Topological Localization of Monomeric C-reactive Protein Determines Proinflammatory Endothelial Cell Responses

Hai-Yun Li,Jing Wang,Yue-Xin Wu,Lin Zhang,Zu-Pei Liu,János G Filep,Lawrence A Potempa,Yi Wu,Shang-Rong Ji

doi:10.1074/jbc.m114.555318

Abstract

The activation of endothelial cells (ECs) by monomeric C-reactive protein (mCRP) has been implicated in contributing to atherogenesis. However, the potent proinflammatory actions of mCRP on ECs in vitro appear to be incompatible with the atheroprotective effects of mCRP in a mouse model. Because mCRP is primarily generated within inflamed tissues and is rapidly cleared from the circulation, we tested whether these discrepancies can be explained by topological differences in response to mCRP within blood vessels. In a Transwell culture model, the addition of mCRP to apical (luminal), but not basolateral (abluminal), surfaces of intact human coronary artery EC monolayers evoked a significant up-regulation of MCP-1, IL-8, and IL-6. Such polarized stimulation of mCRP was observed consistently regardless of EC type or experimental conditions (e.g. culture of ECs on filters or extracellular matrix-coated surfaces). Accordingly, we detected enriched lipid raft microdomains, the major surface sensors for mCRP on ECs, in apical membranes, leading to the preferential apical binding of mCRP and activation of ECs through the polarized induction of the phospholipase C, p38 MAPK, and NF-κB signaling pathways. Furthermore, LPS and IL-1β induction of EC activation also exhibited topological dependence, whereas TNF-α did not. Together, these results indicate that tissue-associated mCRP likely contributes little to EC activation. Hence, topological localization is an important, but often overlooked, factor that determines the contribution of mCRP and other proinflammatory mediators to chronic vascular inflammation.

Highlights

Monomeric C-reactive protein may contribute to atherogenesis by inducing endothelial activation
Polarized Induction of endothelial cell (EC) Responses by Cys-mutated Monomeric C-reactive protein (mCRP)— We examined whether the effects of Cys-mutated mCRP on ECs depend on topological localizations by using the Transwell model, in which EC monolayers were cultured on porous membrane filters with their apical and basolateral surfaces exposed to different chambers (Fig. 1B)
ECs respond strongly to apical but weakly to basolateral stimulation of Cys-mutated or reduced mCRP, suggesting that tissue-resident mCRP may be inefficient in activating ECs

Summary

Background

Monomeric C-reactive protein (mCRP) may contribute to atherogenesis by inducing endothelial activation. The serum level of CRP is closely associated with the risk and prognosis of many chronic inflammatory diseases, including cardiovascular disease [2] and cancer [3] It is still hotly debated whether this protein plays any significant role in the underlying pathological processes [1, 3, 4]. MCRP is proposed to be the major CRP isoform that functions as a regulator of local inflammation, whereas native pentameric CRP may serve as the precursor of mCRP and a systemic marker of inflammation [7, 9, 10] Because both reduced and Cys-mutated mCRP are potent activators of endothelial cells (ECs) [15, 16], it appears plausible that mCRP would contribute to the initiation of atherosclerosis, in which EC dysfunction is one of the earliest events [17]. We investigated the impact of distinct localizations of mCRP with respect to EC on EC function and detected profound EC responses only with apically applied, Cys-mutated mCRP

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION