Danying Liao1, Heng Mei2, Yu Hu2, Debra K Newman3, Peter J Newman4. 1. Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI, United States; Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. 2. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. 3. Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI, United States; Department of Pharmacology, Medical College of Wisconsin, Milwaukee, United States; Department of Microbiology, Medical College of Wisconsin, Milwaukee, United States; The Cardiovascular Center, Medical College of Wisconsin, Milwaukee, United States. 4. Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI, United States; Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China; Department of Pharmacology, Medical College of Wisconsin, Milwaukee, United States; Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, United States; The Cardiovascular Center, Medical College of Wisconsin, Milwaukee, United States. Electronic address: peter.newman@bcw.edu.
Abstract
AIMS: PECAM-1 is an abundant endothelial cell surface receptor that becomes highly enriched at endothelial cell-cell junctions, where it functions to mediate leukocyte transendothelial migration, sense changes in shear and flow, and maintain the vascular permeability barrier. Homophilic interactions mediated by the PECAM-1 extracellular domain are known to be required for PECAM-1 to perform these functions; however, much less is understood about the role of its cytoplasmic domain in these processes. MAIN METHODS: CRISPR/Cas9 gene editing technology was employed to generate human endothelial cell lines that either lack PECAM-1 entirely, or express mutated PECAM-1 missing the majority of its cytoplasmic domain (∆CD-PECAM-1). The endothelial barrier function was evaluated by Electric Cell-substrate Impedance Sensing, and molecular mobility was assessed by fluorescence recovery after photobleaching. KEY FINDINGS: We found that ∆CD-PECAM-1 concentrates normally at endothelial cell junctions, but has the unexpected property of conferring increased baseline barrier resistance, as well as a more rapid rate of recovery of vascular integrity following thrombin-induced disruption of the endothelial barrier. Fluorescence recovery after photobleaching analysis revealed that ∆CD-PECAM-1 exhibits increased mobility within the plane of the plasma membrane, thus allowing it to redistribute more rapidly back to endothelial cell-cell borders to reform the vascular permeability barrier. SIGNIFICANCE: The PECAM-1 cytoplasmic domain plays a novel role in regulating the rate and extent of vascular permeability following thrombotic or inflammatory challenge.
AIMS: PECAM-1 is an abundant endothelial cell surface receptor that becomes highly enriched at endothelial cell-cell junctions, where it functions to mediate leukocyte transendothelial migration, sense changes in shear and flow, and maintain the vascular permeability barrier. Homophilic interactions mediated by the PECAM-1 extracellular domain are known to be required for PECAM-1 to perform these functions; however, much less is understood about the role of its cytoplasmic domain in these processes. MAIN METHODS: CRISPR/Cas9 gene editing technology was employed to generate human endothelial cell lines that either lack PECAM-1 entirely, or express mutated PECAM-1 missing the majority of its cytoplasmic domain (∆CD-PECAM-1). The endothelial barrier function was evaluated by Electric Cell-substrate Impedance Sensing, and molecular mobility was assessed by fluorescence recovery after photobleaching. KEY FINDINGS: We found that ∆CD-PECAM-1 concentrates normally at endothelial cell junctions, but has the unexpected property of conferring increased baseline barrier resistance, as well as a more rapid rate of recovery of vascular integrity following thrombin-induced disruption of the endothelial barrier. Fluorescence recovery after photobleaching analysis revealed that ∆CD-PECAM-1 exhibits increased mobility within the plane of the plasma membrane, thus allowing it to redistribute more rapidly back to endothelial cell-cell borders to reform the vascular permeability barrier. SIGNIFICANCE: The PECAM-1 cytoplasmic domain plays a novel role in regulating the rate and extent of vascular permeability following thrombotic or inflammatory challenge.