Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking

Abstract

Background: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain—an intracellular modulatory protease—interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. Methods: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. Results: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 subsets. This occurs via the proteolytic degradation of MEKK1 and the subsequent inhibition of TGF (transforming growth factor)-β1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-β type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. Conclusions: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

How Lymphatic Endothelial Cells Destabilize Regulatory T Cells

“…Qingkang Lyu, Klaus Ley I n this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Miyazaki et al 1 investigated the role of calpain in regulatory T lymphocyte stability and trafficking under hypercholesterolemia. The authors demonstrated that lysophosphatidic acid (LPA) induced overexpression of calpain in lymphatic endothelial cells (LECs).…”

“…Furthermore, Miyazaki et al 1 explored a possible causative relationship of calpain activation in atherosclerosis. The authors first showed that calpastatin expression was reduced in LECs in hypercholesterolemic mice.…”

The multiple roles of lysophosphatidic acid in vascular disease and atherosclerosis

“…Miyazaki et al [26 ▪▪ ] studied the calpain systems in lymphatic endothelial cells (LECs). Calpains are a family of Ca 2+ -dependent, nonlysosomal proteases [27 ▪ ].…”

“…Activated calpains can selectively degrade the inhibitor of κB (IκB) causing the activation of NFκB [27 ▪ ]. In vitro , Miyazaki et al [26 ▪▪ ] demonstrated that LPA downregulated calpastatin and upregulated calpain 2 in lymph node-derived murine endothelial cells that have the majority of LEC markers and properties (SVEC4-10 cells) [26 ▪▪ ]. In cocultures of SVEC4-10 cells and CD4 + T cells, it was found that knocking down the calpain-s1 gene ( Capns1 ), which encodes a common regulatory subunit of conventional calpains, expanded Foxp3+ Tregs, and in the presence of LPA, upregulated Treg-associated cytokines IL-10 and TGF-β1 [26 ▪▪ ].…”

“…In cocultures of SVEC4-10 cells and CD4 + T cells, it was found that knocking down the calpain-s1 gene ( Capns1 ), which encodes a common regulatory subunit of conventional calpains, expanded Foxp3+ Tregs, and in the presence of LPA, upregulated Treg-associated cytokines IL-10 and TGF-β1 [26 ▪▪ ]. The Tgfb1 gene and its protein TGF-β1 were upregulated in LPA-stimulated Capns1 knockdown SVEC4-10 cells [26 ▪▪ ]. Antagonists of TGF- β receptor 1 reversed the Treg expansion suggesting that LPA and Capns1 knockdown in LECs may stabilize Tregs by increasing TGF- β1 production [26 ▪▪ ].…”