Diabetes concomitant to aortic stenosis is associated with increased expression of NF-κB and more pronounced valve calcification

Abstract

Aims/hypothesis: Type 2 diabetes has been demonstrated to predispose to aortic valve calcification. We investigated whether type 2 diabetes concomitant to aortic stenosis (AS) enhances valvular inflammation and coagulation activation via upregulated expression of NF-κB, with subsequent increased expression of bone morphogenetic protein 2 (BMP-2).

Methods: In this case-control study, 50 individuals with severe isolated AS and concomitant type 2 diabetes were compared with a control group of 100 individuals without diabetes. The median (IQR) duration of diabetes since diagnosis was 11 (7-18) years, and 36 (72%) individuals had HbA_1c ≥48 mmol/mol (≥6.5%). Stenotic aortic valves obtained during valve replacement surgery served for in loco NF-κB, BMP-2, prothrombin (FII) and active factor X (FXa) immunostaining. In vitro cultures of valve interstitial cells (VICs), isolated from obtained valves were used for mechanistic experiments and PCR investigations.

Results: Diabetic compared with non-diabetic individuals displayed enhanced valvular expression of NF-κB, BMP-2, FII and FXa (all p ≤ 0.001). Moreover, the expression of NF-κB and BMP-2 positively correlated with amounts of valvular FII and FXa. Only in diabetic participants, valvular NF-κB expression was strongly associated with serum levels of HbA_1c, and moderately with fructosamine. Of importance, in diabetic participants, valvular expression of NF-κB correlated with aortic valve area (AVA) and maximal transvalvular pressure gradient. In vitro experiments conducted using VIC cultures revealed that glucose (11 mmol/l) upregulated expression of both NF-κB and BMP-2 (p < 0.001). In VIC cultures treated with glucose in combination with reactive oxygen species (ROS) inhibitor (N-acetyl-L-cysteine), the expression of NF-κB and BMP-2 was significantly suppressed. A comparable effect was observed for VICs cultured with glucose in combination with NF-κB inhibitor (BAY 11-7082), suggesting that high doses of glucose activate oxidative stress leading to proinflammatory actions in VICs. Analysis of mRNA expression in VICs confirmed these findings; glucose caused a 6.9-fold increase in expression of RELA (NF-κB p65 subunit), with the ROS and NF-κB inhibitor reducing the raised expression of RELA by 1.8- and 3.2-fold, respectively.

Conclusions/interpretation: Type 2 diabetes enhances in loco inflammation and coagulation activation within stenotic valve leaflets. Increased valvular expression of NF-κB in diabetic individuals is associated not only with serum HbA_1c and fructosamine levels but also with AVA and transvalvular gradient, indicating that strict long-term glycaemic control is needed in AS patients with concomitant type 2 diabetes. This study suggests that maintaining these variables within the normal range may slow the rate of AS progression.

Keywords: Aortic stenosis; Bone morphogenetic protein 2; Coagulation factors; Diabetes mellitus; Inflammation; NF-κB; Oxidative stress.

Fig. 1

The expression of NF-κB and BMP-2 within stenotic aortic valves in participants with AS and concomitant type 2 diabetes compared with participants with AS without diabetes. (a) Representative microphotographs of valvular NF-κB and BMP-2 expression (red arrowheads indicate aortic side of the leaflet; yellow arrowheads indicate the immunopositive area of expression). Scale bar, 200 μm. (b, c) Box plots showing valvular expression of NF-κB (b) and BMP-2 (c). Values are medians (IQR). **p<0.01 and ***p<0.001 vs non-DM; ^†††p<0.001 vs DM with HbA_1c <48 mmol/mol (<6.5%). (d, e) Associations between valvular expression of NF-κB and BMP-2 in participants with AS with (d) and without (e) concomitant diabetes. DM, AS with concomitant type 2 diabetes; Non-DM, AS without concomitant diabetes

Fig. 2

Associations between valvular NF-κB expression and serum markers of glycaemic control in participants with AS and concomitant type 2 diabetes. Scatterplots represent the correlation between valvular NF-κB expression and serum levels of glucose (a), valvular NF-κB expression and serum concentrations of HbA_1c (b), and valvular NF-κB expression and serum levels of fructosamine (c)

Fig. 3

The expression of valvular FII and FXa within stenotic aortic valves in participants with AS and concomitant type 2 diabetes compared with participants with AS without diabetes. (a) Representative microphotographs of valvular FII and FXa expression (red arrowheads indicate aortic side of the leaflet; yellow arrowheads indicate the immunopositive area of expression). Scale bar, 200 μm. (b) Bar graph showing valvular expression levels of FII and FXa. Values are medians (IQR).***p<0.001 vs non-DM. (c–f) The scatterplots show correlations between valvular NF-κB and FII (c), NF-κB and FXa (d), BMP-2 and FII (e), and BMP-2 and FXa (f) in participants with AS and concomitant type 2 diabetes. DM, AS with concomitant type 2 diabetes; Non-DM, AS without concomitant diabetes

Fig. 4

Associations between valvular expression of inflammatory, calcification and coagulation factors and disease severity in participants with AS and concomitant type 2 diabetes. The scatterplots show correlations between valvular NF-κB and AVA (a), valvular NF-κB and PG_max (b), valvular BMP-2 and AVA (c), valvular BMP-2 and PG_max (d), valvular FII and AVA (e), valvular FII and PG_max (f), valvular FXa and AVA (g), and valvular FXa and PG_max (h)

Fig. 5

Plasma levels of TF and FVIIa-AT in participants with AS and concomitant type 2 diabetes. (a) Bar graphs showing plasma levels of TF and FVIIa-AT in diabetic participants with HbA_1c <48 mmol/mol (<6.5%) and HbA_1c ≥48 mmol/mol (≥6.5%). Values are medians (IQR). *p<0.05 vs DM with HbA_1c <48 (mmol/mol). (b–d) The scatterplots show correlations between serum levels of fructosamine and plasma concentrations of FVIIa-AT (b), serum concentrations of HbA_1c and plasma levels of TF (c) and serum levels of fructosamine and plasma levels of TF (d). DM, type 2 diabetes

Fig. 6

The influence of glucose (11 mmol/l) and specific inhibitors of ROS (NAC) and transcription pathway NF-κB (BAY 11-7082 [BAY]) on the expression of NF-κB and BMP-2 in VICs isolated from aortic stenotic valves obtained during surgery. (a) Representative microphotographs of immunostaining in VIC cultures. Scale bar, 20 μm. (b) Relative expression of RELA in VIC cultures after stimulation. Data are presented as mean±SD