Recombinant Human Matrix Metalloproteinase-2 Impairs

Antioxidant treatment protects against matrix metalloproteinases activation and cardiomyocyte injury during acute pulmonary thromboembolism

O Sousa-Santosa, EM Neto-Nevesa, KC Ferraz, CS Cerona, E Rizzia, RF Gerlach, and JE Tanus-Santos a,*

a Department of Pharmacology, Faculty of Medicine of Ribeirao Preto,

b Department of Pharmacology, State University of Campinas, Campinas, Brazil

c Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900; Ribeirao Preto, SP, Brazil, 14049-900

Short title: Antioxidant therapy in pulmonary thromboembolism

*Address requests for reprints to corresponding author:

Jose E. Tanus-Santos, MD, PhD.

Department of Pharmacology

Faculty of Medicine of Ribeirao Preto

University of Sao Paulo

Av. Bandeirantes, 3900

14049-900 Ribeirao Preto, SP, Brazil

FAX: [phone]

Phone: [phone]

E-mail: [e-mail]; [e-mail]

ACKNOWLEDGMENTS:

This study was funded by Fundação de Aparo a Pesquisa do Estado de São Paulo (FAPESP-Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil), and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil).

Summary:

Background and purpose: Increased reactive oxygen species (ROS) promote matrix metalloproteinases (MMPs) activities and may underlie cardiomyocyte injury and degradation of cardiac troponin I (cTI) during acute pulmonary thromboembolism (APT). We examined whether pretreatment or therapy with tempol (a ROS scavenger) prevents MMPs activation and cardiomyocyte injury of APT.

Experimental approach: Anesthetized sheep received tempol infusion (1.0 mg/kg/min i.v.) or saline starting 30 min before or 30 min after APT (autologous blood clots). Control animals received saline. Hemodynamic measurements were performed. MMPs were studied in the right ventricle (RV) by gelatin zymography, fluorimetric activity assay, and in situ zymography. ROS levels were determined in the RV and cTI were measured in serum samples.

Key results: APT increased the pulmonary arterial pressure and pulmonary vascular resistance by 146% and 164%, respectively. Pretreatment or therapy with tempol attenuated these increases. While APT increased RV +dP/dtmax, tempol infusions had no effects. APT increased RV MMP-9 (but not MMP-2) levels. In line with these findings, APT increased RV MMPs activities, and this finding was confirmed by in situ zymography. APT increased RV ROS levels and tempol infusion, before or after APT, blunted APT-induced increases in MMP-9 levels, MMPs activities, in situ MMPs activities, and ROS levels in the RV. cTI concentrations increased after APT, and tempol attenuated these increases.

Conclusion and implications: RV oxidative stress after APT increases RV MMPs activities leading to degradation of sarcomeric proteins including cTI. Antioxidant treatment may prevent MMPs activation and protect against cardiomyocyte injury after APT.

Keywords: acute pulmonary thromboembolism, oxidative stress, right ventricle; tempol, matrix metalloproteinases; cardiac troponin I.

Abbreviations:

ANOVA, analysis of variance; APT, acute pulmonary thromboembolism; CI, cardiac index; cTI, in cardiac troponin I; DHE, dihydroethidium; HR, heart rate; MAP, mean arterial pressure; MPAP, mean pulmonary artery pressure; MMP, matrix metalloproteinase; PVRI, pulmonary vascular resistance index; ROS, reactive oxygen species; RV, right ventricle; SVRI, systemic vascular resistance index; TIMPs; tissue inhibitors of MMPs;

INTRODUCTION

Acute pulmonary thromboembolism (APT) is a critical condition associated with high morbidity and mortality. Whereas it is clear that emboli migrate to the lungs, obstruct the pulmonary vessels and cause acute right ventricle (RV) failure and circulatory shock (Wood, 2002), there are few accepted therapeutic interventions that may help to protect the heart during APT. In this respect, recent experimental studies showed that increased matrix metalloproteinases (MMPs) activities may play a role in the hemodynamic alterations (Fortuna et al., 2007; Palei et al., 2005; Souza-Costa et al., 2007; Souza-Costa et al., 2005) and in the cardiomyocyte injury after APT (Neto-Neves et al., 2011). Indeed, these studies have shown that doxycycline (a non specific MMPs inhibitor) attenuated APT-induced increases in pulmonary vascular resistance (Fortuna et al., 2007; Palei et al., 2005; Souza-Costa et al., 2007; Souza-Costa et al., 2005) and increases in cardiac troponin I (cTI) concentrations (Neto-Neves et al., 2011), thus indicating that MMPs inhibition may exert protective effects against cardiomyocyte injury associated with APT.

MMPs are a group of enzymes involved in the turnover of many components of the extracellular matrix (Van den Steen et al., 2002). However, it is now becoming clear that MMPs affect many other relevant mediators involved in a variety of functions and pathogenic mechanisms leading to cardiac dysfunction (Chow et al., 2007; Schulz, 2007). For example, MMPs degrade cTI, myosin light chain-1, titin and other sarcomeric and cytoskeletal proteins, and impair myocardial contractile function (Ali et al., 2010; Cheung et al., 2000; Kandasamy et al., 2010; Lalu et al., 2005; Sawicki et al., 2005; Schulz, 2007; Wang et al., 2002). Importantly, cTI levels increase in proportion to the severity of APT and to the increases in circulating MMP-9 levels (Uzuelli et al., 2008), thus predicting adverse outcomes associated with RV dysfunction (Horlander et al., 2003).

A major factor promoting MMPs expression and activity is oxidative stress, and this alteration