Screening analysis of proteolytic enzymes and their inhibitors in the leaflets of epoxy-treated bioprosthetic heart valves explanted due to dysfunction

Abstract

Bioprosthetic heart valves (BHVs) are known for their lower thrombogenicity rates and excellent hemodynamic parameters similar to native valves. However, the lifespan of these medical devices is limited to 15 years due to the structural valve degeneration. One of the mechanisms underlying functional impairment and calcification of BHVs includes proteolytic degradation of biomaterials. However, proteases found in xenogeneic BHVs tissue remain poorly studied. In this study using the dot blot assay, we have performed a screening analysis of proteolytic enzymes and their inhibitors in the leaflets of five BHVs explanted due to their dysfunction. Five aortic valves (AVs) explanted due to calcific aortic valve disease were studied as a comparison group. The results of the study have demonstrated that at least 17 proteases and 19 of their inhibitors can be found in BHVs. In the AVs 20 proteases and 21 their inhibitors were identified. Small quantitative differences were noted between proteomic profiles of the BHVs and AVs. Matrix metalloproteinases (MMPs) were expressed in BHVs and AVs at comparable levels, but the level of tissue inhibitors of metalloproteinases-1/-2 and RECK protein in implant tissues was lower than in natural valves. Probably, excessive activity of MMPs cannot be counterbalanced by their inhibitors in BHVs and therefore MMPs can degrade prosthetic biomaterial. Moreover, the detection of a wide range of proteolytic enzymes and their inhibitors in the degenerated BHVs suggests the existence of several pathophysiological pathways that can lead to structural valve degeneration.Bioproteznye klapany serdtsa (BKS) kharakterizuiutsia nizkoĭ trombogennost'iu i prevoskhodnymi gemodinamicheskimi parametrami, priblizhennymi k takovym estestvennykh klapanov. Odnako sroki funktsionirovaniia étikh meditsinskikh izdeliĭ ogranicheny v srednem 15 godami, poskol'ku ikh biologicheskaia sostavliaiushchaia podverzhena strukturnoĭ degeneratsii. Odnim iz mekhanizmov, otvetstvennykh za razrushenie i kal'tsifikatsiiu BKS, iavliaetsia proteoliticheskaia degradatsiia biomateriala. V nastoiashchee vremia spektr proteaz, vstrechaiushchikhsia v ksenotkaniakh implantatov, ostaetsia slaboizuchennym. Posredstvom dot-blottinga nami proizveden skriningovyĭ analiz proteoliticheskikh fermentov i ikh ingibitorov v stvorkakh piati BKS, éksplantirovannykh po prichine disfunktsiĭ. V kachestve gruppy sravneniia ispol'zovali piat' aortal'nykh klapanov (AK), udalennykh iz-za kal'tsiniruiushchego aortal'nogo stenoza. Rezul'taty issledovaniia pokazyvaiut, chto v BKS prisutstvuet po men'sheĭ mere 17 proteaz i 19 ikh ingibitorov, togda kak v AK vyiavleno 20 i 21 soedinenie sootvetstvenno. Proteomnye profili nativnykh i proteznykh stvorok imeli nebol'shie kolichestvennye razlichiia. V chastnosti, matriksnye metalloproteinazy (MMP) byli ékspressirovany v BKS i AK na sopostavimykh urovniakh, togda kak produktsiia tkanevykh ingibitorov metalloproteinaz-1/-2 i RESK-belka v proteznykh stvorkakh okazalas' nizhe, chem v estestvennykh. Éto ukazyvaet na to, chto aktivnost' MMP v BKS, veroiatno, ne reguliruetsia ingibitorami, spetsifichnymi dlia étogo semeĭstva fermentov, vsledstvie chego oni mogut rasshchepliat' skleroproteiny ksenotkani, vyzyvaia postepennoe razrushenie proteznogo biomateriala. Obnaruzhenie v éksplantirovannykh BKS shirokogo spektra proteoliticheskikh fermentov i ikh ingibitorov predpolagaet sushchestvovanie mnogikh patofiziologicheskikh puteĭ, kotorye sposobny privodit' k strukturnoĭ degeneratsii implantatov.