Elsevier

Thrombosis Research

Volume 176, April 2019, Pages 85-94
Thrombosis Research

Full Length Article
RhoA-Rho associated kinase signaling leads to renin-angiotensin system imbalance and angiotensin converting enzyme 2 has a protective role in acute pulmonary embolism

https://doi.org/10.1016/j.thromres.2019.02.016Get rights and content

Highlights

  • RAS activation was correlated with clinical severity in APE patients.

  • RhoA-ROCK signaling regulated RAS components in rPAECs and an APE rat model.

  • ROCK inhibitors and ACE2 activator had a protective role in an APE rat model.

Abstract

Introduction

Acute pulmonary embolism (APE) is a cardiovascular disease with high morbidity and mortality. Although the anatomical obstruction of the pulmonary vascular bed initiates APE, recent studies have suggested that vasoconstrictors in the renin-angiotensin system (RAS) play a role in the severity of APE.

Materials and methods

We performed a 5-year retrospective clinical study to analyze the key RAS components in APE patients, including angiotensin converting enzyme (ACE), ACE2, angiotensin II (Ang II) and angiotensin 1-7(Ang(1-7)). The role of RhoA-Rho associated kinase (ROCK) signaling in regulating RAS vasoconstrictors was detected in rat pulmonary artery endothelial cells and in an APE rat model.

Results

In clinical study, we found that the levels of RAS vasoconstrictors were correlated with the clinical classification of APE patients, ACE and Ang II were unregulated, whereas ACE2 and Ang(1-7) were downregulated in the high-risk group compared to the healthy volunteers. In animal study, we found that activated RhoA-ROCK signaling was responsible for the imbalance in RAS vasoconstrictors both in vitro and in vivo, and further evidence indicated that ROCK inhibitors (Y27632 or HA1077) and an ACE2 activator (Resorcinol naphthalein) restored the dysregulated RAS vasoconstrictors significantly and had a protective role in an APE rat model.

Conclusions

Our study revealed that RhoA-ROCK signaling leads to RAS imbalance in APE patients, and ACE2 activation might be a novel therapeutic target in APE treatment.

Introduction

Acute pulmonary embolism (APE) is a leading cause of vascular death worldwide after myocardial infarction and stroke [1,2]. The clinical classification of the severity of APE is based on the estimated APE-related early mortality risk, and the high-risk group has a higher 30-day mortality than other groups [2]. The major reason for APE is the anatomical obstruction of the pulmonary vascular bed, which leads to an increase in pulmonary vascular resistance (PVR) and interferes with blood gas exchange [3]. APE induced vasoconstriction also contributes to the increased PVR [4,5].

Previous studies have suggested that the renin-angiotensin system (RAS), which regulates blood pressure and fluid balance, is involved in the pathogenesis of various pulmonary diseases, including pulmonary hypertension (PH) [6,7], lung fibrosis [8], and lung injury [9]. Vasoconstrictive factors in RAS are suggested to be involved in the pathogenesis of pulmonary embolism [10,11]. Angiotensin converting enzyme (ACE) and its homologue ACE2 are key regulators in the RAS. ACE cleaves angiotensin I (AngI) to generate AngII, which is one of the major vasoconstrictors in the RAS [12]. ACE2 counter-regulates the RAS by converting AngII into the vasodilator Ang(1-7) [13,14]. Decreased expression of ACE2 is associated with pulmonary vascular diseases [15]. Likewise, pharmaceutically activated ACE2 can restore overactivated RAS in the endothelium [[16], [17], [18]].

RhoA is a member of the Rho family, a small GTP-binding protein, and its downstream effector Rho-associated kinase (ROCK) plays an important role in the pathogenesis of pulmonary vasoconstriction and vascular remodeling [19]. RhoA-ROCK signaling is activated by vasoconstrictors such as AngII [20] and endothelin-1 [21]. Specific ROCK inhibitors such as Y-27362 [22] and HA-1077 (Fasudil) [23] can reverse sustained vasoconstriction induced by many kinds of agonists [24,25]. Little is known about the characteristics of RhoA-ROCK signaling in APE.

Previously, we observed that ACE was a direct target of hypoxia-inducible factor 1α (HIF-1α), and the accumulated AngII was a key mediator in the downregulation of ACE2 by HIF-1α [26]. Furthermore, dysregulation of the ACE/ACE2 ratio enhanced the proliferation and migration of pulmonary artery smooth muscle cells and contributed to the pathogenesis of hypoxic PH [27,28]. Overexpression of ACE2 attenuated the elevation in right ventricular systolic pressure (RVSP) and pulmonary vascular remodeling and protected animals from cardiovascular and pulmonary disorder in PH [29,30]. However, whether ACE or ACE2 contributes to the severity of APE and the therapeutic potential for APE is still unknown. We hypothesized that activated RhoA-ROCK signaling in APE was responsible for dysregulation of the ACE/ACE2 ratio.

In this study, we evaluated ACE and ACE2 levels in APE patients with different clinical classifications through a retrospective clinical study. We also investigated the role of RhoA-ROCK signaling in the regulation of ACE and ACE2 in rat pulmonary artery endothelial cells and in an APE rat model.

Section snippets

Study population and risk assessment

One hundred and fourteen patients with an initial diagnosis of APE were enrolled into this study from January 2009 and December 2014 at Sir Run Run Shaw Hospital, Medical School of Zhejiang University. The diagnosis of APE followed the 2014 European Society of Cardiology (ESC) guidelines [2] and was mainly based on clinical features, blood examination and imaging. Exclusion criteria included a history of malignancy, cor pulmonale, interstitial lung disease, severe COPD, tuberculosis,

Dysregulation of the RAS in APE patients

To investigate the correlations among RAS components in different clinical classifications for APE patients, we collected plasma samples from 82 APE patients and 30 healthy volunteers for a retrospective study. APE patients were categorized into four groups: a low risk group (n = 32), an I-low risk group (n = 17), an I-high risk group (n = 23) and a high risk group (n = 10), according to their PE-related risk and clinical status and comorbidities on admission as recommended by the 2014 ESC

Discussion

In this study, we investigated that high-risk APE patients had increased ACE and AngII but decreased ACE2 and Ang-(1-7) levels in their circulating system in a retrospective clinical study. In an animal study, we revealed that the activation of RhoA-ROCK signaling was related to the dysregulation of the RAS vasoconstrictive factors both in vitro and in vivo. ROCK inhibitors or an ACE2 activator restored the dysregulation in the RAS and had a protective role in an APE rat model.

The principle

Conclusions

In summary, our study evaluated RAS dysregulation in APE patients with different clinical severities. We found that RhoA-ROCK signaling led to an ACE/ACE2 imbalance in APE rast, and revealed the potential value of ROCK inhibitors and ACE2 activator for APE treatment.

Funding

This work was supported by National Natural Science Foundation of China [grant number 81570043]; Zhejiang Provincial Natural Science Foundation of China [grant numbers LQ18H010001, LY19H010003]; Zhejiang Provincial Medical Science and Technology Foundation of China [grant number 2017KY415]. The funders have no role in study design, no role in the collection, analysis and interpretation of data, no role in the writing of the report, and no role in the decision to submit the article for

Acknowledgements

We appreciate the patients and healthy volunteers and their families for participating in this study.

Ethics approval and consent to participate

The study was approved by the ethics committee of Sir Run Run Shaw Hospital, Medical school of Zhejiang University, Zhejiang, China. All subjects have given written informed consent. All methods were performed according to the approved documents. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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    These two authors contributed equally to this work.

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