CRISPR-mediated In Situ Deletion Of The PECAM-1 Cytoplasmic Domain:Effect On Border Localization And Endothelial Cell Junctional Integrity

Objective:PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of hematopoietic cells, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that the cytoplasmic tail of PECAM-1 has a potential multifunctional role in endothelial cells. The cytoplasmic domain of PECAM-1 is required for PECAM-1 to function as a mechanosensory complex in endothelial cells, to confer cytoprotection to proapoptotic stimuli, and to interact with other junctional adhesion proteins and cytoskeleton molecules. The above functions seem to be independent of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Given the abundance of residues in the PECAM-1 cytoplasmic domain, I hypothesize that other residues within the PECAM-1 cytoplasmic domain may be responsible for the above functions. Therefore, the goal of my project is to determine whether the cytoplasmic domain of PECAM-1 plays a role in endothelial cell-cell border localization and endothelial barrier function to confer junctional integrity to the vasculature.Methods:PECAM-1 null immortalized human umbilical vein endothelial cells (iHUVECs) were generated using CRISPR gene editing technology (PE-KO), two sgRNAs were designed to achieve in situ deletion of the PECAM-1 cytoplasmic tail, resulting in iHUVEC variant cells referred to as (?CD-PECAM-1) iHUVECs. Since endothelial cells have low transfection efficiency, Lentiviruses harboring these two CRISPR guides and Cas9 nuclease cDNA were transduced into iHUVECs. After puromycin enrichment, transduced iHUVECs were sorted into single clones, which were characterized by FACS analysis using antibodies specific for the extracellular and cytoplasmic domains of PECAM-1. The function of ?CD-PECAM-1 was then characterized using confocal microscopy, Electric Cell-substrate Impedance Sensing (ECIS-a real-time, label-free, impedance-based method to study the barrier function of endothelial cells grown in tissue culture), and PECAM-1/IgG binding for its ability to support border localization, barrier function and homophilic interactions.Results:Two unique lines of iHUVECs-one completely lacking PECAM-1, and the other expressing ACD-PECAM-1-were successfully generated and characterized Confocal microscopy showed ACD-PECAM-1 can localize to the border of confluent endothelial cells. iHUVECs expressing ACD-PECAM-1 exhibited higher baseline resistance compared to WT PECAM-1-expressing cells, PE-KO had significantly lower baseline resistance. ACD-PECAM-1-expressing iHUVECs showed much faster rate of recovery following thrombin-induced disruption of endothelial cell barrier function, while PECAM-1-null iHUVECs had a slower rate of recovery. Finally, ACD-PECAM-1 expressed on the surface of iHUVECs showed significantly lower affinity for PECAM-1/IgG compared with WT PECAM-1-expressing cells.Conclusions:The cytoplasmic domain of PECAM-1 does not appear to be required for the ability of this cell adhesion and signaling receptor to become enriched at endothelial cell-cell borders. Interestingly, ACD-PECAM-1 appears to have lower homophilic binding affinity for other PECAM-1 molecules, leading to enhanced baseline barrier function and faster restoration of endothelial cell junctional integrity following thrombin challenge. Taken together, these data are consistent with the cytoplasmic domain forming connections with elements of the endothelial cell cytoskeleton that serve to restrict its mobility within the plane of the endothelial plasma membrane that when freed, results in a mobile receptor with increased ability to support the integrity of the endothelial cell-cell junction.