CRISPR‐Cas9 knock out of gravin variant 1 impairs in vitro angiogenic sprouting

Abstract

Angiogenesis is a highly regulated physiological process that is controlled by several signaling pathways including Src dependent and cAMP dependent pathways. Currently, the mechanisms underlying the coordination and integration of these signaling pathways are unknown, but based on several knockdown studies, we postulate that gravin (AKAP12), which is expressed as two variants in endothelial cells, may serve as a scaffold to coordinate Src and cAMP dependent signaling during angiogenesis. To investigate this hypothesis, we are using gene editing and protein expression approaches to identify the role of each gravin variant and the role of the Src and PKA binding domains in gravin in angiogenesis. Here, we report on the effect of knocking out gravin variant 1 using CRISPR‐Cas9 on angiogenic sprouting in a 3D‐collagen gel model. Immortalized human umbilical vein endothelial cells (HUVEC‐TERT2) were transfected with a CRISPR‐Cas9 construct in which Cas9 was targeted to exon 3. An RFLP assay designed to identify changes in an HpyCH4III site at the targeted double stranded break point identified a positive clone which lacked the restriction site. Subsequent sequencing revealed a single base deletion at the targeted site in exon 3 of the AKAP12 gene. Reverse transcriptase PCR showed a partial loss of variant 1 mRNA and sequencing of the RT‐PCR product confirmed the single base deletion. Western blotting demonstrated loss of variant 1 in the CRISPR‐Cas9 modified cells while immunofluorescence microscopy using an anti‐gravin antibody that recognized the C‐terminal region of both variants revealed approximately a 50% decrease in fluorescence intensity in CRISPR‐Cas9 treated cells. This CRISPR modified cell line, expressing only variant 2 of gravin, showed altered cell morphology and behavior compared to wild type cells. When cultured on collagen gels, variant 1 knockout cultures showed significantly fewer sprouts than wild type cultures. Confocal microscopy revealed that fewer variant 1 knockout cells migrated into collagen gels, resulting in shorter sprouts with fewer branches, and that these cells displayed fewer filopodia than wild type cells. These results provide evidence that variant 1 of gravin plays a role in invasive cell migration and angiogenic sprouting, which are essential cellular events that regulate angiogenesis. Future studies will investigate the mechanism by which variant 1 contributes to angiogenic sprouting and the role that each variant of gravin plays in Src and cAMP dependent signaling in endothelial cell migration, adhesion and angiogenic sprouting.Support or Funding InformationSupported by NIH grants P30GM103329, P20GM103442 and P20GM113123This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.