Annexin A2 inhibits the migration of PASMCs stimulated with HPS rat serum by down-regulating the expression of paxillin

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Highlights

  • Annexin A2 (ANXA2) plays regulatory role in Hepatopulmonary syndrome (HPS) associated pulmonary vascular remodelling (PVR).

  • HPS rat serum increased the migration of pulmonary artery smooth muscle cells (PASMCs) and expression of ANXA2.

  • After silencing ANXA2 with siRNA, HPS serum-induced up-regulation of paxillin expression was reversed.

  • Down-regulation of ANXA2 significantly inhibited the migration of PASMCs.

Abstract

Hepatopulmonary syndrome (HPS) has been classically associated with intrapulmonary vasodilatation (IPVD) and pulmonary vascular remodelling (PVR), which are the key pathophysiological components of HPS and concerned frequently in the studies of HPS. Little is known about the relevance of pulmonary artery smooth muscle cells (PASMCs) migration or the molecular mechanisms of PVR in HPS. Annexin A2 (ANXA2) plays crucial role in HPS-associated PVR and might activate the activity of paxillin which as a regulatory protein participates in the regulation of PASMCs function in PVR. In addition, it has been identified that ANXA2 could influence the cells migration by some important signaling pathways in many diseases, including lung cancer, pulmonary hypertensionand and liver cancer. In this study, we performed scratch wound motility assay, modified boyden chamber, reverse transcription PCR, western blot and co-immunoprecipitation to determine the role of ANXA2 on HPS-associated PVR. We found that HPS rat serum from a common bile duct ligation (CBDL) rat model enhanced the migration of PASMCs and increased the expression of ANXA2 in PASMCs. We reported that ANXA2 and paxillin could form a co-immunoprecipitation. After silencing ANXA2 with siRNA, we found that the up-regulation of paxillin expression, induced by the HPS rat serum, was reversed. Additionally, we found that down-regulation of ANXA2 could significantly inhibit the migration of PASMCs. These findings indicated that down-regulation of ANXA2 by siRNA results in the inhibition of the aberrant dysregulation of paxillin and migration of PASMCs, which suggesting a potential therapeutic effect on HPS-associated PVR.

Introduction

Hepatopulmonary syndrome (HPS) is a chronic hepatic complication characterised by a defect in arterial oxygenation induced by intrapulmonary vasodilatation (IPVD) in the setting of chronic liver disease [1], [2]. Multiple circulating cytokines and growth factors are released by a diseased liver and contribute to the occurrence of IPVD, which leads to development of hypoxemia. Hypoxic conditions contribute to numerous pulmonary artery smooth muscle cells (PASMCs) migrating from membrane to endometrial of pulmonary vascular, where they proliferate abnormally. These are the main pathophysiological changes of pulmonary vascular remodelling (PVR) [3].

Previous studies on HPS-associated PVR mainly focused on PASMC proliferation. Some proteins, including Annexin A2 (ANXA2) and paxillin, play key regulatory roles in PASMC proliferation [4], [5], [6]. However, so far, PASMC migration has not been a major focus in HPS-associated PVR research. A number of studies have shown that chronic exposure to hypoxia leads to the migration of PASMCs [3], [5], [7]. Therefore, the pathophysiological mechanisms of increased PASMC migration in HPS-associated PVR require an in-depth study. In addition, previous studies have demonstrated that PASMC migration occurs before the proliferation of these cells [8]. We hypothesised that regulating the migration of PASMCs may be a key upstream target for PVR treatment and provide a new approach for HPS prevention.

ANXA2 is found in many tissues and participates in the regulation of numerous cell functions. It is a key regulator of cell migration and invasion in several cell types. Previous studies have demonstrated that the expression of ANXA2 was increased significantly in PASMCs stimulated with HPS rat serum. Furthermore, it was reported that ANXA2 influenced the proliferation of PASMCs treated with HPS rat serum through the ERK and NF-κB pathways. Most of the time, proliferation occurred after migration [9], [10], [11]. It was noted that ANXA2 may play a key regulatory role in the increased PASMC migration observed in HPS-associated PVR.

It has been reported that paxillin is a key regulator of adhesion, migration and apoptosis of PASMCs, which it regulates via the AKT and ERK1/2 kinases [6]. A number of studies have reported that Annexin A1 (ANXA1) regulates integrin β1 protein expression and promotes gastric cancer cell invasiveness through the formyl peptide receptor/ERK/integrin β1 pathway. Integrins also recruit and regulate paxillin expression in multiple cells [12], [13]. ANXA1 and ANXA2 share similar functions, given their structural similarities. Thus, ANXA2 and paxillin could regulate the expression of cytoskeletal proteins, which results in the migration and adhesion of PASMCs [14], [15], [16].

The purpose of this work was to examine the role of ANXA2 in regulating the migration of PASMCs following exposure to HPS rat serum and to identify the underlying mechanism [17], [18]. We established the HPS model, detected the expression of ANXA2 in PASMCs, and found increased migration of PASMCs after treatment with HPS rat serum. We also detected an interaction between ANXA2 and paxillin. Our data showed that the expression of ANXA2 in PASMCs increased significantly, while the migration of PASMCs was noticeably enhanced when the cells were stimulated with HPS rat serum. In addition, ANXA2 and paxillin co-immunoprecipitated. Upon knockdown of ANXA2 expression, we observed decreased paxillin expression and a reduction in PASMC migration.

Section snippets

HPS rat model and cell culture

All procedures performed on the rats were conducted according to guidelines from the National Institutes of Health. The study protocol was approved by the committee on Animal Research of Southwest Hospital. Sprague Dawley (SD) rats weighing 180–220 g, which were obtained from the Laboratory Animal Center of the Third Military Medical University, were used in this study. The HPS rat model was used in our previously published study successfully [19]. The protocol utilised here was similar to the

HPS rat serum enhanced the migration of PASMCs, as identified by the scratch wound motility assay and the modified boyden chamber

The results of scratch wound motility assay indicated that treatment with normal rat serum did not increase the distance that PASMCs migrated, which remained consistently low at each time-point. However, the distance PASMCs migrated increased significantly when the PASMCs had been stimulated with HPS rat serum. The distance increased in a time-dependent manner (Fig. 1A). The modified Boyden chamber results showed that HPS rat serum promoted transmembrane migration of PASMCs compared with

Discussion

Previous studies and our initial study demonstrated that HPS rat serum induces PASMC proliferation and that hypoxia enhances the migration of PASMCs [21], [22]. These are key pathophysiological components of PVR associated with HPS [23]. It has been reported that the migration of vascular smooth muscle cells (VSMCs) in some organs or tissues is a key early change in vascular remodelling [8], [9].

Our data indicated that HPS rat serum enhanced PASMC migration dramatically. Recent research has

Conflicts of interest

The authors have no conflict of interest.

Acknowledgments

This work is supported by grant nos. 30700347, 30872448, 81170053, 81170414 and 81270510 from the National Science Foundation of China (NSFC).

References (27)

  • A. Miyamoto et al.

    Effect of chronic methylene blue administration on hypoxemia in rats with common bile duct ligation

    Hepatol. Res.

    (2010)
  • J. Zeng et al.

    Role of annexin A1 in human pulmonary arterial smooth muscle cells proliferation induced by hypoxia

    Chin. J. Anesthesiol.

    (2011)
  • Y. Chen et al.

    Changes in expression of annexin A2 in lung tissues in rats with hepato-pulmonary syndrome

    Chin. J. Anesthesiol.

    (2013)
  • 1

    Yang Chen and Xinrong Wen contributed equally to this study.

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