Influence of cold preservation on the cytoskeleton of cultured pulmonary arterial endothelial cells.

Abstract

In donor lungs preserved for transplantation, pulmonary arterial endothelial cells become thin and partially detached from the basement membrane at 4 degrees C, recovering slowly after transplantation. These changes have now been modeled in vitro. Porcine pulmonary arterial endothelial cell monolayers were incubated at 4 degrees C for 2 or 4 h, rewarmed to 37 degrees C, and incubated for up to 24 h. Responses were studied using wound healing assays, bead phagocytosis, immunostaining of cytoskeletal components, and quantification of actin by SDS-PAGE and immunoblotting. Cooling caused cessation of cell movement and phagocytosis associated with depolymerization of the cytoskeleton. Depolymerization of microtubules was complete after 2 h but 14.6% of actin filaments remained (SDS-PAGE) after 4 h at 4 degrees C. Loss of actin stress fibers paralleled the disappearance of vinculin/talin co-labeling focal adhesions. However, a fiber network at the inner surface of the cell membrane labeling for talin was stable at 4 degrees C. After rewarming, the rate of cell movement and phagocytosis immediately returned to normal. Actin filaments and thin stress fibers were present by 1 h, although poorly organized, and actin had increased to 68.2% of control. Many small vinculin/talin focal adhesions had formed. Microtubules redeveloped by 1 h. The cytoskeleton of cultured human pulmonary arterial endothelial cells showed similar changes. In conclusion, the cytoskeletal changes help explain in vivo observations. On rewarming, the endothelial cells appeared to recover rapidly, but the abnormal appearance of the reformed cytoskeleton suggests an interim period of instability which may have metabolic and mechanical consequences.