International Journal of Cardiology 179 (2015) 79–81

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International Journal of Cardiology

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Letter to the Editor

Metalloproteinases 2 and 9 in different stages of chronic Chagas disease☆

Marianna Garcia-Saldivia a, Gabriel Lopez-Mendez a, Lisbeth Berrueta b,c, Siham Salmen b,Jose H. Donis a, Diego F. Davila a,⁎a Instituto de Investigaciones Cardiovasculares, Universidad de Los Andes, Hospital Universitario de Los Andes, Mérida, Venezuelab Instituto de Inmunologia Clinica, Universidad de Los Andes, Hospital Universitario de Los Andes, Mérida, Venezuelac Division of Preventive Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA

☆ This research was supported by Grant M-1047-13Científico, Humanístico y Tecnológico, Universidad de(CDCHT).⁎ Corresponding author at: Instituto de Investigacione

de Los Andes. Merida, Venezuela.E-mail address: diegod@ula.ve (D.F. Davila).

http://dx.doi.org/10.1016/j.ijcard.2014.10.0610167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved

a r t i c l e i n f o

Article history:

Received 16 October 2014Accepted 20 October 2014Available online 22 October 2014

Keywords:Chronic Chagas' DiseaseEchocardiographyVentricular geometryMatrix metaloproteinasesHeart failure

dependent pathways. These pathways, present in serum samples,would result in enhanced levels of active MMP-2 [7]. However, oppositeeffects of plasma, as having a higher inhibitory activity against MMP-2than serum, have been reported [8]. The serum levels of MMP-2 andMMP-9 of our patients in the indeterminate stage, measured byWesternblot, were similar to those of the normal controls.MMP-2was significant-ly elevated in the chagasic and non-chagasic patients with compensatedheart failure (Fig. 1) [4]. Western blot, a method for analyzing zymogenssuch as caspasas and metalloproteinases, allows for the detection ofdifferent metalloproteinase forms, such as pro-enzyme and activeones [9,10]. Thus, the MMP-2 andMMP-9 values of our Venezuelan pa-

Chronic Chagas' disease comprises an indeterminate and a cardiacstage. Patients in the former have no clinical, electrocardiographic or ra-diologic abnormalities. However, 20 to 40% of these patients may have,as demonstrated by echocardiography, left ventricular wall motion ab-normalities and normal systolic function [1,2]. Clinicians and re-searchers consider this particular stage as transitional and amenableto preventive treatment. Therefore, several investigations have beenconducted to identify predictive biomarkers of progression of this dis-ease. To this purpose, matrix metaloproteinases 2 (MMP-2) and 9(MMP-9) were studied in patients with positive serology for Chagas'disease from different endemic areas of South America [3–6].

Bautista-Lopez and Schulz [7] have raised several relevant questionsconcerning the methodology used to measure matrix metaloproteinases,as well as the results of their investigation in Colombian patients [3] com-pared to our results in Venezuelan patients [4] and to Brazilian patients[5]. Increased levels of MMP-2 and MMP-9 were found in the indetermi-nate stage of the disease and in patients with electrocardiographic abnor-malities. Although, it is acknowledged that, only about 50% of thesepatients had augmented levels of both metaloproteinases; the authorsrightly point out the possible limitations imposed by processing serum

-07-B. Consejo de DesarrolloLos Andes, Mérida, Venezuela

s Cardiovasculares. Universidad

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versus plasma samples, due to the presence of thrombin and plasmin-

tients, measured by a broadly used and highly sensitive method, do notappear to be early biomarkers of disease progression in chronic Chagas'disease [4].

Studies in Brazilian [5] and Bolivian patients [6] have used methodsother than Western blot, to measure metalloproteinases. MMP-2 andMMP-9 in the Brazilian patients were measured in serum samples byMilliplex assay (Tissue inhibitor of metalloproteinase + free MMPs)and zimography (Only enzyme forms). Both methods failed to show el-evated levels ofMMP-9, in the indeterminate stage of the disease.More-over, gelatin zymography showed that MPP-9 levels of patients in theindeterminate stage are significantly lower than those of non-infectedcontrols. MMP-2 values by Milliplex assay were not different fromnon-infected controls (Fig. 2) [5]. MMP-2 and MMP-9 were also deter-mined in serum samples from Bolivian patients who were in differentstages of chronic Chagas disease and compared to non-chagasic cardiacpatients, by means of Milliplex MAP HumanMMPMagnetic Bead Panel2. MMP-2 and MMP-9 were increased only in the advanced stages ofchronic Chagas and non-chagas cardiac diseases [5,6].

MMP-9 was not augmented in our chagasic and non-chagasicpatients with compensated heart failure [4]. Possible explanations, forthese apparently contradictory results, could be the clinical status andthedegree of cardiac remodeling of the patients. Increasing serum levelsof MMP-9 are associated with concurrent deterioration of ventricularfunction and clinical status [11–14]. Thus, the compensated status ofour chagasic and non-chagasic patients with eccentric ventricular hy-pertrophy could explain the lower levels of MMP-9 and still increasedvalues of MMP-2. It should be emphasized that, the MMP-2–MMP-9ratio did differ between Bolivian chagasic patients with and withoutabnormal electrocardiograms [6]. Finally, although follow up clinical in-vestigations indicate that, most chagasic patients in the indeterminate

Fig. 1. Serum levels of metalloproteinase 2 (MMP-2) from normal controls and chagasic andnon-chagasic patientswith normal left ventricular geometry andwith eccentric left ventricularhypertrophy.MMP-2 25 kDa from chagasic patientswith normal left ventricular geometrywassimilar to controls, but significantly increased in the chagasic and non-chagasic patientswitheccentric hypertrophy. Values are M ± SD. Optical densities. (N. Geometry = Normal leftventricular geometry, E. hypertrophy = Left ventricular eccentric hypertrophy.Adapted fromGarcia-SaldiviaM, Lopez-Mendez G, Berrueta L, Salmen S, Donis JH, Davila DF.Left ventricular geometry and matrix metalloproteinases 2 and 9 in chronic Chagas heartdisease. Int J Cardiol. 2014;176:565–566. With permission from Elsevier.

Fig. 2. Serum levels of MMP-2 andMMP-9 in Brazilian patients in the indeterminate (gray bars)Both methods failed to show elevated levels of MMP-9, in the indeterminate stage of the diseasthan those of non-infected controls. MMP-2 levels by Milliplex assay, of patients in the indeterquartile ranges. (OD = Optical densities).Fares RC, Gomes J de A, Garzoni L., et al. Matrix Metalloproteinases 2 and 9 Are Differentially ExpImmun 2013;81:3600–3608). With permission, American Society of Microbiology.

80 M. Garcia-Saldivia et al. / International Journal of Cardiology 179 (2015) 79–81

stage of the disease, may have a normal life expectancy [1,2]; about 7 to20% of these chagasic patients, with normal echocardiograms, showmyocardialfibrosis inmagnetic resonance imaging studies [15,16]. Con-sequently, further investigations should be performed in order to deter-mine the role of metaloproteinases 2 and 9 in the pathogenesis andtransition of chagasic patients, from the indeterminate stage to the ad-vanced stages of the disease [17].

References

[1] M.C. Nunes, A.A. Carmo, M.O. Rocha, A.L. Ribeiro, Mortality prediction in Chagasheart disease, Expert. Rev. Cardiovasc. Ther. 10 (2012) 1173–1184.

[2] A. Rassi Jr., A. Rassi, S.G. Rasi, Predictors ofmortality in chronic Chagas disease. A sys-tematic review of observational studies, Circulation 115 (2007) 1101–1108.

[3] N.L. Bautista-López, C.A. Morillo, P. López-Jaramillo, et al., Matrix metalloproteinases2 and 9 as diagnostic markers in the progression to Chagas cardiomyopathy, Am.Heart J. 165 (2013) 558–566.

[4] M. Garcia-Saldivia, G. Lopez-Mendez, L. Berrueta, S. Salmen, J.H. Donis, D.F. Davila,Left ventricular geometry and matrix metalloproteinases 2 and 9 in chronic Chagasheart disease, Int. J. Cardiol. 176 (2014) 565–566.

[5] R.C. Fares, J. de A. Gomes, L. Garzoni, et al., Matrixmetalloproteinases 2 and 9 are dif-ferentially expressed in patients with indeterminate and cardiac clinical forms ofChagas disease, Infect. Immun. 81 (2013) 3600–3608.

[6] E.E. Okamoto, J.E. Sherbuk, E.H. Clark, et al., Biomarkers in Trypanosoma cruzi-infected and uninfected individuals with varying severity of cardiomyopathy inSanta Cruz, Bolivia, PLoS Negl. Trop. Dis. 8 (2014) e3227.

[7] N.L. Bautista-Lopez, R. Schulz, Matrix metalloproteinases 2 and 9 as diagnostic toolsin Chagas cardiomyopathy, Int. J. Cardiol. (2014) [in press].

[8] B.H. Koo, M.Y. Park, O.H. Jeon, D.S. Kim, Regulatory mechanism of matrixmetalloprotease-2 enzymatic activity by factor Xa and thrombin, J. Biol. Chem.284 (2009) 23375–23385.

[9] S. Salmen, H. Montes, A. Soyano, D. Hernández, L. Berrueta, Mechanisms of neutro-phil death in human immunodeficiency virus-infected patients: role of reactive ox-ygen species, caspases and map kinase pathways, Clin. Exp. Immunol. 150 (2007)539–545.

and cardiac stages (dark-gray bars) measured byMilliplex assay (A) and Zimography (B).e. Moreover, gelatin zymography showed that MPP-9 serum levels are significantly lowerminate stage, were not different from controls. Values are expressed as median and inter-

ressed in Patients with Indeterminate and Cardiac Clinical Forms of Chagas Disease. Infect

81M. Garcia-Saldivia et al. / International Journal of Cardiology 179 (2015) 79–81

[10] M.D. Rojas, D.L. Peterson, L. Barboza, et al., Programmed hepatocytes cell death associ-ated with FLIP downregulation in response to extracellular PreS1/2, J. Med. Virol. 86(2014) 496–504.

[11] C.V. Thomas, M.L. Coker, J.L. Zellner, J.R. Handy, A.J. Crumbley III, F.G. Spinale, Increasedmatrix metalloproteinase activity and selective upregulation in LV myocardium frompatients with end-stage dilated cardiomyopathy, Circulation 9 (1998) 1708–1715.

[12] F.G. Spinale, M.L. Coker, L.J. Heung, et al., A matrix metalloproteinase induction/acti-vation system exists in the human left ventricular myocardium and is upregulatedin heart failure, Circulation 102 (2000) 1944–1949.

[13] Y.Y. Li, Y. Feng, C.F. McTiernan, et al., Downregulation of matrix metalloproteinasesand reduction in collagen damage in the failing human heart after support with leftventricular assist devices, Circulation 104 (2001) 1147–1152.

[14] A.T. Yan, R.T. Yan, F.G. Spinale, et al., Plasma matrix metalloproteinase-9 level is cor-related with left ventricular volumes and ejection fraction in patients with heartfailure, J. Card. Fail. 12 (2006) 514–519.

[15] A1. Regueiro, A. García-Álvarez, M. Sitges, et al., Myocardial involvement in Chagas dis-ease: insights from cardiac magnetic resonance, Int. J. Cardiol. 165 (2013) 107–112.

[16] C.E. Rochitte, P.F. Oliveira, J.M. Andrade, et al., Myocardial delayed enhancement bymagnetic resonance imaging in patients with Chagas disease: a marker of diseaseseverity, J. Am. Coll. Cardiol. 46 (2005) 1553–1558.

[17] N. Geurts, G. Opdenakker, P. Van den Steen,Matrixmetaloproteinases as therapeutictargets in protozoan parasitic infections, Pharmacol. Ther. 133 (2012) 257–279.