Author + information
- Received February 1, 2016
- Revision received June 22, 2016
- Accepted July 12, 2016
- Published online October 11, 2016.
- S0735109716349245-5c1504ee193b6976eb5fd760d7a8e3d4Erik W. Holy, MD, PhDa,∗ (, )
- S0735109716349245-09ffe53e96b77a1cd182e53e73770454Alexander Akhmedov, PhDb,
- S0735109716349245-be9cedc537c0246ee11fd29c5c3858f0Thimoteus Speer, MD, PhDc,
- S0735109716349245-1960527a51a393e52a31f255b0c19de2Giovanni G. Camici, PhDb,
- S0735109716349245-d98e42b76d3422aae938a1664239e885Stephen Zewinger, MDc,
- S0735109716349245-1485f411baefcfcbfd7699244449eae4Nicole Bonetti, MDb,
- S0735109716349245-a1e58bbe34a187b12562cc81accb77c2Jürg H. Beer, MDd,
- S0735109716349245-43f492ccdaf05add8e5bc27100284e27Thomas F. Lüscher, MDa,b and
- S0735109716349245-a35e6f69faaa8e6d4295ad8ddd288cbdFelix C. Tanner, MDa,b
- aUniversity Heart Center Zurich, University Hospital Zürich, Zürich, Switzerland
- bCenter of Molecular Cardiology, University of Zürich, Zürich, Switzerland
- cDepartment of Internal Medicine 4, Saarland University Hospital, Homburg, Germany
- dDepartment of Medicine, Cantonal Hospital Baden, Baden, Switzerland
- ↵∗Reprint requests and correspondence:
Dr. Erik W. Holy, University Heart Center, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland.
Background Carbamylation alters low-density lipoprotein (LDL) structure and is thought to promote vascular inflammation and dysfunction in patients with chronic kidney disease (CKD).
Objectives This study sought to determine whether carbamylated LDL (cLDL) exerts prothrombotic effects in vascular cells and platelets and whether cLDL enhances arterial thrombus formation in vivo.
Methods LDL was isolated from healthy subjects or patients with CKD by sequential ultracentrifugation. Ex vivo carbamylation of LDL from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analyzed in a murine carotid artery photochemical injury model. Protein expression and mRNA levels were analyzed by Western blotting, flow cytometry, and real-time PCR. Platelet aggregation was measured by impedance aggregometry.
Results Intravenous administration of cLDL in mice accelerated arterial thrombus formation compared to treatment with native LDL (nLDL) or vehicle. Tissue lysates of mouse carotid arteries revealed that cLDL induced the expression of TF, PAI-1, and LOX-1 mRNA in vascular cells. In human aortic smooth muscle and endothelial cells, cLDL induced TF and PAI-1 expression. In contrast, nLDL had no effect on either cell type. While nLDL and cLDL had no aggregatory effect on resting platelets, cLDL enhanced platelet aggregation in response to different agonists. This effect was mediated by mitogen-activated protein kinase p38 phosphorylation and LOX-1 translocation to the surface. LDL isolated from patients with CKD mimicked the prothrombotic effects of cLDL on vascular cells, platelets, and thrombus formation in vivo.
Conclusions We found that cLDL induces prothrombotic effects in vascular cells and platelets by activation of the LOX-1 receptor and enhances thrombus formation in vivo. This observation reveals a new mechanism underlying the increased incidence of acute thrombotic events observed in patients with CKD and may lead to the development of new lipid-targeting therapies in this population.
Chronic kidney disease (CKD) is associated with a significant cardiovascular disease (CVD) burden. Studies have linked reduced renal function to a higher incidence of CV mortality (1). In fact, CVD is the leading cause of death in patients with CKD, particularly patients undergoing hemodialysis. In these patients, the incidence of fatal acute thrombotic events is 10-fold higher than in the general population (2,3). Largely, this increase in CV mortality occurs independently of conventional risk factors, suggesting that CKD constitutes an independent risk factor for acute thrombotic events such as myocardial infarction and ischemic stroke (4,5). Despite this evidence, the underlying pathophysiological mechanisms remain incompletely understood.
Lipid-lowering therapies stand at the forefront of primary and secondary preventions of CV events (6), yet incidence of CV events in patients with CKD remains high despite the plasma cholesterol reduction achieved by lipid-lowering therapies (7). Recent studies revealed that carbamylation is a post-translational protein modification triggered by urea-derived cyanate, and patients with CKD display markedly increased levels of carbamylated low-density lipoproteins (cLDL) (8).
Carbamylation of proteins may also be driven enzymatically at sites of inflammation, even in the absence of renal disease by the phagocyte-derived inflammatory mediator myeloperoxidase (9). The levels of plasma protein carbamylation correlate with the incidence of major CV events (9,10). Ex vivo cLDL, as well as high-density lipoproteins (HDL), have been shown to induce proatherogenic effects in vascular cells (11–15).
In this study, we hypothesized that cLDL promotes thrombus formation and represents a link between CKD and CV events.
A detailed description of Methods is provided in the Online Appendix.
Briefly, native LDL was incubated with potassium cyanate (20 mg/mg LDL) for 6 h, followed by extensive dialysis against phosphate-buffered saline (pH 7.4, 100 μM EDTA) for 36 h. Unmodified LDL was dialyzed in the same manner.
LDL was isolated from plasma of healthy human donors or patients with CKD undergoing hemodialysis by density gradient. In patients with CKD, blood samples for LDL isolation were retrieved at the time of hemodialysis. Isolation of LDL from mice was performed by ultracentrifugation similarly to isolation of human LDL particles.
Carotid artery thrombosis model
C57Bl6 mice were treated with cLDL (2 mg/kg), native low-density lipoprotein (nLDL) (2 mg/kg), or the vehicle. For experiments performed with LDL isolated from patients with CKD or healthy controls, similar concentrations of LDL were used (2 mg/kg). Mice were anesthetized, and thrombus formation was induced by photochemical injury in the exposed right carotid artery 24 h after treatment. From the onset of injury, blood flow was monitored, and cyclic flow variations were recorded. Stable vessel occlusion was defined as a blood flow below 0.1 ml/min for at least 1 min.
Human aortic smooth muscle (AoSMC) and human aortic endothelial cells (HAEC) were cultured as previously described (16). Protein expression and mRNA levels were analyzed by Western blotting and real-time PCR.
Aggregation studies were performed in citrated whole blood (1 part sodium citrate 3.2%; 9 parts whole blood) or washed platelets isolated from healthy volunteers. Aggregation was displayed as a function of time (Aggro/Link software, Chrono-Log, Havertown, Pennsylvania). Samples were incubated with nLDL (100 μg/ml) or cLDL (100 μg/ml), followed by stimulation with ADP, thrombin, or collagen.
All data are mean ± SD. Statistical comparisons were performed with unpaired Student t test, Kruskal-Wallis 1-way analysis of variance followed by post hoc Dunn’s multiple comparisons test, or 1-way ANOVA with post hoc Tukey’s multiple comparisons testing, as appropriate.
After LDL particles had been modified ex vivo, the extent of carbamylation was determined by carbamoyl-dl-lysine amino acid analysis. In ex vivo carbamylated samples, 0.28 ± 0.06 pmol carbamoyl-dl-lysine per μg of LDL was obtained, whereas no carbamoyl-dl-lysine was detectable in nLDL preparations (p < 0.01) (Online Figures 1A and 1B). No differences in lipid peroxidation, as assessed by malondialdehyde content, were observed between the cLDL and nLDL preparations (Online Figure 1C).
Arterial thrombus formation was studied in carotid arteries of 11- to 12-week-old male C57Bl6 mice treated intravenously with nLDL or cLDL for 24 h. Amino acid analysis confirmed elevated levels of LDL carbamylation in cLDL-treated mice (0.14 ± 0.17 vs. 0.002 ± 0.005 pmol carbamoyl-dl-lysine per μg of LDL; p < 0.01). Time to stable thrombotic occlusion was accelerated in animals treated with cLDL versus controls treated with vehicle (p < 0.01) (Figure 1A) but not in mice treated with nLDL. Embolization of a formed thrombus and restored blood flow above 0.1 ml/min also occurred less often in mice treated with cLDL than in controls (p < 0.05) (Figure 1B). Importantly, no significant differences in initial blood flow were observed (data not shown), and clotting assays excluded significant changes in pro-thrombin or activated partial thromboplastin time between the groups (p = 0.21; and p = 0.37, respectively; data not shown).
Real-time PCR analysis demonstrated increased tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in carotid arteries from cLDL-treated animals compared to nLDL-treated mice or controls (p < 0.05) (Figures 1C and 1D). This effect was paralleled by increased TF and PAI-1 activity in arterial tissue (p < 0.05) (Figures 1E and 1F) as well as in plasma samples (p < 0.05 for both) (Online Figures 2A and 2B). This increase in TF activity resulted in increased thrombin generation, reflected by higher thrombin-antithrombin complex levels in animals treated with cLDL versus nLDL (p < 0.01) (Online Figure 2C). In contrast to tissue lysates, no difference in TF or PAI-1 mRNA levels was observed in peripheral leukocytes isolated from mice treated with either nLDL or cLDL (Online Figures 2D and 2E).
Only cLDL (10 to 300 μg/ml), but not nLDL (10 to 300 μg/ml), markedly increased basal TF expression in AoSMC in a concentration-dependent manner (p < 0.05) (Figures 2A and 2B). Similarly, cLDL strongly induced PAI-1 expression in AoSMC (p < 0.05) (Figures 2C and 2D). Analysis of mRNA expression demonstrated that cLDL-enhanced TF and PAI-1 expression at the transcriptional level in this cell type (p < 0.05) (Figures 2E and 2F).
In HAEC, cLDL produced similar concentration-dependent increases in basal TF (p < 0.05) (Online Figure 3B) and PAI-1 expression (p < 0.05) (Online Figure 2D), whereas nLDL had no effect (Online Figures 3A and 3C).
Neither nLDL nor cLDL caused any cytotoxic effects as determined by lactate dehydrogenase release in any cell types at any of the concentrations used (data not shown).
Analysis of reactive oxygen species (ROS) production by electro spin resonance spectroscopy demonstrated that treatment with cLDL (but not nLDL) induced ROS generation in the arterial tissue lysates as well as in whole blood (n = 6; p < 0.05) (Figures 3A and 3B). To investigate whether cLDL-induced ROS generation in AoSMCs contributes to TF and PAI-1 induction, AoSMCs were incubated with the ROS scavengers polyethylene glycol (PEG)-catalase and PEG-superoxide dismutase (SOD) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI) prior to stimulation with cLDL or nLDL. None of these treatments prevented the induction of TF and PAI-1 expression by cLDL in AoSMC, suggesting this effect is independent of increased ROS generation (p < 0.05) (Figures 3C and 3D).
To determine whether LOX-1 mediates the prothrombotic effects of cLDL observed in AoSMC, LOX-1 mRNA expression levels were assessed and small interfering RNA (siRNA) knockdown experiments performed. Real-time PCR analysis demonstrated that treatment with cLDL induced LOX-1 mRNA in carotid artery tissue lysates (p < 0.05) (Online Figure 4A) but not in leukocytes (p = 0.29) (Online Figure 4B). Experiments performed in cultured AoSMCs confirmed that cLDL increased LOX-1 mRNA expression (p < 0.01) (Online Figure 4C). Inhibition of LOX-1 by siRNA specifically blunted the induction of TF and PAI-1 protein expression (p < 0.01) (Figures 4A and 4B). Moreover, cLDL but not nLDL, enhanced activation of PI3k/p110α in AoSMCs (n = 6; p < 0.05) (Figure 4C). Similarly, blockade of LOX-1 inhibited cLDL-induced PI3k/p110α activation (p < 0.05) (Figure 4D) in AoSMCs.
Recently, we identified GTPase RhoA as a downstream target of PI3k/p110α in AoSMCs. Accordingly, stimulation of AoSMCs with cLDL induced activation of the GTPase RhoA (p < 0.05) (Figure 4E) and inhibition of PI3k/p110α by specific siRNA prevented cLDL-induced activation of the GTPase RhoA (n = 6; p < 0.01) (Figure 4F). Furthermore, cLDL induced a transient phosphorylation of the mitogen-activated protein (MAP) kinases ERK and p38 (p < 0.05) (Figures 4G and 4H), but not JNK (data not shown). Overexpression of the dominant negative mutant Rho19 inhibited the cLDL-induced p38 and ERK phosphorylation, confirming that the GTPase RhoA is an upstream regulator of MAP kinase activation in cLDL-induced signaling in AoSMCs (p < 0.01) (Figures 4I and 4J). Finally, stimulation with cLDL activated the transcription factor NFκB as determined by RelA/p65 DNA binding activity (p < 0.01) (Figure 4K).
cLDL also activated the scavenger receptor LOX-1 in endothelial cells and analysis of LOX-1 mRNA expression in cultured HAEC confirmed this previous observation (p < 0.01) (Online Figure 4D). Similar to AoSMC, knockdown of LOX-1 mRNA by siRNA also prevented cLDL-induced TF and PAI-1 expression in HAEC (p < 0.05 for both) (Online Figures 4E and 4F).
Analysis of platelet aggregation was performed in whole blood and washed platelets from healthy subjects. Incubation of unstimulated whole blood samples with cLDL (100 μg/ml) or nLDL (100 μg/ml) had no effect on platelet activation (data not shown). However, incubation with cLDL (100 μg/ml), in contrast to nLDL (100 μg/ml), markedly enhanced ADP-triggered platelet aggregation (p < 0.05) (Figures 5A and 5B). Furthermore, cLDL-enhanced platelet aggregation in whole blood samples stimulated with other agonists, such as thrombin or collagen (p < 0.05) (Figure 5C). To confirm whether the effect of cLDL observed in thrombin-activated platelets was independent of thrombin-fibrinogen interaction, aggregation experiments were repeated in the absence of fibrinogen in washed platelets. In contrast to nLDL, whole blood cLDL-enhanced thrombin-induced platelet aggregation (Online Table 1). To analyze whether the LOX-1 receptor also mediates the prothrombotic effect of cLDL in platelets, similar to what is seen in vascular cells, LOX-1 surface expression was analyzed by flow cytometry. While LOX-1 is barely expressed on the surface of resting platelets, activation with ADP induced a rapid 1.5- to 2-fold increase in LOX-1 surface expression (p < 0.05 vs. 0 min) (Figure 5D). Inhibition of ADP-induced p38 MAP-kinase phosphorylation with the inhibitor SB302580 suppressed LOX-1 translocation to the cell surface (Figure 5D). Blockade of LOX-1 with an inhibitory antibody abrogated the prothrombotic effect of cLDL on ADP-induced platelet aggregation (p < 0.05) (Figure 5E). The effect of cLDL on platelets was paralleled by an increase in ADP-induced platelet p38 phosphorylation (p < 0.05) (Figure 5F) and incubation of platelets with a LOX-1-blocking antibody (anti-LOX-1) prevented p38 activation by cLDL (Figure 5F) suggesting a positive feedback loop mechanism underlying the effect of cLDL on activated platelets.
To confirm the physiological relevance of these observations, we investigated the degree of carbamylation and potential prothrombotic effects of LDL isolated from patients with end-stage renal disease (ESRD) (Online Table 2). Amino acid analysis demonstrated that LDL from patients with CKD, but not from age-matched healthy controls, displayed markedly increased levels of carbamoyl-dl-lysine (p < 0.001) (Figure 6A). In vivo time to stable thrombotic occlusion was accelerated in animals treated with LDL from patients with CKD as compared to LDL from healthy subjects (p < 0.05) (Figure 6B). Additionally, analysis of cyclic flow variations revealed that thrombus embolization was less frequent in mice treated with LDL isolated from patients with ESRD, suggesting more stable thrombus formation in these mice compared to controls or mice treated with healthy donor LDL (p < 0.05) (Figure 6C). LDL from patients with CKD induced expression of TF and PAI-1 in AoSMC (p < 0.05) (Figures 6D and 6E). Finally, platelet aggregation in response to ADP stimulation was significantly increased in platelets incubated with LDL from patients with CKD (p < 0.05) (Figure 6F).
For the first time, we demonstrated that LDL modified by carbamylation in vitro and in vivo in patients with CKD exerted potent prothrombotic effects on endothelial and vascular smooth muscle cells as well as platelets via the LOX-1 receptor (Central Illustration). This led to activation of PI3K/p110α, GTPase RhoA, the MAP kinases p38 and ERK, and eventually of the transcription factor NFκB, which then induces expression of TF and PAI-1, enhancing arterial thrombus formation and carotid artery occlusion in vivo.
Accumulation of LDL in the arterial intima is a critical step in the initiation of atherosclerotic lesions, as it modulates vascular hemostasis and triggers local inflammation as well as arterial thrombus formation (17). In the vessel wall, LDL is susceptible to post-translational modification by interaction with free radicals as well as with enzymes, such as myeloperoxidase, lipoxygenase, or phospholipase A2 (18). Such post-translational modifications generate structurally modified LDL particles exhibiting proinflammatory and proatherogenic properties by scavenger receptors (17,18).
Urea- or myeloperoxidase-driven carbamylation of lipoprotein lysine residues were recently recognized as a new form of lipoprotein modification altering the structure and function of LDL and HDL particles (19,20). Experimental studies have also revealed that cLDL and cHDL induce leukocyte-endothelial cell interaction, endothelial dysfunction, and cholesterol accumulation in macrophages by scavenger receptors (10–13,15,21). Moreover, not only are serum levels of carbamylated proteins and carbamyllysine increased in patients with coronary artery disease and CKD, they also correlate with all-cause mortality and the incidence of CV events in these patient populations (9,10).
Thrombus formation on top of ruptured atherosclerotic plaque is the key step in the development of acute CV events. Typically, plaques with high TF content and a thin fibrous cap are prone to rupture. Colocalization of TF with lipid-rich areas strongly suggests that LDL particles are implicated in inducing TF expression in atherosclerotic plaques (21). Consistent with this, modified lipoproteins, such as oxidized LDL, promote the expression and activity of TF and PAI-1 in human vascular cells (22–24).
Our study demonstrated that cLDL potently induced expression of TF and PAI-1 in cultured endothelial and vascular smooth muscle cells, the major source of TF and PAI-1 in the vessel wall. Most importantly, this effect occurred at concentrations of cLDL reported in patients with chronic renal failure (8,10).
The scavenger receptor LOX-1 is the predominant endothelial receptor for cLDL (10,11). Binding cLDL to LOX-1 enhances the expression of adhesion molecules on the endothelial surface, mediating cLDL’s deleterious effects on endothelial dysfunction (10,11). In AoSMCs, LOX-1 is also a receptor for oxidized LDL (25,26), but it remains unknown whether LOX-1 functions as a receptor for cLDL in this cell type. The current experiments demonstrated that LOX-1 knockdown prevented cLDL-mediated TF and PAI-1 induction in AoSMC and HAEC. Thus, LOX-1 acted as a functional receptor for cLDL in these cells, extending its known role in atherosclerosis to prothrombotic effects. Of note, cLDL but not nLDL enhanced TF and PAI-1 expression at the transcriptional level involving p38, ERK, and NFκB. Both MAP kinases and NFκB are important regulators of TF and PAI-1 expression in vascular cells (27–30). Additionally, cLDL induced activation of the GTPase RhoA in AoSMC and incubation with a dominant negative mutant prevented cLDL-induced p38 and ERK activation, confirming GTPase RhoA as an upstream regulator of p38 and ERK in these cells. The roles of RhoA activation and NFκB regulation of TF and PAI-1 have previously been described (16,27). Recently PI3K/p110α was identified as a crucial regulator of TF and PAI-1 expression by GTPase RhoA and NFκB, and pharmacological inhibition of PI3K/p110α prevented arterial thrombosis by down-regulating TF and PAI-1 expression and activity within the arterial wall (16). Thus, our results supported the critical role of PI3K/p110α in regulating TF and PAI-1 expression in AoSMCs and demonstrated that targeting PI3K/p110α prevents cLDL-induced TF and PAI-1 expression as well as RhoA activation. Hence, PI3K/p110α represents a novel therapeutic target to prevent expression of prothrombotic mediators in vascular cells and, in particular, in atherosclerotic lesions in which cLDL induce a proinflammatory state.
The current study investigated for the first time the impact of cLDL on platelet aggregation in whole-blood samples and washed platelets. Although cLDL alone had almost no effect on platelet activation (data not shown), it significantly enhanced platelet aggregation in response to agonists such as ADP, collagen, or thrombin. Previous studies showed that LOX-1 is barely expressed on the surface of resting platelets, but stimulation with agonists, such as thrombin or ADP, induced rapid translocation of LOX-1 from intracellular granules to the plasma membrane leading to enhanced binding of oxidized LDL to platelets (31). To test whether this mechanism would also account for the effect of cLDL on platelet aggregation observed here, LOX-1 expression was analyzed by flow cytometry. Within 1 min, ADP rapidly induced LOX-1 expression on the surface of activated platelets. The MAP kinase p38 is an important regulator of platelet activation as it mediates the signals necessary for platelets to aggregate in response to oxLDL, ADP, thrombin, and collagen (32–34). Inhibition of p38 phosphorylation prevented the induction of LOX-1 surface expression, suggesting that p38 mediates activation-dependent LOX-1 expression in platelets. Blockade of LOX-1 further confirmed the critical role of the LOX-1 receptor in mediating the enhancing effect of cLDL on agonist-induced platelet aggregation. Analysis of the molecular pathways revealed that cLDL enhances phosphorylation of the p38 MAP-kinase in activated platelets, an effect mediated by LOX-1.
Altogether, these results supported the concept that, while resting platelets do not express LOX-1 on their surface, they will once activated, enabling binding of modified lipoproteins, such as cLDL, thereby enhancing platelet aggregation. Thus, cLDL appears to potentiate platelet response to agonists rather than induce aggregation directly themselves; therefore, cLDL may propagate thrombus formation once platelets are activated. Indeed, accumulating evidence supports the role of LOX-1 in regulating platelet activation: LOX-1 expression colocalizes with platelets in coronary endarterectomy specimens isolated from patients with acute coronary syndrome, and LOX-1 plays an important role in ADP-induced platelet integrin binding to fibrinogen (31,35).
cLDL induces endothelial dysfunction by generating ROS by activation of NADPH-oxidase and endothelial nitric oxide synthase uncoupling (10). Because AoSMCs generate ROS by activation of NADPH oxidase upon stimulation with oxLDL and oxidative stress induces both TF and PAI-1 expression, we investigated whether ROS generation mediates the effect of cLDL on TF and PAI-1 expression in AoSMCs. Treatment with the ROS scavengers SOD and catalase, as well as pharmacological inhibition of NADPH, did not prevent cLDL-induced expression of TF and PAI-1, demonstrating that the prothrombotic effect of cLDL in AoSMC occurred in a ROS-independent manner.
To study the in vivo impact of our in vitro findings, we compared the effect of cLDL and nLDL on thrombus formation in a mouse model of arterial thrombosis. Treatment with cLDL at a concentration of 2 mg/kg of body weight accelerated time to thrombotic occlusion, while occlusion time was not affected in animals treated with nLDL or vehicle. Previous studies demonstrated that cLDL accumulates in the arterial vessel wall at this concentration inducing relevant vascular effects (11).
In line with the results obtained in cultured human cells, acceleration of thrombotic occlusion in cLDL-treated mice was paralleled by an increase in TF and PAI-1 in arterial tissue but not in peripheral leukocytes. These in vivo results further supported the interpretation that cLDL exerts prothrombotic effects by induction of TF and PAI-1 in arterial tissue (17,36). In the studied photochemical injury model, time to stable occlusion, defined as sustained absence of blood flow (<0.1 ml/min), is commonly considered a primary endpoint. However, before achieving sustained occlusion of the carotid artery, several episodes of clot formation and subsequent embolization are observed. These cyclic flow variations reflect unstable flow patterns and thrombus instability attributable to thrombolytic activity. Therefore, tracking and analyzing these variations may provide physiologically meaningful data regarding frequency and magnitude of thrombus formation, in addition to time to occlusion. Analysis of flow variations demonstrated that in animals treated with cLDL or LDL isolated from patients with CKD, not only was time to occlusion significantly accelerated but also embolization of a thrombus and restoration of blood flow above 0.1 ml/min occurred less often in those mice.
Cell culture and in vivo experiments demonstrated that LDL isolated from patients with CKD enhanced TF, PAI-1, platelet aggregation, and injury-induced thrombus formation similar to ex vivo carbamylated LDL. As expected, almost no carbamoyl-dl-lysine could be detected in age-matched healthy controls. Notably, the levels of carbamylation of cLDL used for cell culture experiments were similar to those measured in patients, which strongly supports the clinical relevance of the current results.
As for every translational and experimental study, limitations need to be considered. Although animal studies and in vitro experiments are extremely helpful in understanding underlying pathophysiological mechanisms and may direct clinical trials in the future, one must be very careful in translating those results to humans. In the current study, analysis of LDL samples was performed in patients with ESRD only and whether LDL from patients with moderate or severe CKD exerts similar prothrombotic effects remains unknown.
Our data indicated that cLDL induced important prothrombotic effects in vascular cells and platelets by activation of the LOX-1 receptor. Ultimately these effects translated into enhanced arterial thrombus formation in an established in vivo model of thrombosis. These findings revealed new potential mechanisms underlying cardiovascular events in particular in patients with severe renal failure and may lead to new lipid and LOX-1 targeting strategies in those patients.
COMPETENCY IN MEDICAL KNOWLEDGE: In patients with end-stage renal disease, carbamylation-induced alteration of LDL structure promotes vascular dysfunction and exerts prothrombotic effects by activation of the LOX-1 receptor.
TRANSLATIONAL OUTLOOK: Future studies should explore the mechanisms underlying cardiovascular events in patients with CKD and assess the therapeutic potential of strategies targeting the LOX-1 receptor.
For an expanded Methods section as well as supplemental figures and tables, please see the online version of this article.
The study was supported by grants from the Swiss National Science Foundation and the Swiss Heart Foundation.
Dr. Beer has received grant support and compensation as advisor, speaker, and CME from Bayer, Boehringer-Ingelheim, Sanofi, Pfizer, and Bristol-Myers Squibb. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Drs. Holy and Akhmedov contributed equally to this work.
- Abbreviations and Acronyms
- aortic smooth muscle cell
- chronic kidney disease
- carbamylated low-density lipoprotein
- human aortic endothelial cell
- low-density lipoprotein
- lectin-like oxidized LDL receptor 1
- MAP kinase
- mitogen-activated protein kinase
- nuclear factor kappa B
- native low-density lipoprotein
- plasminogen activator inhibitor type 1
- tissue factor
- Received February 1, 2016.
- Revision received June 22, 2016.
- Accepted July 12, 2016.
- American College of Cardiology Foundation
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