Biochemical and Biophysical Research Communications
Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages
Introduction
Atherosclerosis is a multifactorial and progressive pathological process in large and medium sized arteries, characterized by formation of atherosclerotic plaques in the vessel wall. The exact mechanism leading to plaque formation and progression is still unclear, but monocyte infiltration and macrophage-mediated vascular inflammation seem to play important roles [1]. In vivo, macrophage polarization is a dynamic process, but distinctions can be made between the two extremes classically activated macrophages (M1), which are pro-inflammatory, and alternatively activated macrophages (M2), which are anti-inflammatory. Inside the plaques, macrophages become exposed to various microenvironmental stimuli, which drive their specific activation and polarization [2], and the balance between the two subtypes correlates with plaque progression and regression [3]. Thus, macrophage polarization can be of critical importance to disease progression.
Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF-induced blood clotting. It is an essential inhibitor of the coagulation system and as such, an important factor in both benign and pathological thrombus formation. TFPI is mainly synthesized in endothelial cells (ECs), but is also expressed by macrophages, smooth muscle cells (SMCs), and T-cells in atherosclerotic plaques [4], [5] where it is upregulated compared to in normal vessels [4], [6]. Furthermore, animal studies have shown increased atherosclerotic burden and thrombosis in ApoE−/- mice with heterogeneous TFPI ± deficiency [7] and decreased plaque formation in high-fat diet fed ApoE−/- mice overexpressing TFPIα in SMCs [8], suggesting that TFPI plays a protective role in atherothrombosis and atherogenesis [9].
The endoplasmic reticulum (ER) is a membranous organelle present in all eukaryotic cells that is involved in post translational processing of proteins and lipid biosynthesis. Disturbances related to ER function have been associated with a number of human diseases, including atherosclerosis [10]. Prolonged ER stress results in activation of the unfolded protein response (UPR), which can lead to activation of inflammatory pathways and apoptosis in macrophages [11], contributing to plaque progression.
Recently, we demonstrated that cholesterol crystals (CC), a hallmark of advanced atherosclerotic plaques, induced the expression of TFPI in polarized human monocyte-derived macrophages (MDMs) [6]. As cholesterol has been shown to be able to induce ER stress in macrophages [12], we aimed to investigate the potential role of ER stress in the induction of TFPI expression by CC and the function of TFPI. In the present study, we show that the upregulation of TFPI by CC was dependent on ER stress induction. CC also induced an ER stress-dependent pro-inflammatory response in human M2-polarized MDMs, which was attenuated by TFPI. Moreover, the ER stress marker CCAAT-enhancer-binding protein homologous protein (CHOP) was detected in human carotid endarterectomies, in the same area as TFPI. These results show that CC induce TFPI expression and an inflammatory response in M2 macrophages through activation of the ER stress pathway, and suggest a novel role of TFPI in protecting against the CC-mediated inflammatory response in these cells.
Section snippets
Reagents
RPMI 1640 medium, fetal bovine serum (FBS) and phosphate buffered saline (PBS) were from Gibco (Paisley, Renfrewshire, UK). RIPA buffer (R0278), formalin solution (4% formaldehyde, HT5011), bovine serum albumin (BSA), phorbol-12 myristate 13-acetate (PMA, P8139), 4-phenylbutyric acid (PBA, P21005), and lipopolysaccharide (LPS) were from Sigma Aldrich (St. Louis, MO, USA). IFN-γ and IL-4 were from Peprotech Nordic (Stockholm, Sweden). Recombinant human GM-CSF and M-CSF were from R&D systems
CC induce TFPI expression in human MDMs dependent on ER stress
As cholesterol is known to induce ER stress, we speculated if activation of the ER stress pathway was involved in the CC-induced TFPI expression previously observed in polarized human MDMs. The presence of ER stress was verified by measuring the levels of the ER stress markers CHOP and BiP in polarized MDMs. Distinct macrophage polarization was obtained, as confirmed by qRT-PCR of M1 and M2 markers [6]. CHOP mRNA levels were significantly upregulated in the M2-polarized macrophages, by 3-fold,
Discussion
Atherosclerotic plaques that have a high degree of macrophage-induced inflammation together with a large lipid-rich core are more prone to rupture [14], [15], causing exposure of TF expressing cells and pathologic thrombus formation. TFPI is the natural inhibitor of TF induced thrombus formation and is thus implicated in many diseases including atherosclerosis [16], where it is believed to have a protective effect. We have recently found that TFPI expression was increased in human macrophages
Conflict of interest
None.
Funding
This work was supported by the Oslo University Hospital and the University of Oslo (Oslo, Norway), and funded by research grants from the Norwegian Research Council (NRC, Oslo, Norway, Grant # 24973), South-Eastern Norway Regional Health Authority (Hamar, Norway, Grant # 2014090), and the Henrik Homans Minde endowment (Oslo, Norway).
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Equal contribution.
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Present address: Institute of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway.