Prolyl hydroxylase 3 overexpression accelerates the progression of atherosclerosis in ApoE−/− mice

https://doi.org/10.1016/j.bbrc.2016.03.058Get rights and content

Highlights

  • Our study demonstrated that PHD3 is involved in the progression of atherosclerosis in ApoE−/− mice for the first time.

  • PHD3 is associated with the expression of inflammatory cytokines such as ICAM-1, VCAM-1, MCP-1, IL-1β and TNF-αin vivo and in vitro.

  • We found that PHD3 could activate the MAPK signaling pathway in HUVECs with ox-LDL treatment.

Abstract

PHD3 belongs to the family of 2-oxoglutarate and iron-dependent dioxygenases and is a critical regulator of HIF-1α. Its expression is increased in cardiovascular diseases such as cardiomyopathy, myocardial ischemia-reperfusion injury, and congestive heart failure. However, the association between PHD3 and atherosclerosis has not been clearly elucidated.

In the present study, we investigated the potential effect and mechanism of PHD3 in apolipoprotein E–deficient (ApoE–/–) mice. Murine PHD3 lentivirus and shRNA –PHD3 lentivirus were constructed and injected intravenously into ApoE–/– mice fed on a high fat diet. The aortic atherosclerotic lesion area was larger with PHD3 over-expression. With increased PHD3 levels, macrophages and smooth muscle cells were enhanced. The apoptosis of atherosclerotic plaques revealed an increase when PHD3 was elevated. Furthermore, the expression of intercellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), monocyte chemotactic protein 1 (MCP-1), interleukin-1beta (IL-1β) and tumor necrosis factor-α(TNF-α) were upregulated with PHD3 over-expression. In vitro, we explored the specific signaling pathway of PHD3 in HUVECs. PHD3 over-expression is associated with activation of ERK1/2 and JNK phosphorylation of MAPK signaling pathway. PHD3 inhibition decreased the apoptosis of HUVECs treated with ox-LDL (50 μg/ml). Our study suggests that PHD3 is not only a regulator of HIF-1α but also an active participant in atherogenesis.

Introduction

Atherosclerosis is a complex pathological process, which plays a pivotal role in the progression of cardiovascular disease (CVD), accounting for one of the major mortalities throughout the world [1]. Recent decades, it has gained ascendency that atherosclerosis is a chronic inflammatory disease mainly involving in large and medium-sized elastic and muscular arteries [2], [3]. Endothelial dysfunction is viewed as the initial step of atherosclerosis, and the subsequent production of chemokines and adhesion molecules beckon monocytes to migrate into intima, where they differentiate into macrophages, and then foam cells come into being when engorged with lipids [4]. Previous studies led to the confirmation that inflammation involves in all phases of atherosclerosis progression and triggers the clinical thrombotic complications [5], [6], [7], [8], [9], [10], [11]. Thus, identifying the mechanisms of cytokines leading to atherosclerosis could provide a novel insight for preventive and therapeutic strategies and favor the atherosclerotic events.

The prolyl hydroxylase 3 (PHD3) belongs to the family of 2-oxoglutarate and iron-dependent dioxygenases, which could hydroxylate the proline residues of hypoxia inducible factor 1α (HIF-1α) causing proteasomal degradation via combination with the von Hippel-Lindau protein (pVHL) ubiquitin ligase under normoxia [12], [13], [14]. In hypoxia condition, inhibition of PHDs activity results in the stabilization of HIF-1α. Aggregated HIF-1α together with HIF-1β forms dimers and then translocate into the nucleus where a series of genes transcription are activated [15], [16]. PHD3 is abundantly expressed in heart, muscle and great vessels [17], and is evenly distributed in the cytoplasm and nucleus [18].

In addition to its role in regulating oxygen tension, PHD3 is identified to exert other functions. Previous studies lead to the confirmation that PHD3 is intensively related to various cardiac diseases. PHD3 expression is increased in cardiovascular diseases such as cardiomyopathy [19], myocardial ischemia-reperfusion injury [20] and congestive heart failure [21]. Inhibition of PHD3 could lead to a hypofunctional sympathoadrenal system and reduced blood pressure [22]. The rat ortholog gene of human EGLN3 is SM20 and its expression is upregulated in the neointima in a model of rat aortic balloon injury [23]. Besides, PHD3 is considered as a pro-apoptotic protein and could regulate apoptosis of various cell types in HIF-independent pathways [24], [25], [26]. In rats, SM20 is abundantly expressed in the intimal smooth muscle cells [27]. Furthermore, PHD3 identifies pro-inflammatory macrophages in vivo and in vitro [28]. Kiss et al. found that PHD3 plays an eminent role in macrophage differentiation, polarization and consequently, pro-inflammatory behavior [29]. Thus, it reminds us that PHD3 may exert an effect on cardiovascular tissues. Hence, we hypothesize PHD3 contributes to the progression of atherosclerosis. Apoe−/− mice and cells of cardiovascular origin were used to identify the underlying mechanism for PHD3 to accelerate atherosclerosis.

Section snippets

Animals

All experimental procedures were performed in accordance with guidelines for Institutional Animal Care and were approved by The Animal Ethics Committee of Shandong University. Male ApoE−/− mice (n = 100) of eight-week-old were randomly divided into the following 5 groups (n = 20 per group): (1) control group), (2) high-fat diet (HFD) group, (3) lentivirus-PHD3 group (lentivector at the dose of 2× 107 TU/mouse), (4) shRNA-PHD3 group (flentivector at the dose of 2× 107 TU/mouse), and (5) NC group

Effect of PHD3 on atherosclerotic lesions in ApoE−/− mice

Atherosclerotic plaque involvement in ApoE–/– mice was mainly confined to the aortic arch region throughout the entire aorta. En face analysis of aortic tissues stained with Oil Red O from ApoE−/− mice with injection of lentivirus-PHD3 revealed larger atherosclerotic lesions compared with NC group (P < 0.05), while atherosclerotic lesion area decreased with in shRNA-PHD3 group compared with NC group (P < 0.05) (Fig. 1A). In addition, the atherosclerotic lesions in cross-sections of the aortic

Discussion

The present study revealed the findings that PHD3 is involved in the progression of atherosclerotic lesions in ApoE−/− mice and inhibition of PHD3 attenuates the formation of plaques in the aortic sinus and entire aorta. In addition, atherosclerotic lesions in ApoE−/− mice injected with shRNA-PHD3 lentivirus exert decreased inflammatory reaction, which is reflected by reduced infiltration of macrophages and smooth muscle cells; diminished collagen fibers; declined expression of inflammatory

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgment

This project was supported by the National Natural Science Foundation of China (No. 81370325).

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