[Screening analysis of proteolytic enzymes and their inhibitors in the leaflets of epoxy-treated bioprosthetic heart valves explanted due to dysfunction]. / Skriningovyi analiz proteoliticheskikh fermentov i ikh ingibitorov v stvorkakh époksiobrabotannykh bioproteznykh klapanov serdtsa, éksplantirovannykh po prichine disfunktsii.

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.

Endothelial Dysfunction in the Context of Blood-Brain Barrier Modeling.

Here, we discuss pathophysiological approaches to the defining of endothelial dysfunction criteria (i.e., endothelial activation, impaired endothelial mechanotransduction, endothelial-to-mesenchymal transition, reduced nitric oxide release, compromised endothelial integrity, and loss of anti-thrombogenic properties) in different in vitro and in vivo models. The canonical definition of endothelial dysfunction includes insufficient production of vasodilators, pro-thrombotic and pro-inflammatory activation of endothelial cells, and pathologically increased endothelial permeability. Among the clinical consequences of endothelial dysfunction are arterial hypertension, macro- and microangiopathy, and microalbuminuria. We propose to extend the definition of endothelial dysfunction by adding altered endothelial mechanotransduction and endothelial-to-mesenchymal transition to its criteria. Albeit interleukin-6, interleukin-8, and MCP-1/CCL2 dictate the pathogenic paracrine effects of dysfunctional endothelial cells and are therefore reliable endothelial dysfunction biomarkers in vitro, they are non-specific for endothelial cells and cannot be used for the diagnostics of endothelial dysfunction in vivo. Conceptual improvements in the existing methods to model endothelial dysfunction, specifically, in relation to the blood-brain barrier, include endothelial cell culturing under pulsatile flow, collagen IV coating of flow chambers, and endothelial lysate collection from the blood vessels of laboratory animals in situ for the subsequent gene and protein expression profiling. Combined with the simulation of paracrine effects by using conditioned medium from dysfunctional endothelial cells, these flow-sensitive models have a high physiological relevance, bringing the experimental conditions to the physiological scenario.