The Scaffolding Protein Gravin/AKAP12 Plays a Role in Endothelial Cell Migration and Angiogenic Sprouting

Angiogenesis and vascular integrity are regulated by a wide array of pro‐ and anti‐angiogenic factors through a network of interconnected and sometimes opposing pathways that include Src and PKA dependent signaling. How this complex network of signaling pathways is organized and integrated in space and time is poorly understood, but scaffolding proteins may play a role by serving as a platform to target multiple signaling proteins to specific compartments. We postulate that the A‐kinase anchoring protein AKAP12/gravin serves such a role. Gravin, a multivalent scaffolding protein that is expressed as two variants in endothelial cells (EC), binds several signaling proteins, including PKA and Src, which are known to regulate EC migration and angiogenesis. Studies in our lab show that gravin is upregulated in migrating ECs at the wound edge in wounded monolayers and that gravin knockdown with antisense oligonucleotides inhibits this migration. Studies by others also show that gravin knockdown and overexpression alter PKA dependent permeability across EC monolayers. To further investigate a role for gravin in angiogenesis, we conducted knockdown and knockout experiments using a 3D collagen model of angiogenic sprouting. In knockdown experiments, primary ECs transfected with antisense and missense oligonucleotides were cultured overnight on 3D collagen gels and sprout number was counted. Knockdown of both gravin variants resulted in a decrease in the density of angiogenic sprouts. Next, we undertook a gene editing approach to begin determining the role of each of these variants in angiogenic sprouting and EC migration. Using CRISPR Cas9, we created a variant 1 knockout cell line by deleting a single base in exon 3 which created a premature stop codon just downstream of the start site. Western blotting demonstrated loss of this variant. These cells, expressing only variant 2 of AKAP12 (gravin V.2), expressed half as much gravin as wild type (WT) cells and showed altered cell morphology and behavior compared to WT cells. Gravin V.2 cells had a slower growth curve in culture than WT cells. When cultured on collagen gels, gravin V.2 cultures showed significantly fewer sprouts than WT cultures. Confocal microscopy also revealed that fewer gravin V.2 cells migrated into collagen gels, resulting in shorter sprouts with fewer branches, and that these cells displayed fewer filopodia than WT cells. Interestingly, gravin V.2 cells migrated faster on 2D surfaces and through Transwell membranes than WT cells. These findings are consistent with the hypothesis that gravin variants play a role in regulating EC migration and angiogenic sprouting. CRISPR Cas9 gene editing studies are underway to further understand the role of each variant and the role of binding domains for proteins such as Src and PKA in regulating the mechanisms underlying cell migration, sprouting and tubulogenesis. Support or Funding Information Supported by NIH grants P30GM103329, P20GM103442 and P20GM113123 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .