Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

Abstract

ObjectivesIncreased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1.Methods and ResultsPlasma LDL samples isolated from asymptomatic hypercholesterolemic (LDL cholesterol [LDL-C] levels, 154.6±20 mg/dL; n = 7) patients and normocholesterolemic (LDL-C levels, 86.1±21 mg/dL; P<0.001; n = 7) control individuals were chromatographically resolved into 5 subfractions, L1-L5. The L5 percentage (L5%) and the plasma L5 concentration ([L5] = L5% × LDL-C) in the patient and control groups were 8.1±2% vs. 2.3±1% (P<0.001) and 12.6±4 mg/dL vs. 1.9±1 mg/dL (P<0.001), respectively. In hypercholesterolemic patients treated with atorvastatin for 6 months (10 mg/day), [L5] decreased from 12.6±4 mg/dL to 4.5±1.1 mg/dL (P = 0.011; n = 5), whereas both [L5] and L5% returned to baseline levels in 2 noncompliant patients 3 months after discontinuation. In cultured human aortic ECs (HAECs), L5 upregulated CRP expression in a dose- and time-dependent manner up to 2.5-fold (P<0.01), whereas the least electronegative subfraction, L1, had no effect. DiI-labeled L1, internalized through the LDL receptor, became visible inside HAECs within 30 seconds. In contrast, DiI-labeled L5, internalized through LOX-1, became apparent after 5 minutes. L5-induced CRP expression manifested at 30 minutes and was attenuated by neutralizing LOX-1. After 30 minutes, L5 but not L1 induced reactive oxygen species (ROS) production. Both L5-induced ROS and CRP production were attenuated by ROS inhibitor N-acetyl cysteine.ConclusionsOur results suggest that CRP, L5, and LOX-1 form a cyclic mechanism in atherogenesis and that reducing plasma L5 levels with atorvastatin disrupts the vascular toxicity of L5.

Interplay between CRP, Atherogenic LDL, and LOX-1 and Its Potential Role in the Pathogenesis of Atherosclerosis

“…Although Fc␥Rs were initially postulated to mediate the effects of CRP outside of complement activation, evidence has indicated that both LOX-1 and Fc␥Rs mediate some effects of CRP (30 ). Importantly, OxLDL and plasma L5 isolated from hypercholesterolemic patients have been shown to induce the release of CRP from human aortic endothelial cells in a LOX-1-dependent manner, whereas native LDL has no effect (32,33 ).…”

“…Thus, we have proposed that CRP and LOX-1 form a cyclic mechanism with OxLDL or L5 in atherogenesis (Fig. 2), whereby increased levels of atherogenic LDL in patients with cardiovascular risks induce endothelial cells to express CRP, which in turn increases the expression of LOX-1 to promote the uptake of each respective lipoprotein into endothelial cells (33 ). To support the notion that LOX-1 and CRP potentially function together in cardiovascular pathogenesis, we have shown a simple comparison between CRP and LOX-1 structure and function in Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol62/issue2.…”

“…As mentioned above, increased levels of CRP have been detected in the supernatant of cultured human aortic endothelial cells treated with OxLDL or L5. L5 has also been shown to increase the expression of the CRP transcriptional activator TNF-␣ in endothelial cells (22,33 ). For studies with OxLDL, CRP was detected in the supernatant of cultured cells by using liquid chromatography combined with multiple-reaction monitoring mass spectrometry (MRM-MS).…”

Human electronegative LDL induces mitochondrial dysfunction and premature senescence of vascular cells in vivo

“…The most electronegative subfraction of LDL can be obtained using fast‐protein liquid chromatography with anion‐exchange columns to fractionate human LDL into five subfractions with increasing electronegativity, called L1 to L5 (Chen et al., 2003; Ke et al., 2011). The most electronegative subfraction, L5, is the only LDL subfraction that can induce endothelial dysfunction and atherogenic responses in cultivated arteries and cultured vascular cells or impair normal differentiation of endothelial progenitor cells (Chen et al., 2007; Chu et al., 2013; Lu et al., 2008; Stancel et al., 2016). These biochemical properties of L5 have been attributed to its lipid and protein composition, enzymatic activities, and structural features (Ke, Stancel, Bair, & Chen, 2014; Ke et al., 2016).…”

Estudo da interação de peptídeos mimotopos da LDL eletronegativa com células endoteliais e macrófagos

“…O reconhecimento da LDLox via LOX-1 se dá pelo domínio lectina-like, com grande importância dos resíduos de aminoácidos básicos presentes nesta região, promovendo uma interação eletrostática com a carga negativa da LDLox (CHEN et al, 2001). A LDL (-) também é um ligante de LOX-1, promovendo a produção de espécies reativas de oxigênio pelas células endoteliais e aumento da expressão de mediadores inflamatórios (CHU et al, 2013).…”