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Mechanism of cholesterol transport to the front of migrating endothelial cells treated with angiogenic growth factor

Plasma membrane microviscosity (PMMV) is a critical determinant of endothelial cell (EC) migration. We have reported that angiogenic growth factors induce polarization of PMMV in motile EC. Here we investigate the mechanism(s) responsible for motility‐related membrane polarization. Free cholesterol distribution in the plasma membrane was determined by filipin stain. Relocalization of free cholesterol to the front of migrating EC suggests that polarization results from the movement of non‐raft cholesterol. Immunocytochemical studies indicate that endoplasmic reticulum (ER) and Golgi membranes are not involved in trafficking of newly synthesized cholesterol to the cell front. Trapping of caveolin‐1 in the ER by progesterone, restricted cholesterol to the perinuclear region and inhibited growth factor‐stimulated migration. Caveolin‐1 knock‐down with siRNA gave a similar result, and confocal microscopy showed impaired stability of the F‐actin network at the leading edge of EC. We have evidence that P‐glycoprotein, an ABC transporter that binds caveolin‐1 and is associated with membrane cholesterol movement, is involved in PMMV polarization during EC migration. Cyclosporin A, a P‐glycoprotein inhibitor, prevents cholesterol trafficking to the front and abrogates growth factor‐stimulated motility. Our results suggest a mechanism in which caveolin‐1 carries free cholesterol to the plasma membrane at the rear of the migrating EC, and P‐glycoprotein directs it to the front to polarize PMMV and facilitate angiogenic growth factor‐stimulated EC migration.