Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease - PubMed (original) (raw)

. 2011 Jul;121(7):2693-708.

doi: 10.1172/JCI42946. Epub 2011 Jun 23.

Kathrin Heinrich, Lucia Rohrer, Carola Doerries, Meliana Riwanto, Diana M Shih, Angeliki Chroni, Keiko Yonekawa, Sokrates Stein, Nicola Schaefer, Maja Mueller, Alexander Akhmedov, Georgios Daniil, Costantina Manes, Christian Templin, Christophe Wyss, Willibald Maier, Felix C Tanner, Christian M Matter, Roberto Corti, Clement Furlong, Aldons J Lusis, Arnold von Eckardstein, Alan M Fogelman, Thomas F Lüscher, Ulf Landmesser

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Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease

Christian Besler et al. J Clin Invest. 2011 Jul.

Abstract

Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.

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Figures

Figure 1

Figure 1. Effects of HDL from healthy subjects, patients with sCAD or ACS on endothelial cell NO production, as determined by ESR spectroscopy analysis.

(A) HDL from healthy subjects and patients with sCAD or ACS was isolated by sequential ultracentrifugation, and the effects of HDL (50 μg/ml) on endothelial cell NO production were analyzed by ESR spectroscopy analysis (n = 25 per group). Data are expressed as percent change versus buffer-treated cells; data points for each study participant are shown. (B) Representative ESR spectra of the NO-Fe(DETC)2 signal in endothelial cells treated with HDL from healthy subjects and patients with sCAD or ACS. (C) Increasing physiological concentrations of HDL (25, 50, and 100 μg/ml) from healthy subjects stimulated endothelial NO production that was not observed with increasing concentrations of HDL from patients with sCAD or ACS (n = 12 per group). (D) Effects of HDL2 (d = 1.063–1.125 g/ml) and HDL3 (d = 1.125–1.21 g/ml) from patients with sCAD or ACS and healthy subjects, separated by sequential ultracentrifugation, on endothelial cell NO production (n = 12–15 per group).

Figure 2

Figure 2. Effects of HDL from healthy subjects and patients with sCAD and ACS on endothelial Akt activation, eNOS-activating/inhibiting phosphorylation, and endothelial superoxide production.

(A) Effects of HDL (50 μg/ml) on phosphorylation of Akt at Ser473, (B) Akt-dependent activating phosphorylation of eNOS at Ser1177, and (C) inhibitory phosphorylation of eNOS at Thr495 in HAECs, as detected by Western blot analysis (n = 10–12 per group). (D) Effects of HDL on endothelial superoxide production (n = 25 per group; data points for all study participants are shown) and (E) NAD(P)H oxidase activity, as measured by ESR spectroscopy (n = 8–10 per group). (F) Representative ESR spectra of NAD(P)H oxidase activity in HAECs treated with buffer (basal) and with HDL from healthy subjects or patients with sCAD or ACS.

Figure 3

Figure 3. Critical role of eNOS in the effects of HDL on TNF-α–stimulated endothelial NF-κB activation and VCAM-1 expression.

(A) Inhibition of eNOS-mediated endothelial NO production by siRNA knockdown of eNOS or treatment with L-NAME (1 mM) prevented the effects of HDL (50 μg/ml) from healthy subjects on endothelial NF-κB activation (as examined by TNF-α–stimulated DNA binding of NF-κB subunit p65; n = 8–10 per group). Scr., scrambled. (B) In contrast to HDL from healthy subjects, HDL from patients with sCAD and ACS failed to inhibit TNF-α–stimulated endothelial NF-κB activation (n = 10–12 per group). (C) Representative images (original magnification, ×20) showing that HDL from a healthy subject, but not from patients with sCAD or ACS, inhibited endothelial nuclear translocation of NF-κB subunit p65, as detected by fluorescence microscopy of TNF-α–stimulated HAECs. (D) Inhibition of eNOS-mediated NO production by siRNA knockdown of eNOS or treatment with L-NAME prevented the effects of HDL from healthy subjects on TNF-α–stimulated VCAM-1 expression in HAECs, as detected by Western blot analysis (n = 8–10 per group). (E) Effect of HDL from healthy subjects and patients with sCAD or ACS on TNF-α–stimulated VCAM-1 expression in HAECs (n = 25 per group; data points for all study participants are shown).

Figure 4

Figure 4. Role of eNOS for the effects of HDL on TNF-α–stimulated endothelial monocyte adhesion.

(A) Inhibition of eNOS-mediated NO production by siRNA-specific knockdown of eNOS or treatment with L-NAME (1 mM) prevented the potent inhibitory effects of HDL from healthy subjects on endothelial cell monocyte adhesion (n = 6–8 per group). Endothelial cells were incubated with HDL (50 μg/ml) from healthy subjects, and binding of CFSE-labeled human monocytes was analyzed after 3 hours by fluorescence microscopy (HAECs were stimulated with TNF-α; 5 ng/ml). (B) Representative photographs (original magnification, ×10) of endothelial monocyte adhesion obtained by fluorescence microscopy are shown. (C) Effects of HDL isolated from patients with sCAD and ACS as compared with HDL from healthy subjects on TNF-α–stimulated endothelial monocyte adhesion (n = 25 per group; data points for all study participants are shown).

Figure 5

Figure 5. Impaired endothelial repair capacity and specific endothelial binding of HDL from patients with sCAD or ACS as compared with healthy subjects.

(A) HDL from healthy subjects and patients with sCAD or ACS was injected in nude mice following carotid artery injury, and re-endothelialized area was determined after 3 days by staining with Evan’s blue (n = 6–8 per group). (B) Representative photographs of carotid arteries from nude mice stained with Evan’s blue. (C) HDL from healthy subjects and patients with sCAD or ACS was labeled with sodium iodide I-125, and binding of 125I-HDL to endothelial cells was examined (n = 8–10 per group). (D) Effect of siRNA-specific knockdown of SR-BI on endothelial binding of HDL from healthy subjects and patients with CAD (n = 4–5 per group).

Figure 6

Figure 6. Differential effects of HDL from healthy subjects and patients with sCAD or ACS on endothelial LOX-1 and PKCβII activation — role of PKCβII activation in the altered effects of HDL on endothelial Akt and eNOS-activating/inhibiting phosphorylation in patients with CAD.

(A) Effect of HDL from healthy subjects and patients with sCAD and ACS on PKCβII-activating phosphorylation at Ser660 and (B) membrane translocation of PKCβII in endothelial cells (n = 8–12 per group). (C) Incubation of endothelial cells with the nonselective inhibitor of PKCβI and PKCβII isoforms LY379196 and CGP53353, a highly selective inhibitor of PKCβII, restored the ability of HDL from patients with CAD to stimulate endothelial NO production and (D) the activating eNOS phosphorylation at Ser1177 (n = 8–10 per group). (E) Pretreatment of endothelial cells with an anti–LOX-1 blocking antibody prevented the increase in PKCβII phosphorylation at Ser660 (n = 6–8 per group). (F) Moreover, incubation of endothelial cells with an anti–LOX-1 blocking antibody improved the capacity of HDL from patients with CAD, but not from healthy subjects, to stimulate endothelial NO production (n = 8–10 per group).

Figure 7

Figure 7. Critical role of HDL-associated MDA in the impaired capacity of HDL to increase endothelial NO production and in the increased stimulation of PKCβII by HDL.

(A) Protein-bound MDA content in HDL from healthy subjects and patients with sCAD or ACS was measured by spectrophotometry, as described in more detail in Methods (n = 12–14 per group). (B) Mass spectrometry analysis of MDA-lysine adducts in HDL from healthy subjects and patients with CAD (n = 4 per group). (C) Effect of MDA-modified HDL on endothelial NO production, as measured by ESR spectroscopy (n = 8 per group). (D) Inhibition of endothelial LOX-1 with a specific blocking antibody partially prevented the decrease in endothelial NO production in response to MDA-modified HDL (12.5 mol MDA/mol apoA-I; n = 6 per group). (E) Modification of HDL by MDA (12.5 mol MDA/mol apoA-I; n = 6 per group) resulted in an increased activation of endothelial PKCβII that was prevented by a specific blocking antibody against endothelial LOX-1 (n = 6–10 per group).

Figure 8

Figure 8. Activity and content of HDL-associated PON1 in healthy subjects and patients with sCAD or ACS — effect of inhibition of PON1 on HDL-associated MDA content and phosphorylation of PKCβII at Ser660.

(A) Paraoxonase and (B) arylesterase activities of HDL-associated PON1 isolated from healthy subjects and patients with sCAD or ACS, as measured by UV spectrophotometry (n = 25 per group). (C) PON1 content in HDL isolated from healthy subjects and patients with sCAD or ACS was determined by Western blot analysis (n = 25 per group). (D) Effect of the specific PON1 inhibitor hydroxyquinoline (HQ, 200 μM) on protein-bound MDA content and (E) MDA-lysine adducts in HDL, as detected by spectrophotometry and mass-spectrometry analysis, respectively (n = 4–6 per group). (F) Effect of HDL pretreated with the PON1 inhibitors HQ or EDTA (5 mM) on phosphorylation of PKCβII at Ser660 was detected by Western blot analysis (n = 8–10 per group).

Figure 9

Figure 9. Effect of inhibition of HDL-associated PON1 on eNOS-activating signaling pathways, endothelial cell NO production, endothelial cell inflammatory activation, and endothelial repair.

Effect of supplementation of HDL from CAD patients with recombinant PON1 on the capacity of HDL to stimulate endothelial NO production. (A) HDL-associated PON1 was inhibited either by hydroxyquinoline (200 μM) or EDTA (5 mM), and the effect of HDL on the activating eNOS phosphorylation at Ser1177 and (B) the inhibitory eNOS phosphorylation at Thr495 was examined by Western blot analysis (n = 6–7 per group). (C) Inhibition of HDL-associated PON1 resulted in a loss of the capacity of HDL to stimulate endothelial NO production, as measured by ESR spectroscopy (n = 6–8 per group). (D) Furthermore, treatment of HDL from healthy subjects with PON1 inhibitors impaired the capacity of HDL to inhibit TNF-α–stimulated endothelial monocyte adhesion (n = 8–10 per group). (E) Importantly, inhibition of PON1 in HDL from healthy subjects prevented stimulation of endothelial repair after carotid artery injury in nude mice (n = 6 per group). (F) Supplementation of HDL from patients with CAD with purified PON1 (pPON1; 5 U/100 μg HDL protein) partially restored the capacity of HDL to stimulate endothelial NO production as measured by ESR spectroscopy (n = 6–9 per group).

Figure 10

Figure 10. Effects of HDL isolated from _Pon1_-deficient mice or their wild-type littermates on endothelial cell NO production, superoxide production, and endothelial inflammatory activation.

(A) In contrast to HDL from wild-type littermates, HDL isolated from _Pon1_-deficient mice inhibited rather than stimulated NO production in mouse aortic endothelial cells, as detected by ESR spectroscopy analysis (n = 5 per group). (B) In addition, HDL from _Pon1_-deficient mice failed to inhibit basal and TNF-α–stimulated superoxide production in mouse aortic endothelial cells (ESR spectroscopy analysis, n = 5–7 per group). (C) Whereas HDL from wild-type littermates potently inhibited endothelial inflammatory activation, HDL from _Pon1_-deficient mice did not inhibit TNF-α–stimulated VCAM-1 expression and (D) endothelial monocyte adhesion (n = 5–6 per group). (E) Supplementation of HDL from _Pon1_-deficient mice with purified PON1 (5 U/100 μg HDL protein) partially restored the capacity of HDL to stimulate endothelial NO production as measured by ESR spectroscopy (n = 5–6 per group).

Comment in

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