathway in this process is unclear. This study was conducted to evaluate the potential involvement of the transcellular pathway in EMAP-II-induced opening of the BTB and to identify the associated mechanisms. Evans blue extravasation test was used to measure changes in BTB permeability after EMAP-II (80 ng/kg) administration in a rat model of C6 glioma. Changes in the quantity of pinocytotic vesicles in rat brain microvascular endothelial cells (BMECs) were observed using transmission electron microscopy. Reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry assays were performed to detect the expression of the caveolar structural proteins, caveolin-1 and caveolin-2, in BMECs. Alterations in the expression of phospho (p)-Src, p-caveolin-1, and p-caveolin-2 and the activity of RhoA also were measured. Effects of tyrosine kinase inhibition on EMAP-II-induced RhoA/Rho kinase activations and tyrosine kinase, RhoA, or Rho kinase inhibition on EMAP-II-induced caveolin-1 and caveolin-2 phosphorylation were determined by inhibition studies. One hour after EMAP-II administration, the quantity of pinocytotic vesicles in BMECs increased markedly, consistent with changes in BTB permeability. The expression levels of caveolin-1, caveolin-2, p-caveolin-1, and p-caveolin-2 in BMECs also were significantly increased at 1 h. The peak expression level of p-Src and the peak activity of RhoA occurred at 0.25 and 0.5 h, respectively. Inhibition of tyrosine kinase significantly diminished the activities of RhoA and Rho kinase induced by EMAP-II. In addition, EMAP-II-induced phosphorylation of caveolin-1 and caveolin-2 was completely blocked by inhibition of tyrosine kinase, RhoA, or Rho kinase. We suggest that low-dose EMAP-II can induce BTB hyperpermeability via the transcellular pathway, which is associated with phosphorylation and upregulation of caveolin-1 and caveolin-2 and involves the tyrosine kinase/RhoA/Rho kinase signaling pathway.