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Title: Factors modulating the progression of aortic stenosis - association with inflammation, activation of the coagulation/fibrinolysis system and calcification


Background Aortic stenosis (AS) is the most common cause of acquired valvular heart disease in individuals over 65 years of age, with no available pharmacological treatment. The pathomechanism of AS is a complex and tightly regulated process involving the activation of multiple molecular, cellular, and tissue pathways. Numerous studies indicate that elevated levels of lipoproteins, oxidized phospholipids (OxPL), or coexisting type 2 diabetes (DMT2) may accelerate the development of AS. However, the underlying mechanisms by which these factors influence inflammation, coagulation and fibrinolysis pathways, and aortic valve calcification have not been fully elucidated. Aims To investigate the mechanisms involving factors favouring AS progression, such as hyperglycemia and increased oxidative stress, with particular emphasis on their potential associations with inflammation, coagulation activation, hypofibrinolysis, and calcification in patients with severe AS. Methods and Results In Publication 1, 76 patients with severe AS (without DMT2) and 50 patients with AS and DMT2 (AS-DM) were studied. Valvular expression of advanced glycation end products (AGEs) and receptor for AGEs (RAGE) was evaluated by immunofluorescence. Levels of AGEs and soluble isoform of RAGE (sRAGE) in serum were assessed using ELISA tests. AS-DM patients exhibited increased accumulation of AGEs and RAGE wi ; thin stenotic aortic valves and in serum compared to patients without DMT2. Furthermore, in the AS-DM group, the expression of AGEs and RAGE, as well as their serum levels, correlated with glycosylated hemoglobin (HbA1c) levels. Importantly, in AS-DM patients, valvular expression of AGEs and serum AGE levels were associated with disease severity. Publication 2 evaluated 100 patients with AS (without DMT2) and 50 patients with AS and DMT2 (AS-DM). Valvular expression of nuclear factor kappa B (NF-κB), bone morphogenetic protein-2 (BMP-2), prothrombin (FII), and activated factor X (FXa) was assessed by immunofluorescence. The expression of NF-κB and BMP-2 in valve interstitial cells (VICs) in vitro cultures stimulated with glucose was evaluated at the protein and mRNA (RELA) levels. Mechanistic studies were performed using a reactive oxygen species inhibitor (NAC) or NF-κB pathway inhibitor (BAY 11-7082). Increased expression of NF-κB, BMP-2, and coagulation proteins was observed in AS-DM valves compared to those without DM. Valvular expression of NF-κB and BMP-2 positively correlated with FII and FXa expression. Only in patients with DM, valvular NF-κB expression was associated with HbA1c and blood fructosamine levels, as well as AS severity. In vitro experiments demonstrated that high glucose concentrations increased NF-κB and BMP-2 expression, while the use of ROS (NAC) o ; r NF-κB (BAY 11– 7082) inhibitors significantly reduced their expression. Analysis of mRNA (RELA) expression in VICs confirmed these results. In Publication 3, 50 patients with AS and lipoprotein(a) [Lp(a)] concentration ≥50 mg/dl were evaluated, along with 20 patients with AS and Lp(a) <50 mg/dl. Valvular OxPL expression was assessed using immunofluorescence. The levels of OxPL, plasminogen activator inhibitor type 1 (PAI-1) antigen, and thrombin-activated fibrinolysis inhibitor (TAFI) were evaluated using ELISA tests. Clot lysis time (CLT) in plasma was determined turbidimetrically. AS patients with Lp(a) ≥50 mg/dl had increased OxPL valvular expression and higher serum levels of OxPL compared to patients with AS and Lp(a) <50 mg/dl. Valvular OxPL expression correlated with serum OxPL and Lp(a) levels. Only in patients with Lp(a) ≥50 mg/dl, serum OxPL levels correlated with CLT, PAI-1 and TAFI concentrations, as well as the severity of AS. Multifactorial regression analysis showed that higher levels of OxPL were associated with prolonged CLT in patients with AS and Lp(a) ≥50 mg/dl. In Publication 4, 75 patients with severe AS were examined. Valvular lipid accumulation was assessed histochemically, while the expression of PAI-1 and NF-κB was evaluated using immunofluorescence. Valves obtained from autopsy donors served as controls. The expression of PAI-1 in V ; ICs cultured under different conditions was assessed at the protein and mRNA (SERPINE1) levels. Mechanistic studies were performed using an inhibitor of PAI-1 activity (TM5275) or an NF-κB pathway inhibitor (BAY 11-7082). VICs culture supernatants were added to PAI-1 – depleted plasma, and CLT was measured using a modified method. The level of PAI-1 antigen in the supernatants was determined using an ELISA test. Valvular expression of PAI-1, which correlated with lipid accumulation, NF-κB expression, and the severity of AS, was observed only in stenotic aortic valves. In vitro, VICs showed high PAI-1 expression. LDL stimulation increased the level of PAI-1 in VICs supernatants and prolonged CLT. Inhibition of PAI-1 activity shortened CLT, while inhibition of the NF-κB pathway reduced PAI-1 expression in VICs, the level of its antigen in supernatants, and shortened CLT. Analysis of mRNA expression (SERPINE1) in VICs confirmed these results. Publication 5 discusses the latest potential therapeutic strategies for inhibiting the progression of AS. Conclusions In patients with AS and concomitant poorly controlled DMT2, uncontrolled diabetes leads to excessive accumulation of AGEs in aortic valves, thereby exacerbating local oxidative stress, inflammation, and the synthesis of valvular calcification mediators, ultimately resulting in faster progression of AS. Hypergly ; cemia adversely affects processes related to AS progression by enhanced in loco activation of the NF-κB pathway. Our study suggests that maintaining HbA1c and fructosamine within the reference range in patients with AS and concomitant DMT2 may retard the rate of AS progression. Patients with severe AS exhibit hypofibrinolysis. High levels of serum Lp(a) are associated with elevated levels of OxPL, and better than Lp(a) predict hypofibrinolysis. Additionally, the overexpression of PAI-1 within stenotic valves, caused by lipid accumulation, is mediated by enhanced activation of the NF-κB pathway and, at least in part, contributes to hypofibrinolysis. Therefore, our results suggest that the NF-κB pathway may be a potential therapeutic target in AS.

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2 - studia doktoranckie

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Rada Dyscypliny Nauki medyczne


Natorska, Joanna

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Jun 4, 2024

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ZB-140242 Jun 4, 2024


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