Lipopolysaccharide-pretreated mesenchymal stem cell-conditioned medium optimized with 10kDa filter attenuates the injury of H9c2 cardiomyocytes in a model of hypoxia/reoxygenation

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function, which is partly attributed to the released paracrine factors. Since such cardioprotection is moderate and transient, it is essential that MSC-CM's effective components are optimized to alleviate myocardial injury. To optimize MSC-CM, MSCs were treated with or without lipopolysaccharides (LPSs) for 48 h (serum-free), and the supernatant was collected. Then, LPS-CM (MSC stimulated by LPS) was further treated with LPS remover (LPS Re-CM) or was concentrated with a 10 kDa cutoff filter (10 kDa-CM). Enzyme-linked immunosorbent assay showed that all the pretreatments increased the levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin growth factor (IGF) except LPS Re-CM; 10 kDa-CM was superior to the other CMs. Cell Counting Kit-8 displayed that the viability of injured H9c2 cells was enhanced with the increase in the MSC-CM concentration. We also found that the 10 kDa-CM significantly alleviated H9c2 hypoxia/reoxygenation (H/R) injury, as evidenced by the increased Bcl-2/Bax ratio, and decreased the levels of lactate dehydrogenase and cardiac troponin. Transmission electron microscopy (TEM), TdT-mediated dUTP nick-end labelling (TUNEL), and hematoxylin and eosin staining (H&E) confirmed that 10 kDa-CM inhibited H/R-induced H9c2 morphological changes. Proteomic analysis identified 41 differentially expressed proteins in 10 kDa-CM, among which anti-inflammation, proangiogenesis, and antiapoptosis were related to cardiac protection. This study indicates that 10 kDa-CM protects H9c2 cardiomyocytes from H/R injury by preserving most of the protective factors, such as VEGF, HGF, and IGF, in MSC-CM. Key words: MSC-CM, H9c2, hypoxia/reoxygenation, paracrine factors, proteomics analysis Le milieu conditionne a partir de cellules souches mesenchymateuses (MC-SSM) permet d'ameliorer la fonction cardiaque, ce qui est partiellement attribuable a la liberation de facteurs paracrines. Comme une telle cardioprotection est moderee et transitoire, il est essentiel d'optimiser les composantes efficaces du MC-SSM pour permettre d'attenuer les lesions cardiaques. Afin d'optimiser les MC-SSM, nous avons mis les SSM en presence ou non de lipopolysaccharides (LPS) pendant 48 heures (sans serum), puis recueilli le surnageant. Ensuite, nous avons plus avant expose le MC-LPS (SSM stimulees a l'aide de LPS) a un dissolvant des LPS (MC-Diss LPS) ou encore nous l'avons concentre a l'aide d'un filtre de 10 kDa (MC-10 kDa). La methode d'ELISA a montre que toutes les expositions prealables ont entraine une augmentation des taux de VEGF, d'HGF et d'IGF a l'exception du dissolvant des LPS; le MC 10 kDa etait superieur aux autres MC. Le CCK-8 a montre que la vitalite des cellules H9c2 lesees s'accentuait avec l'augmentation de la concentration du MC-SSM. Nous avons aussi observe que le MC-10 kDa permettait d'attenuer les lesions des cellules H9c2 par l'hypoxie/reoxygenation (H/R) de facon marquee : nous avons mis en evidence une augmentation du ratio Bcl-2/Bax, de meme que la diminution des taux de LDH et de troponine cardiaque (cTn). Les techniques de microscopie electronique par transmission et de TUNEL ainsi que la coloration a l'H-E ont permis de confirmer que le MC-10 kDa entrainait une inhibition des variations morphologiques entrainees par l'H/R dans les cellules H9c2. Les etudes de proteomique ont permis d'identifier 41 proteines exprimees de maniere differentielle dans le MC-10 kDa, avec lesquelles des phenomenes anti-inflammatoires, angiogeniques et anti-apoptotiques etaient lies a une protection cardiaque. Cette etude montre que le MC-10 kDa permet de proteger les cardiomyocytes de type H9c2 contre des lesions d'H/R par l'intermediaire de facteurs de protection comme le VEGF, l'HGF et l'IGF dans le MC-SSM. Mots-cles : MC-SSM, H9c2, hypoxie/reoxygenation, facteurs paracrines, etudes de proteomique
Introduction Ischemia/reperfusion (I/R) injury is a complication after coronary recanalization in patients with acute myocardial infarction (AMI). It is an important cause of irreversible loss of myocardial tissue and ultimately [...]