Abstract
Tumor necrosis factor α (TNFα) 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) TNFα 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 TNFα-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 TNFα and stabilization of cell–cell junctions through reduced cleavage of vascular endothelial cadherin (VE-cadherin) and maintenance of the stable levels of α- and β-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.
Highlights
The endothelial barrier plays a pivotal role in the regulation of the functioning of the entire circulatory system (Godo and Shimokawa, 2017)
We examined the interactions between rh tumor necrosis factor α (TNFα)-activated primary human coronary artery endothelial cells (pHCAECs) and Jurkat T cells (Figure 1B and Supplementary Video S1)
We showed that activation of endogenous expression of TPM1 itself downregulated posttranslational expression of E-selectin (from 0.308 ± 0.0515, p = 0.0112, and 0.584 ± 0.0753, p = 0.0002, to 0.138 ± 0.0409, respectively, as compared to the cells transfected with CRISPR control and knockout systems and inhibited rh TNFα-induced activation of pHCAECs (Figure 6D)
Summary
The endothelial barrier plays a pivotal role in the regulation of the functioning of the entire circulatory system (Godo and Shimokawa, 2017). Endothelial dysfunction manifests into abnormalities in the anticoagulant and anti-inflammatory properties of cells as well as alterations in vascular growth and vascular remodeling It is associated with phenomena like hypertension and atherosclerosis (Chistiakov et al, 2015; Gimbrone and García-Cardeña, 2016). Reduced activity of endothelial nitric oxide synthase is observed in diabetes and hypertension It promotes vasoconstriction, thrombosis, and infiltration of the immune system cells (Matsushita et al, 2001). Thrombosis, and infiltration of the immune system cells (Matsushita et al, 2001) It is associated with the increased production of proinflammatory factors that induce the excessive proliferation of vascular smooth muscle cells. The increased production of endothelium-derived contracting factors leads to even greater deregulation of vessel wall functioning (Virdis et al, 2010)
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