Apolipoprotein-J prevents angiotensin II-induced apoptosis in neonatal rat ventricular cells

Abstract

Background: Up-regulation of angiotensin II (AngII) occurs in cardiac diseases, such as congestive heart failure, cardiac hypertrophy, myocardial ischemia and atrial fibrillation, which represent major health problems. Evidence from in vivo studies suggests that the level of Apolipoprotein-J (ApoJ) is also elevated but plays a protective role in cardiovascular disease. This study aimed to evaluate the protective effects of ApoJ against cytotoxicity of AngII in neonatal rat ventricular cells (NRVCs).

Methods and results: In culture, NRVCs were damaged by exposure to AngII, and ApoJ overexpression using an adenovirus vector significantly reduced the AngII-induced cell injury. ApoJ also prevented AngII from augmenting Nox2/gp91(phox) expression. The reactive oxygen species (ROS) scavenger, Mn(III)TBAP, showed similar results of attenuating AngII-induced cell damage. Furthermore, ApoJ overexpression increased phosphorylation of Akt, and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 diminished the antioxidant effects of ApoJ, and prevented the protective effect of ApoJ against the cytotoxicity of AngII. Moreover, upregulation of nuclear factor κB (NF-κB) p65 expression and phosphorylation of p38 mitogen-activated protein kinase (MAPK) mediated by AngII in cultured NRVCs were significantly inhibited by overexpression of ApoJ. The p38 MAPK inhibitor SB203580 and the NF-κB inhibitor PDTC protected NRVCs from injury caused by AngII.

Conclusions: ApoJ serves as a cytoprotective protein in NRVCs against cytotoxicity of AngII through the PI3K-Akt-ROS and MAPK/ NF-κB pathways.

Fig. 1

ApoJ overexpression by infection with recombinant adenovirus prevents AngII-induced apoptosis in NRVCs. a Apo J expression after infection by recombinant adenovirus was determined by Western blot (n = 3/group). b Cleavage of caspase-3 was stimulated by AngII and inhibited by ApoJ (n = 3/group). c Control and ApoJ-transduced NRVCs were incubated with AngII for 24 h, and then cell viability was determined by MTT assay (n = 8/group). d Apoptosis of cardiomyocytes was determined by caspase-3/7 activity assay. Apoptosis of control adenovirus infected NRVCs was significantly increased, while ApoJ-overexpressing NRVCs showed resistance to AngII-induced cell injury (n = 8/group). e LY294002, Mn(III)TBAP, SB203580 and PDTC were pretreated in NRVCs for 1 h, and then the cells were incubated with AngII for 24 h. Cell apoptosis was determined by caspase-3/7 activity assay (n = 8/group). **P < 0.01 versus control Ad/PBS group; ^## P < 0.01 versus control Ad/AngII group, ^$ P < 0.05 versus control Ad/AngII group

Fig. 2

ApoJ attenuates the increased ROS production via PI3K-Akt pathway induced by AngII. a Western blot analysis of Nox2/gp91phox expression in NRVCs. Nox2/gp91phox expression was enhanced by AngII in control adenovirus-infected cells, while ApoJ-transduced cells inhibited its expression (n = 3/group). b LY294002 was used to pretreat ApoJ-transduced NRVCs for 1 h, and then the control and ApoJ-transduced NRVCs were incubated with AngII for 24 h. Nox2/gp91phox expression was determined by Western blot (n = 3/group). c AngII increased pAkt expression in control infected NRVCs, and ApoJ further augmented its expression. d Cell apoptosis caused by AngII increased in ApoJ-overexpressing cells treated with LY294002. ^* P < 0.05 versus control Ad/PBS group, ^** P < 0.01 versus control Ad/PBS group; ^# P < 0.05 versus control Ad/AngII group, ^## P < 0.01 versus control Ad/AngII group,^$ P < 0.05 versus ApoJ group

Fig. 3

Effects of ApoJ on expression of p38 kinase and NF-κB p65 in NRVCs. a MAPK p38 expression in NRVCs. Phosphorylated MAPK p38 expression was enhanced by AngII in control infected cells, while ApoJ-transduced cells inhibited its expression (n = 3/group). b Western blot analysis of NF-κB expression in NRVCs. NRVCs were infected with control or ApoJ-expressing adenovirus for 36 h and then incubated with AngII for 24 h. Addition of AngII increased phosphorylated NF-κB expression, while ApoJ-transduced cells significantly decreased its expression (n = 3/group). *P < 0.05 versus control Ad/PBS group; **P < 0.01 versus control Ad/PBS group, ^# P < 0.05 versus control Ad/AngII group, ^## P < 0.01 versus control Ad/AngII group

Fig. 4

Signaling pathway diagram of ApoJ action in NRVCs. 1) In control adenovirus-infected cells, AngII stimulates phosphorylation of MAPK and Akt, which enables the activation of NF-κB and NADPH oxidase and consequently induction of apoptosis in NRVCs. 2) In NRVCs, p38 MAPK, NF-κB, Akt and NADPH oxidase activated by AngII are blocked by overexpression of ApoJ