Heart Care Info - Heart Disease Prevention & Treatment

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

C Besler, K Heinrich, L Rohrer, C Doerries... - J Clin ..., 2011 - Am Soc Clin Investig Christian Besler 1, 2, 3, Kathrin Heinrich 1, 2, 3, Lucia Rohrer 3, 4, Carola Doerries 1, 2, 3, Meliana Riwanto 1, 2, 3, Diana M. Shih 5, Angeliki Chroni 6, Keiko Yonekawa 1, 2, Sokrates Stein 1, 2, 3, Nicola Schaefer 1, 2, 3, Maja Mueller 1, 2, Alexander Akhmedov 1, 2, 3, ...

Low HDL cholesterol levels are associated with an increased risk of CAD and major cardiovascular events (1, 2). Besides promotion of cholesterol efflux from macrophage foam cells (3, 4), stimulation of e NOS-dependent NO production (7–10, 18, 21, 36) and mediation of endothelial repair mechanisms (11–15), as well as antiinflammatory effects (55), have been proposed as potential antiatherogenic properties of HDL. HDL-raising therapies are currently being intensely evaluated for prevention and treatment of CAD.

The present study demonstrates that HDL from patients with either s CAD or an ACS — in contrast to HDL from healthy subjects — fails to stimulate endothelial NO production. Of note, reduced endothelial NO bioavailability is thought to contribute to development and progression of atherosclerosis, at least in part by promoting endothelial inflammatory activation (22, 23). In this respect, the inability of HDL from patients with CAD to stimulate endothelial NO production, as observed in the present study, suggests a loss of this potential antiatherogenic property of HDL (22).

Our findings suggest that stimulation of endothelial NO production is important for HDL’s ability to inhibit inflammatory activation of endothelial cells, i.e., to reduce NF-κB activity, VCAM-1 expression, and monocyte adhesion, which are thought to play a role in the development and progression of atherosclerosis (56, 57). In addition, HDL-induced stimulation of endothelial repair was not observed in e NOS-deficient mice, suggesting a role of e NOS in this process. We therefore examined the mechanisms underlying alterations of these potential endothelial-atheroprotective effects of HDL in patients with CAD.

Endothelial superoxide production, which may limit NO bioavailability (39), was not increased in response to HDLCAD, suggesting that the altered endothelial NO response resulted from an impaired capacity of HDLCAD to stimulate e NOS activation rather than from a stimulation of endothelial superoxide production. In previous studies it has been shown that e NOS activation in response to HDL is mediated via Akt-dependent e NOS phosphorylation at Ser1177 (8). Notably, in the present study we have observed that HDLCAD in contrast to HDL Healthy activates endothelial PKCβII, leading to inhibition of Akt-dependent e NOS-activating phosphorylation at Ser1177 and increased phosphorylation of e NOS at Thr495, which inhibits e NOS activity. Our findings further indicated that increased endothelial PKCβII activation by HDLCAD was involved in the impaired effects of HDLCAD on e NOS-derived NO production and was mediated, at least in part, via the endothelial LOX-1 receptor. Endothelial PKCβ activation has been observed to promote endothelial dysfunction, to inhibit Akt-dependent e NOS-activating phosphorylation at Ser1177, to increase e NOS-inhibiting phosphorylation at Thr495 (45–48, 58), and to promote endothelial inflammatory activation (49).

Endothelial SR-BI mediates specific endothelial binding of HDL (44) and stimulates pathways leading to the activation of e NOS in response to the lipoprotein (7, 8, 11, 17). In the present study we observed that knockdown of endothelial SR-BI by RNA interference reduced specific binding of HDL from both healthy subjects and patients with CAD to a similar extent. These findings are compatible with the notion that the interaction of HDL with SR-BI for endothelial binding of HDL is not significantly impaired in patients with CAD. Moreover, recent studies have observed that HDL-mediated cholesterol efflux contributes to e NOS activation in response to HDL (10, 16, 18). In the present study we did not observe a significant difference in the macrophage cholesterol efflux capacity of circulating HDL from patients with CAD and healthy subjects. These findings are compatible with the concept that the impaired capacity of circulating HDL to stimulate endothelial NO production is related to endothelial LOX-1–dependent PKCβII activation rather than to a major impairment in SR-BI binding or cholesterol efflux. This concept is further supported by the observation that the capacity of HDLCAD to stimulate endothelial e NOS-activating pathways and endothelial NO production could be restored, at least in part, by either endothelial LOX-1 blockade or endothelial PKCβII inhibition.

It is likely that the impairment of the macrophage cholesterol efflux capacity of HDL — which has been reported to occur after more extensive oxidative modification of HDL, for example, in atherosclerotic plaque (32, 33) — is a pathophysiological mechanism that is more operative within the atherosclerotic lesion. Indeed, HDL isolated from atherosclerotic plaques had an impaired capacity to stimulate macrophage cholesterol efflux (32, 33).

In addition, we aimed to identify alterations in HDLCAD leading to the gain of endothelial LOX-1– and PKCβII-activating properties and the subsequent inhibition of endothelial e NOS-activating pathways. In patients with CAD, the MDA content of HDL was increased, and modification of HDL Healthy with MDA impaired the capacity of HDL to stimulate endothelial NO production, and this was at least in part mediated via the endothelial LOX-1 receptor. Notably, PON1 has been observed to prevent MDA formation in HDL (52) and to promote HDL-mediated inactivation of oxidized lipids in LDL (52, 59, 60). In addition, oxidized LDL has been suggested to activate endothelial PKCβ (50). These observations raised the possibility that PON1 may be involved in the gain of endothelial LOX-1– and PKCβII-activating properties of HDLCAD. Furthermore, in a recent study in patients with APS, we observed a relationship between reduced serum PON1 activity and endothelial dysfunction (37). We therefore evaluated whether a reduced PON1 activity may have a causal role in the impaired effects of HDL on endothelial NO production.

In the present study, HDL-associated PON1 activity was markedly reduced in patients with CAD, and we demonstrate that impaired HDL-associated PON1 activity is involved in the gain of endothelial PKCβII-activating properties of HDL, leading to a subsequently impaired capacity to stimulate e NOS-activating pathways and endothelial NO production. Consistent with these findings, HDL isolated from Pon1-deficient mice failed to stimulate endothelial NO production. Furthermore, supplementation of HDL from patients with CAD or Pon1-deficient mice with purified PON1 partially improved the capacity of HDL to stimulate endothelial NO production. These findings suggest a role of HDL-associated PON1 activity in maintaining the endothelial-atheroprotective effects of HDL, at least in part by preventing formation of the lipid peroxidation product MDA. Our studies are in line with the concept that PON1 prevents MDA formation in HDL rather than directly degrading MDA, which may explain why we observed only a partial effect of PON1 supplementation on the capacity of HDLCAD to stimulate endothelial NO production under physiological conditions. Furthermore, it is conceivable that PON1 also acts on other chemical entities in the HDL particle that impact its endothelial effects, which will need to be explored further in future studies.

Notably, in recent studies it has been observed that modification of reconstituted HDL (i.e., apo A-I and phospholipids) with myeloperoxidase leads to an impairment of its cholesterol efflux and antiinflammatory properties, likely due to a modification of apo A-I (61). Given that reconstituted HDL does not contain PON1, the question arises how this can be reconciled with a role of PON1 for the dysfunction of HDL. Importantly, however, a more recent study has described a critical role of apo A-I modification by MDA in the ability of apo A-I to promote cellular cholesterol efflux (33). These findings suggest that although PON1 prevents lipid oxidation of HDL and the subsequent formation of advanced lipid oxidation products, such as MDA, it thereby likely also prevents modifications of HDL-associated proteins such as apo A-I.

Moreover, in experimental studies PON1 has been observed to prevent atherosclerotic lesion development (62, 63), and in recent clinical studies reduced serum activity of paraoxonase was associated with increased cardiovascular risk (64, 65). The present study for the first time to our knowledge provides evidence that HDL-associated PON1 activity plays a role in maintaining the endothelial-atheroprotective effects of HDL, i.e., the capacity to stimulate endothelial NO production. However, the molecular mechanisms leading to PON1 inactivation in HDL from patients with CAD and its causal role in the development and progression of CAD need to be further examined in future studies. Notably, in this respect several studies have suggested a role of myeloperoxidase in modification of proteins in HDL, which has been shown in particular for apo A-I, but may potentially also apply to other HDL-associated proteins (32, 66).

Our findings raise the question of whether the impaired capacity of HDL from patients with CAD to stimulate e NOS-activating pathways and NO production may play a role in development and/or progression of atherosclerosis or is a consequence of the disease process. As mentioned above, numerous studies have suggested that endothelial dysfunction, in particular reduced endothelial NO bioavailability, promotes atherosclerotic lesion development, and endothelial dysfunction has been shown to be a predictor of an increased risk of clinical cardiovascular events at later stages of the disease (23, 67). These observations raise the possibility that the altered effects of HDL on endothelial NO production may favor development and progression of atherosclerotic cardiovascular disease. However, the relationship between altered biological functions of HDL and endothelial dysfunction and cardiovascular risk will have to be characterized in future studies.

In the present study we examined the endothelial effects of HDL in bioassays using cultured endothelial cells. However, we have also observed stimulation of endothelial repair in vivo after arterial injury in mice receiving an injection of HDL from healthy subjects, but not after administration of HDL from patients with CAD. Moreover, these effects of HDL on endothelial repair in vivo were dependent on e NOS activation, since they were not observed in e NOS-deficient mice, suggesting that HDL also activates e NOS-dependent endothelial responses in vivo. In addition, in a previous study we observed improved endothelium-dependent, NO-mediated vasodilation in patients with hypercholesterolemia after infusion of reconstituted HDL (19), further supporting the concept that HDL may activate e NOS-dependent NO production in vivo.

The influence of different non-HDL-targeted medications used in patients with CAD on the effects of HDL on e NOS-dependent pathways is largely unknown and needs to be studied in the future. In a recent study by Ansell et al. (35), it was suggested that the proinflammatory effects of HDL from patients with CAD were attenuated by statin therapy. However, in the present study we did not observe a significant difference in the effects on endothelial NO production between HDL from a subgroup of patients with and a subgroup without a certain medication (i.e., statins, beta blocker, and ACE-I/ARB). In fact, the direct proinflammatory properties of HDL from patients with CAD are likely not related to an e NOS-dependent mechanism, since they were also observed in the present study using HDL from patients with s CAD that did not result in a significant inhibition of endothelial NO production. The mechanisms underlying these proinflammatory properties of HDLCAD need to be further characterized in future studies. A recent study has suggested that the hemoglobin/haptoglobin pathway is involved in the formation of proinflammatory HDL in atherosclerotic mice and patients with CAD (68).

Notably, in a recent large clinical outcome trial testing the effects of the CETP inhibitor torcetrapib, which results in substantially elevated HDL cholesterol levels, a significant increase in cardiovascular events and in total mortality was observed (31). While off-target effects of the drug likely played a role, these findings also raise the possibility that the biological activity of on-treatment HDL in addition to its plasma levels need to be taken into account for the development of HDL-raising treatment strategies. The findings of the present study further support the notion that not only the capacity of HDL-targeted treatment strategies to raise HDL plasma levels, but also their effects on vasoprotective properties of HDL, need to be considered. It is likely that HDL-raising therapeutic strategies are more likely to result in cardiovascular protection when the HDL in treated patients itself exerts vasoprotective effects.

In summary, the present study provides evidence that HDL of patients with either s CAD or ACS fails to stimulate endothelial e NOS-activating pathways and NO production. As a result, the endothelial antiinflammatory and endothelial repair effects of HDL are impaired. Notably, in contrast to HDL from healthy subjects, HDL from patients with CAD is characterized by a gain of stimulatory effects on endothelial LOX-1 and PKCβII that inhibit e NOS-activating pathways. Moreover, our findings suggest that impaired HDL-associated PON1 activity plays a role in endothelial activation of PKCβII by HDL from patients with CAD as well as for impaired effects of HDL from patients with CAD on e NOS-activating pathways and e NOS-dependent endothelial antiinflammatory and repair effects. These findings support the concept that the biological functions of HDL in addition to its plasma levels may have to be taken into account to assess the cardiovascular effects of HDL-raising therapies in patients with CAD.

Patient population and blood sampling. Blood samples were obtained from patients with s CAD or ACS (ST elevation myocardial infarction [STEMI] or non-ST elevation myocardial infarction [NSTEMI]) and healthy subjects (without cardiovascular risk factors) between 40 and 70 years of age who were consecutively recruited into the study at the University Hospital of Zurich. The diagnosis of s CAD and an ACS was made according to guidelines of the American Heart Association (69, 70). In brief, patients with ACS (STEMI and NSTEMI) were recruited if they presented within 12 hours after the onset of symptoms and were in a fasting state for at least 12 hours. Patients were excluded from the study if there was evidence for accompanying infectious, inflammatory or autoimmune disorders, diabetes, advanced kidney or liver failure, neoplastic disorders, and a history of major surgery or trauma within the previous month. Age- and sex-matched healthy control subjects were consecutively enrolled by the Blood Donation Service of the University Hospital Zurich and had no cardiovascular risk factors (according to history, clinical examination, and laboratory tests) or accompanying disorders. All subjects gave written informed consent prior to inclusion in the study, and the study was approved by the local ethics committee (Kantonale Ethik-Kommission, Zurich, Switzerland).

Pon1- and e NOS-deficient mice. Pon1-deficient mice were produced by targeted disruption of exon 1 of the Pon1 gene, as described previously (62), and backcrossed more than 10 times onto the C57BL/6J background. Blood for the isolation of HDL from Pon1-deficient and wild-type mice was collected from the retroorbital plexus after a fasting period of 16 hours and immediately centrifuged to obtain serum. e NOS-deficient mice were purchased from The Jackson Laboratory.

Isolation of HDL. HDL from subjects with s CAD or an ACS, healthy controls, and wild-type and Pon1-deficient mice was isolated by sequential ultracentrifugation (d = 1.063–1.21 g/ml) according to the method of Havel et al. (9, 36, 71) using solid potassium bromide (Merck) for density adjustment. Blood samples were processed within 1 hour after collection. HDL2 and HDL3 (d = 1.110–1.210 g/ml) were separated as described previously (9, 36).

Endothelial monocyte adhesion. CD14-positive monocytes were isolated by Ficoll density gradient centrifugation (Vacutainer CPT, BD) and labeled with CFSE (Molecular Probes, Invitrogen). HAE Cs were preincubated with HDL (50 μg/ml) for 1 hour and stimulated with TNF-α (1 ng/ml) for 3 hours. Afterward, cell culture medium was changed, and monocytes (50,000/well) were added to the HAEC monolayer. After 3 hours, adherent CFSE-labeled monocytes were counted on 4 randomly selected high-power fields using a fluorescence microscope (DM-IRB, Leica).

Detection of nuclear translocation of the NF-κB subunit Rel A/p65 by immuno­cytochemistry and quantification of DNA-bound p65 in nuclear extracts of endothelial cells. Nuclear translocation of the NF-κB subunit Rel A/p65 was detected by staining cells with mouse anti-Rel A/p65 (Santa Cruz Biotechnology Inc.) and analyzing the amount of cytoplasmic Rel A/p65 by an SP2 confocal microscope (Leica). Binding of activated NF-κB in the nuclear extracts to an oligonucleotide containing an NF-κB consensus binding site was detected by a Rel A/p65 transcription factor assay (Trans AM kit, Active Motif).

In vivo endothelial repair assay. Male NRM Inu/nu athymic nude mice, aged 7–10 weeks, were used to allow injection of HDL (15 mg HDL protein/kg body weight). Animals were anesthetized with ketamine (100 mg/kg i.p.) and xylazine (5 mg/kg i.p.). Carotid artery electric injury was performed as described previously (36, 42, 43). In brief, the left common carotid artery was injured with a bipolar microregulator (ICC 50, ERBE-Elektromedizin Gmb H). An electric current of 2 W was applied for 2 seconds to each millimeter of carotid artery over a total length of exactly 4 mm with the use of a size marker parallel to the carotid artery. HDL was injected 3 hours after carotid injury via tail vein injection with a 27-gauge needle. The same volume of PBS was injected into control mice. Three days after carotid injury, endothelial repair was evaluated by staining denuded areas with 50 μl of solution containing 5% Evans blue dye via tail vein injection. The re-endothelialized area was calculated as the difference between the blue-stained area and the injured area by computer-assisted morphometric analysis. Of note, this model has been shown to achieve accurate quantification of endothelial repair (36, 42, 43). All animal protocols were approved by the animal care and use committee (Kantonales Veterinäramt, Zurich, Switzerland).

Characterization of cellular cholesterol efflux capacity of HDL. Total and ABCA1-dependent efflux of [14C]cholesterol to HDL was measured using J774 macrophages treated with or without the c AMP analog 8-(4-chloro­phenylthio)adenosine-3′,5′-cyclic monophosphate (cpt-c AMP). The ABCG1-dependent cholesterol efflux capacity of HDL was measured in HEK293 cells transfected with an ABCG1-expressing plasmid or a mock plasmid.

Inhibition of endothelial LOX-1 by blocking antibody. The endothelial LOX-1 receptor was inhibited by a commercially available blocking antibody (R&D Systems), as described previously (72). In brief, HAE Cs were incubated with the blocking antibody (15 μg/ml) for 2 hours, and afterward endothelial cells were subjected to ESR spectroscopy or Western blot analysis.

Detection of protein-bound MDA in HDL by spectrophotometry. Free and protein-bound MDA in HDL was detected by a commercially available lipid peroxidation assay kit (AL Detect, Enzo Life Sciences) involving an acid hydrolysis step in the presence of butylated hydroxytoluene (BHT) to allow the quantification of Schiff base MDA adducts. Hydrolysis of Schiff base MDA adducts in HDL was carried out after addition of hydrochloric acid at p H 1–2 and heating at 60°C for 80 minutes.

MDA modification of HDL. MDA was freshly prepared by rapid acid hydrolysis of malonaldehyde bis-(dimethyl acetal) and used for in vitro modification of HDL according to previously published protocols (33). Briefly, maloncarbonyl bis-(dimethylacetal) was mixed at 1:10 with 1 M H Cl and incubated for 1 hour at 50°C under gentle agitation. The reaction mixture was dissolved with phosphate buffer (10 m M, p H 7.4), and the concentration of MDA was measured spectrophotometrically, as described previously (33). HDL (0.2 mg protein/ml) was incubated with freshly prepared MDA for 24 hours at 37°C in 50 m M sodium phosphate buffer (p H 7.4) containing 100 μm DTPA.

Statistics. All data are expressed as mean ± SEM. Statistical comparisons were made by the nonparametric Wilcoxon 2-sample test (paired analysis) or the nonparametric Mann-Whitney U test, and a P value less than 0.05 was considered statistically significant. Bonferroni adjustment was performed in the comparisons of the clinical cohorts. All analyses were performed with Graph Pad Prism software (version 4.0).

We thank Bernd Roschitzki (Functional Genomics Center Zurich), Ines Bühler, Simone Kaufmann, and the staff of the Andreas Grüntzig Catheterization Laboratory at the University Hospital Zurich for their excellent technical assistance. This work was supported by two Swiss National Research Foundation grants (310030-122339 and 3100A0-116404/1), the Swiss Heart Foundation, the Zurich Center for Integrative Human Physiology, the Roche Research Foundation, an educational grant from Pfizer, the Leducq Foundation, the European Union (HD Lomics: LSHM-CT-2006-037631), and U.S. Public Health Service grant HL-30568.