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Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis

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Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis Ghada Al-Salih1, Nawwar Al-Attar2,3, Sandrine Delbosc1, Liliane Louedec1, Elisabeth Corvazier1, Ste´phane Loyau1, Jean-Baptiste Michel1,3, Dominique Pidard1,4, Xavier Duval5, Olivier Meilhac1,3,6* 1 INSERM U698, Paris, France, 2 Cardiovascular Surgery Department, Bichat Hospital, AP-HP, Paris, France, 3 University Paris Diderot, Sorbonne Paris Cite´, Paris, France, 4 Institut National des Sciences du Vivant, Centre National de la Recherche Scientifique, Paris, France, 5 INSERM CIC 007, Paris, France, 6 Bichat Stroke Centre, AP-HP, Paris, France Abstract Aims: Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization. Methods and Results: IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). The presence of neutrophil extracellular traps (NETs) associating MPO and externalized nucleosomes, was shown by immunostaining in the VG. Both MPO and cell-free DNA were released in larger amounts by VG than N samples, suggesting bacterial activation of PMNs within the vegetation. Finally, evidence of proteolytic tissue damage was obtained by the release of fragments of extracellular matrix components such as fibrinogen and fibronectin, as well as protease-sensitive receptors such as the uPA receptor. Conclusion: Our data obtained using human IE valves suggest that septic vegetations represent an important source of proteases originating from massive leukocyte recruitment and activation of the host plasminergic system. The latter forms a potential therapeutic target to minimize valvular tissue degradation independently from that induced by bacterial proteases. Citation: Al-Salih G, Al-Attar N, Delbosc S, Louedec L, Corvazier E, et al. (2012) Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis. PLoS ONE 7(9): e45695. doi:10.1371/journal.pone.0045695 Editor: Partha Mukhopadhyay, National Institutes of Health, United States of America Received February 7, 2012; Accepted August 22, 2012; Published September 20, 2012 Copyright: ß 2012 Al-Salih et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction leads to formation of ‘‘clusters’’ protected by layers of fibrin which constitute an important morphological feature of the lesion [3]. Infective endocarditis (IE) is characterized by vegetative lesions Compared with fibrinoid vegetations of IE, the structure and consisting of microbial colonization of a damaged valvular biology of non-infected thrombi, such as those observed in human endocardium (native valve IE), or of an intracardiac prosthesis abdominal aortic aneurysms (AAA), have been studied in more (prosthetic IE). IE is associated with high mortality rates (15–20%), detail. These mural thrombi which maintain a constant interface and high costs, related to the duration of antibiotic therapy and the with the circulating blood, are a source of proteases [4], including need for surgery in half of the patients [1,2]. The infected matrix metalloproteinases (MMPs), leukocyte elastase [5], and vegetation is the elementary lesion of IE. It is formed by successive proteases of the plasminergic system [6]. These host proteases are appositions of fibrinoplatelet thrombi, incorporating pathogens capable of inducing both degradation of extracellular matrix and leukocytes [3]. The characteristics of the bacterial population (matrilysis) and apoptosis of smooth muscle cells subsequent to the that colonizes the vegetation may explain the limited efficacy of loss of cell adherence (anoikis) [5,6,7]. For example, laminin, antibiotics sometimes observed in the treatment of human IE. vitronectin, and fibronectin can be cleaved by plasmin and After the initial phase of bacterial adhesion and colonization, elastase. In IE vegetations, proteolytic and pro-oxidant activities pathogens proliferate within the forming vegetation. The non- associated with leukocyte activation are similar to those observed homogenous distribution of bacteria within the vegetation often in uninfected mural thrombi (i.e. AAA) [4], although the infectious PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45695 Host Proteases in Human Infective Endocarditis nature of thrombi in IE is responsible for a greater biological Conditioned Media activity due to enzymatic activities of the microorganisms The pieces of fresh VG and N tissue samples were weighed and themselves, but also due to the massive recruitment of innate then incubated separately in RPMI (Gibco, Grand Island, NY) immune cells. This recruitment of leukocytes may therefore containing antibiotics for 24 h at 37uC in a ratio of 10 mL per mg constitute an additional deleterious insult, related to the cytotox- of wet tissue [8]. Conditioned media were then collected and icity of proteolytic enzymes or radical forms of oxygen [8]. centrifuged (3000 g for 10 min at 20uC). Supernatants were stored Moreover, bacteria may also represent a source of proteases, at 280uC until further analysis, and the tissue samples were including matrilytic gelatinases [9] that can induce vascular cell frozen. Tissue conditioned media were then used for determina- anoikis [10,11]. They can also be procoagulant (staphylocoagulase) tion of secreted protease activity, and for assessment of the and fibrinolytic (staphylokinase and streptokinase are able to presence of soluble fibrinogen, fibronectin and uPAR fragments. convert plasminogen into plasmin) [12]. The generation of Analyses were performed on all samples available for the each plasmin by bacterial plasminogen activators may also favor experiment, limiting the number of samples in certain cases as weakening of the surrounding tissue and thus promote bacterial detailed in Figure S1. invasion. The aim of this study was to characterize leukocyte-driven Immunohistochemistry and Immunohistofluorescence proteolytic activity as well as proteases associated with thrombus Paraformaldehyde-fixed endocarditic valve samples were em- formation/degradation, and to assess their pathogenic effect on bedded in paraffin, and cut into 5 mm-thick sections. Immunohis- the valvular tissue/myocardium. We thus analyzed the septic tochemistry was performed using primary antibodies targeting thrombi that constitute the vegetations in human endocarditis by platelet-specific marker, GPIb (anti-CD42b; dilution 1:200, histological and biochemical approaches. Immunotech, Marseille, France), as well as primary antibodies targeting components of the fibrinolytic system: tissue-type Methods plasminogen activator (anti-tPA ESP5, dilution 1:1000, American Diagnostica, Greenwich, CT), urokinase-type plasminogen acti- Tissue Samples vator (anti-uPA #3689, dilution 1:500, American Diagnostica), Thirty-nine valve samples were obtained from patients operated uPA receptor (anti-uPAR #3932, dilution 1:1000, American for IE, conforming with the principles outlined in the Declaration Diagnostica), and human plasminogen (anti-Pg Ab-1, dilution of Helsinki. The study was approved by INSERM Ethics 1:20, Neomarker, Fremont, CA). Anti-CD66b (dilution 1:25, Committee. Before surgery, all patients were informed that Immunotech, Marseille, France) to detect granulocytes, anti- surgical wastes would be used for this study. A verbal consent elastase (dilution 1:100, HBT, HM2174, Uden, The Netherlands) was obtained from each patient and a certificate of non-opposition and anti-MMP9 (dilution 1:50, Abcam, ab58803, Cambridge, was signed by the treating physician. This is the standard UK) antibodies were also used. procedure for the use of surgical waste for research purposes Antigen retrieval was performed with Tris/EDTA, pH 9.0 according to the French ethical laws (L. 1211-2 to L. 1211-7, L. (anti-CD42b, anti-tPA, anti-Pg, anti-CD66b, anti-MMP-9) or with 1235-2 and L. 1245.2 - August 6th 2004). (Table 1). citrate 0.1 M, pH 6.0 (anti-uPA, anti-uPAR, anti-elastase) at 95uC, endogenous peroxidase was blocked with 3% (v/v) H2O2 in Dissection of Valves into Vegetation and Adjacent aqueous solution, and nonspecific binding was blocked with 1% Apparently
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