Peripheral blood AKAP7 expression as an early marker for lymphocyte-mediated post-stroke blood brain barrier disruption

O’Connell, Grant C.; Treadway, Madison B.; Petrone, Ashley B.; Tennant, Connie S.; Lucke-Wold, Noelle; Chantler, Paul D.; Barr, Taura L.

In order to test that hypothesis that AKAP7 may be a marker of lymphocyte extravasation, specifically by way of ITGA3-mediated adhesion, qRT-PCR was used to measure the transcriptional levels of AKAP7 splice variants and ITGA3 in primary human lymphocytes which exhibited a strong ability to adhere to culture surfaces coated in a matrix abundant in ITGA3 ligands. Lymphocytes were isolated from the peripheral blood of three healthy donors and expanded for 72 hours yielding healthy, actively proliferating lymphocytes (Fig. 7A ). Lymphocytes were then plated in culture dishes coated in a matrix composed of collagen, laminin, and fibronectin under serum-free conditions and allowed 20 minutes for adherence. Non-adherent lymphocytes were collected from the culture supernatant (Fig. 7B ), and the adherent lymphocytes were then separately harvested (Fig. 7C ). Viability of cells in both fractions was assessed via trypan blue and RNA was extracted for gene expression analysis. Cell viabilities were greater than 95% in both populations in the case of every donor. Expression levels of ITGA3 and the PKA-binding AKAP7 variants were significantly higher on lymphocytes in adherent fractions than those in the non-adherent fractions (Fig. 7D ). Collectively these data suggest that AKAP7 expression is a marker of ITGA3-mediated lymphocyte adhesiveness and is likely elevated in patients who later develop post-stroke BBB disruption as a result of the presence of an invasive lymphocyte population in peripheral blood.