CRISPR-Based Activation of Endogenous Expression of TPM1 Inhibits Inflammatory Response of Primary Human Coronary Artery Endothelial and Smooth Muscle Cells Induced by Recombinant Human Tumor Necrosis Factor α

Tumor necrosis factor alpha (TNF alpha) is one of the most important proinflammatory cytokines, which affects many processes associated with the growth and characteristics of endothelial, smooth muscle, and immune system cells. However, there is no correlation between most in vivo and in vitro studies on its role in endothelial cell proliferation and migration. In this study, we examined the effect of recombinant human (rh) TNF alpha produced in HEK293 cells on primary human coronary artery endothelial cells (pHCAECs) in the context of F-actin organization and such processes as migration and adhesion. Furthermore, we evaluated the possibility of the inhibition of the endothelial inflammatory response by the CRISPR-based regulation of TPM1 gene expression. We showed that TNF alpha-induced activation of pHCAECs was related to the reorganization of the actin cytoskeleton into parallel-arranged stress fibers running along the longer axis of pHCAECs. It allowed for the directed and parallel motion of the cells during coordinated migration. This change in F-actin organization promoted strong but discontinuous cell-cell contacts involved in signalization between migrating cells. Moreover, this form of intercellular connections together with locally increased adhesion was related to the formation of migrasomes and further migracytosis. Stabilization of the actin cytoskeleton through the CRISPR-based activation of endogenous expression of TPM1 resulted in the inhibition of the inflammatory response of pHCAECs following treatment with rh TNF alpha and stabilization of cell-cell junctions through reduced cleavage of vascular endothelial cadherin (VE-cadherin) and maintenance of the stable levels of alpha- and beta-catenins. We also showed that CRISPR-based activation of TPM1 reduced inflammatory activation, proliferation, and migration of primary human coronary artery smooth muscle cells. Therefore, products of the TPM1 gene may be a potential therapeutic target for the treatment of proinflammatory vascular disorders.

Automated summary

TNF alpha plays a role in activating endothelial cells and reorganizing the actin cytoskeleton to facilitate cell migration. The activation of TPM1 gene can stabilize the actin cytoskeleton, inhibit inflammatory response, and maintain stable cell-cell junctions in endothelial cells. This approach may serve as a potential therapeutic target for treating proinflammatory vascular disorders.