Lipopolysaccharide induces endothelial cell apoptosis via activation of Na+/H+ exchanger 1 and calpain-dependent degradation of Bcl-2

https://doi.org/10.1016/j.bbrc.2012.09.023Get rights and content

Abstract

The calcium-dependent protease calpain is involved in lipopolysaccharide (LPS)-induced endothelial injury. The activation of Na+/H+ exchanger (NHE) is responsible to increase intracellular Ca2+ (Cai2+) in cardiovascular diseases. Here we hypothesized that activation of NHE mediates LPS-induced endothelial cell apoptosis via calcium-dependent calpain pathway. Our results revealed that LPS-induced increases in NHE activity are dependent on NHE1 in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with LPS increased the NHE1 activity in a time-dependent manner associated with the increased Cai2+, which resulted in enhanced calpain activity as well as HUVECs apoptosis via NHE1-dependent degradation of Bcl-2.

Graphical abstract

The current study demonstrates that LPS induces vascular endothelial cell apoptosis by triggering calpain-dependent degradation of Bcl-2 via activating NHE1. Inhibition of NHE1 reverses LPS-induced increase of calcium concentration and calpain activity. In addition, inhibition of NHE1 by cariporide or RNA interference blocked the decrease of Bcl-2 degradation caused by LPS. These results strongly suggest that NHE1 is required for LPS-induced endothelial cell apoptosis via calcium-dependent protease calpain.

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Highlights

► LPS-induced increases in NHE activity are dependent on NHE1 in HUVECs. ► LPS increases intracellular calcium level via activation of NHE1. ► LPS-enhanced calpain activity is both NHE1 and calcium-dependent. ► LPS induces NHE1 activation-dependent Bcl-2 degradation. ► NHE1 activation is required for LPS-induced HUVECs apoptosis.

Introduction

Lipopolysaccharide (LPS) is a component of the outer membrane Gram-negative bacteria and elicits inflammatory responses in immune and non-immune cells including endothelial cells [1]. The endothelial cell is a prime target of the LPS molecule, and vascular complications of septic shock due to Gram-negative bacteria are related to endothelial injury [2], [3]. Indeed, LPS-induced systemic organ failure is triggered initially by vascular endothelial injury, characterized by vascular occlusion, perivascular accumulation of leukocytes and cell death [4]. Recently, it has been demonstrated that LPS induces endothelial cell injury by activating calpain [5]. However, the molecular mechanism of LPS-induced activation of the death pathway in endothelial cells is not completely understood.

Na+/H+ exchanger 1 (NHE1), a ubiquitously expressed protein on mammalian plasma membranes [6], is known to involve cell apoptosis [7], [8]. NHE1 exchanges intracellular H+ for extracellular Na+ to regulate intracellular pH (pHi) value and the concentration of intracellular Na+ (Nai+) [9]. The activation of NHE1 increases Nai+ concentration that leads to Ca2+ overload through the Na+/Ca2+ exchanger, which is assumed to be the crucial factor in cell injury [10]. The increased Cai2+ concentration automatically activates calpain, a calcium-dependent protease [11]. Many proteins in cells are the substrates of calpain, including anti-apoptotic family member, Bcl-2 [12]. Previous studies have shown that inhibition of NHE1 has anti-apoptotic effects and LPS-induced apoptosis is dependent on changes in Bcl-2 [7], [8], [13]. These findings support the hypothesis that LPS-induced apoptosis via Ca2+/calpain-dependent Bcl-2 degradation is mediated by NHE1.

In this study, we demonstrate that LPS stimulates a calpain-mediated apoptosis pathway in HUVECs, and the LPS-induced apoptosis in endothelial cells is mediated through activation of NHE1. The study provides new mechanisms underlying LPS-induced apoptosis in endothelial cells.

Section snippets

Materials

Zllal (benzyloxycarbonyl-leucyl-leucinal) was purchased from Biomol Research Laboratories (Plymouth Meeting, USA). Calcium chelator, BAPTA, was purchased from Invitrogen. 2-carboxyethyl-5(6)-carboxyfluorescein (BCECF) was purchased from Calbiochem (Billerica, USA). Pan-Caspase Inhibitor, Z-VAD-FMK, was purchased from KAMIYA BIOMEDICAL (Seattle, USA). Cariporide and other chemicals were purchased from Sigma (Shanghai, China). Anti-Bcl-2, β-actin, caspase-3 and 2nd antibodies were purchased from

LPS-induced increases in NHE activity are dependent on NHE1 in HUVECs

Early studies have shown that LPS induces endothelial cell apoptosis and endothelial dysfunction, which contribute to cardiovascular disease [21]. The NHE plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy [6]. We first determined whether LPS activates NHE in HUVECs. As shown in Fig. 1A, LPS increased NHE activity beginning at 4 h. The increased NHE activity induced by LPS reached the peak at 16 to 24 

Discussion

The current study demonstrates that LPS induces vascular endothelial cell apoptosis by triggering calpain-dependent degradation of Bcl-2 via activating NHE1. Inhibition of NHE1 reverses LPS-induced increase of calcium concentration and calpain activity. In addition, inhibition of NHE1 by cariporide or RNA interference blocked the decrease of Bcl-2 degradation caused by LPS. These results strongly suggest that NHE1 is required for LPS-induced endothelial cell apoptosis via calcium-dependent

Acknowledgments

We thank Dr Humphrey (Department of Medicine, University of Oklahoma Health Sciences Center) for critically reading this manuscript. This work was funded by Jilin University grant (450060445662, 430504001043, and 430505010272 to Qisheng Peng).

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