Effect of irreversibly glycated LDL in human vascular smooth muscle cells: lipid loading, oxidative and inflammatory stress

Abstract

The major complication of diabetes is accelerated atherosclerosis, the progression of which entails complex interactions between the modified low-density lipoproteins (LDL) and the cells of the arterial wall. Advanced glycation end product-modified-LDL (AGE-LDL) that occurs at high rate in diabetes contributes to diabetic atherosclerosis, but the underlying mechanisms are not fully understood. The aim of this study was to assess the direct effect of AGE-LDL on human vascular smooth muscle cells (hSMC) dysfunction. Cultured hSMC incubated (24 hrs) with human AGE-LDL, native LDL (nLDL) or oxidized LDL (oxLDL) were subjected to: (i) quantification of the expression of the receptors for modified LDL and AGE proteins (LRP1, CD36, RAGE) and estimation of lipid loading, (ii) determination of NADPH oxidase activity and reactive oxygen species (ROS) production and (iii) evaluation of the expression of monocyte chemoattractant protein-1 (MCP-1). The results show that exposure of hSMC to AGE-LDL (compared to nLDL) induced: (a) increased NADPH oxidase activity (30%) and ROS production (28%) by up-regulation of NOX1, NOX4, p22phox and p67phox expression, (b) accumulation of intracellular cholesteryl esters, (c) enhanced gene expression of LRP1 (160%) and CD36 (35%), and protein expression of LRP1, CD36 and RAGE, (d) increased MCP-1 gene expression (160%) and protein secretion (300%) and (e) augmented cell proliferation (30%). In conclusion, AGE-LDL activates hSMC (increasing CD36, LRP1, RAGE), inducing a pro-oxidant state (activation of NADPHox), lipid accumulation and a pro-inflammatory state (expression of MCP-1). These results may partly explain the contribution of AGE-LDL and hSMC to the accelerated atherosclerosis in diabetes.

Bilberries exert an anti-atherosclerotic effect in lipid-loaded macrophages

“…Published data show that oxLDL, but not native LDL, upregulate the expression of NOX4/p22 phox in human macrophages, mediated by MEK1/ERK pathway, upstream messengers of NF-κB signaling, but the upstream components of this signaling mechanism in macrophages have not yet been clearly identified [5]. Our experiments showed that BE inhibits NF-κB in oxLDLloaded macrophages, a signaling pathway responsible for the inhibition of p22 phox and NOX4 expression, similar to the one described in human aortic smooth muscle cells [4]. Unlike the other subunits of Nox complex, NOX1 and NOX2, which demand regulatory subunits for their activation, NOX4 generates ROS constitutively, and changes in its levels may directly affect Nox activity [26].…”

“…The generation of increased intracellular reactive oxygen species (ROS) was associated with oxLDL uptake by macrophages [3]. Moreover, oxLDL is known to induce the expression of NADPH oxidase subunits NOX4/p22 phox and NOX2/p47 phox /p22 phox in human smooth muscle cells [4] and macrophages [5,6], thus generating intracellular oxidative stress. Macrophages are key mediators in the immune response and their physiological protective function is harmfully activated in the atherosclerotic process [7].…”

“…LDL and HDL 3 fractions were isolated from human plasma of healthy donors from Blood Transfusion Center Bucharest by using sequential flotation ultracentrifugation on an Optima LE-80 ultracentrifuge (Beckman Coulter, CA, USA) [4]. Cu-oxidized LDL (oxLDL) was prepared and characterized as previously described [4].…”

“…Samples were amplified in triplicate by using specific primers for the genes of interest and β-actin (as housekeeping gene), as previously reported [4,13], in a StepOne Plus RealTime PCR System (Applied Biosystems, Carlsbad, CA, USA); relative quantification was achieved by using the Fit-Point method [17] based on SYBR-Green I detection (Invitrogen, Carlsbad, CA, USA).…”

Are oxidised low-density lipoproteins the true inducers of inflammation in atherosclerosis?

“…In addition, the presence of AGES and glycosylated LDL in the lesion will be detected by RAGE expressed on macrophages and promote inflammation. Thus, the presence of other forms of modified LDL inside atheromas will be enough to justify a dangerous and accelerated evolution of atheromas in people with diabetes, smokers and patients with chronic kidney disease [99,127,128]. Several canonical ligands of TLRs can potentially be present in the lesions and mediate the polarisation of macrophages towards M1 functions.…”