Abstract
Aims
OxPL are pro-inflammatory and may mediate atherogenesis, thrombosis and endothelial dysfunction. We studied the histological presence and temporal increases in oxidized phospholipids on apolipoprotein B-100 particles (OxPL/apoB), lipoprotein (a) [Lp(a)] and biomarkers of oxidized lipoproteins in subjects with chronic total coronary occlusions (CTO) with sudden cardiac death (SCD) and following percutaneous coronary intervention (PCI).
Methods
Eight subjects with SCD and CTO and 33 patients with successful PCI of CTO were included. Blood samples were drawn before PCI, immediately post-PCI, at 6 and 24 h, at 3 days and at 1 week. Plasma levels of OxPL/apoB, Lp(a), IgG and IgM autoantibodies to malondialdehyde (MDA) low-density lipoprotein and apoB-immune complexes were measured in all samples and compared with previous data from 141 patients undergoing PCI of non-CTO vessels.
Results
Immunohistochemistry of coronary CTOs revealed OxPL and MDA-like epitopes, particularly in areas of recanalized and organized thrombus and neovascularization. Following PCI, OxPL/apoB and Lp(a) levels, expressed as percent change from baseline levels before PCI, rose gradually and progressively over the next 7 days. In contrast, levels of OxPL/apoB and Lp(a) in non-CTO vessels rose immediately post PCI and then dropped rapidly to baseline within 24 h.
Conclusions
CTOs contain immunohistological evidence of OxPL and MDA-like epitopes. Successful PCI of CTOs results in a slower increase in OxPL/apoB and Lp(a) but higher increase in IgM immune complexes compared to non-CTO vessels. Pro-inflammatory oxidation-specific epitopes may impact development of CTOs and affect outcomes following PCI that can be evaluated in larger clinical trials.
1
Introduction
Oxidized low-density lipoprotein (OxLDL) is present in atherosclerotic lesions of animal models and humans and directly influences a multitude of atherogenic responses . In particular, unstable plaques appear to be preferentially enriched in OxLDL and lipoprotein (a) [Lp(a)] , and OxLDL and oxidized phospholipids on apolipoprotein B-100 particles in plasma (OxPL/apoB) have been shown to be associated with acute coronary syndromes and endothelial dysfunction .
Chronic total coronary occlusions (CTOs), defined as occlusions of at least six weeks duration, are encountered in approximately one third of patients undergoing coronary angiography for known or suspected coronary disease . Very little is known about the pathobiology of such lesions. Fibro-calcific lesions gradually replace cholesterol and foam cell-laden intimal plaques as CTOs remodel, while a high incidence of plaque calcification has been noted in CTOs with a relative increase in both the frequency and severity of calcification with advancing lesion age . We have previously documented a significant increase in plasma OxPL/apoB immediately post PCI, suggesting that OxPL may be released and/or generated as a result of PCI-induced plaque disruption . Furthermore, we have recently documented significant increase in a variety of oxidized phospholipids in debris captured in both saphenous and carotid distal protection devices . Since such OxPL are pro-inflammatory, are pro-thrombotic, activate endothelial cells and quench nitric oxide, their release from atherosclerotic lesions or their passage through filter devices may results in peri- and post-procedural clinical complications. As a first step in starting to define their clinical role in patients undergoing PCI, in the current study, we hypothesized that CTOs would contain evidence of oxidative biomarkers and that differences in oxidation biomarkers would be present following successful PCI of CTO. This may presumably be due to differences in plaque characteristics in CTO vessels compared to lesions associated with non-CTO lesions. Accordingly, we immunologically detected the presence of oxidation-specific epitopes in CTOs of subjects with sudden cardiac death and measured a comprehensive panel of OxLDL markers, including OxPL/apoB, apoB-immune complexes (apoB-IC), OxLDL autoantibodies and Lp(a), which is a carrier of OxPL , before and serially up to 1 week after PCI of CTO vessels and compared the results to PCI of non-CTO vessels.
2
Methods
2.1
Patients
2.1.1
Human atherosclerotic lesions of coronary CTO
Hearts of patients ( n =8) who had died suddenly with coronary artery disease and had the presence of CTOs were obtained as previously described in detail .
2.1.2
PCI cohort
The patient cohort was derived from 33 patients who underwent successful elective PCI of a CTO vessel at St. Michael’s Hospital in Toronto from Jan 2004 to Dec 2005. Drug-eluting stents were implanted in 12 patients and bare-metal stents in 21. Exclusion criteria included recent (<2 weeks) myocardial infarction (MI) or unstable angina (defined as rest pain with ST-segment changes), bypass graft lesions, or concurrent illnesses such as cancer, rheumatoid arthritis, or inflammatory bowel disease. Venous blood samples were collected in EDTA-containing tubes pre-PCI, immediately post-PCI, and at 6 h, 24 h, 3 days and 1 week post-PCI. Blood samples were stored in aliquots at −80°C until all assays were performed. The St. Michael’s Hospital Internal Review Board approved the study protocol and all patients gave written informed consent to participate in the study.
2.2
PCI controls
The control group was derived from a single-center prospective study of 141 patients, recruited from January 1994 to January 1997, with stable angina undergoing elective uncomplicated PCI to a non-CTO vessel. This cohort has been previously described in detail and the OxPL and OxLDL biomarker data used in our study for comparison purposes were published previously . As patient recruitment for this study was performed in the mid to late 1990s, most patients underwent balloon angioplasty with adjunctive stenting reserved for suboptimal balloon angioplasty results and/or the presence of significant (type C or greater) dissections (41 patients, 29%). Restenosis rates differed between these two subsets of patients: 37% for percutaneous transluminal coronary angioplasty and 17% for stents, but otherwise, the groups were considered similar enough to pool the data for a baseline summary and for analysis of blood plasma tests. In addition, most patients were not being treated with statins in this study. Venous blood was obtained in a similar manner to the patient group .
2.3
Histological preparation
Formalin-fixed, paraffin-embedded coronary segments were cut at 5-μm thick sections, mounted on charged slides, and stained with hematoxylin and eosin and the modified Movat pentachrome method as previously described .
2.4
Antibodies
Two unique monoclonal antibodies, E06 and Ik17 were used in this study to assess the presence of OxPL and MDA-like epitopes. E06 is a natural IgM murine monoclonal antibody cloned from apoE -/- mice that binds to the phosphocholine head group of oxidized (OxPL), but not native, phospholipids . IK17 is a fully human Fab fragment generated with phage-display library technology that binds to a unique epitope present on both MDA-LDL and Cu-OxLDL .
2.5
Immunohistochemistry
Formalin-fixed paraffin sections (5 μm) were incubated overnight at 4°C with primary antibody E06 at 1:400 dilution. The detection of primary antibody was achieved using the biotinylated link antibody LSAB2 System-HRP DAB kit (Dako, Carpenteria, CA, USA) with appropriate secondary antibodies directed to mouse IgM. Histologic sections for antibody staining against IK17 were initially incubated overnight with non-immune goat anti-human IgG (GAH, Vector, BA-3000) at a dilution of 1:100 in 2% goat serum to reduce non-specific background staining. For IK17 immunostaining, IK17 was diluted 1:600 in 2% goat serum and incubated for one hour at room temperature (RT). Primary labeling was then visualized using an alkaline phosphatase-labeled goat anti-human secondary antibody (dilution 1:200, Sigma A3813) for 1 h at RT and visualized with Vector Red (Vector SK-5100). The degree of E06 and IK17 positivity was assessed qualitatively.
2.6
Determination of OxPL/apoB, OxLDL autoantibody titers, apolipoprotein B-100 immune complexes, Lp(a) and high-sensitivity C-reactive protein (hsCRP) levels
Chemiluminescence enzyme-linked immunosorbent assay was used to measure OxPL and OxLDL markers as previously described in detail . OxPL/apoB is a measure of the content of OxPL per apoB-100 particle (OxPL/apoB) using the murine monoclonal antibody E06, which specifically binds to the phosphorylcholine head group of oxidized, but not native, phospholipids. A 1:50 dilution of plasma in phosphate-buffered saline is added to microtiter wells coated with the monoclonal antibody MB47, which specifically binds apoB-100 particles. Under these conditions, a saturating amount of apoB-100 is added to each well and consequently an equal number of apoB-100 particles are captured in each well for all assays. The content of OxPL per apoB-100 is then determined with biotinylated E06 as previously described . Plasma titers of immunoglobulin G (IgG) and immunoglobulin M (IgM) malondialdehyde (MDA-LDL) (1:200 plasma dilution) autoantibodies and IgG and IgM apoB-IC previously were measured as previously described Lp(a), which has previously been shown to preferentially bind OxPL compared to other lipoproteins, was also measured at each time point as previously described . hsCRP levels were assessed at four time points (before PCI, after PCI and at 3 and 7 days).
2.7
Statistical analysis
Assay results were expressed as percent changes in markers relative to the baseline levels for each subject. This approach was taken mainly to provide assay measures that could be compared between CTO and non-CTO subjects as this assay’s evolving methodology was expressed in relative light units rather than a standard curve and absolute levels between studies cannot be easily compared. However, the assay is internally validated and high and low standards are used on microtiter well plates to minimize variability. Changes were analyzed in SAS 9.0 using a generalized linear model (mixed procedure) with an unstructured covariance. Two sets of analysis were performed. In the first analysis of variance (ANOVA), CTO and non-CTO data were analyzed separately; percentage changes in markers post-PCI were compared over time. In a second ANOVA, percent changes in markers of CTO and non-CTO vessels were compared at each time point (post PCI, 6 h, 24 h, 3 days and 7 days). The probability of each comparison was estimated by calculating a Fisher probability for the comparison. The p values for comparisons between CTO and non-CTO vessels at each time point are the ones shown in our figures. A p value of less than .05 was considered statistically significant.
2
Methods
2.1
Patients
2.1.1
Human atherosclerotic lesions of coronary CTO
Hearts of patients ( n =8) who had died suddenly with coronary artery disease and had the presence of CTOs were obtained as previously described in detail .
2.1.2
PCI cohort
The patient cohort was derived from 33 patients who underwent successful elective PCI of a CTO vessel at St. Michael’s Hospital in Toronto from Jan 2004 to Dec 2005. Drug-eluting stents were implanted in 12 patients and bare-metal stents in 21. Exclusion criteria included recent (<2 weeks) myocardial infarction (MI) or unstable angina (defined as rest pain with ST-segment changes), bypass graft lesions, or concurrent illnesses such as cancer, rheumatoid arthritis, or inflammatory bowel disease. Venous blood samples were collected in EDTA-containing tubes pre-PCI, immediately post-PCI, and at 6 h, 24 h, 3 days and 1 week post-PCI. Blood samples were stored in aliquots at −80°C until all assays were performed. The St. Michael’s Hospital Internal Review Board approved the study protocol and all patients gave written informed consent to participate in the study.
2.2
PCI controls
The control group was derived from a single-center prospective study of 141 patients, recruited from January 1994 to January 1997, with stable angina undergoing elective uncomplicated PCI to a non-CTO vessel. This cohort has been previously described in detail and the OxPL and OxLDL biomarker data used in our study for comparison purposes were published previously . As patient recruitment for this study was performed in the mid to late 1990s, most patients underwent balloon angioplasty with adjunctive stenting reserved for suboptimal balloon angioplasty results and/or the presence of significant (type C or greater) dissections (41 patients, 29%). Restenosis rates differed between these two subsets of patients: 37% for percutaneous transluminal coronary angioplasty and 17% for stents, but otherwise, the groups were considered similar enough to pool the data for a baseline summary and for analysis of blood plasma tests. In addition, most patients were not being treated with statins in this study. Venous blood was obtained in a similar manner to the patient group .
2.3
Histological preparation
Formalin-fixed, paraffin-embedded coronary segments were cut at 5-μm thick sections, mounted on charged slides, and stained with hematoxylin and eosin and the modified Movat pentachrome method as previously described .
2.4
Antibodies
Two unique monoclonal antibodies, E06 and Ik17 were used in this study to assess the presence of OxPL and MDA-like epitopes. E06 is a natural IgM murine monoclonal antibody cloned from apoE -/- mice that binds to the phosphocholine head group of oxidized (OxPL), but not native, phospholipids . IK17 is a fully human Fab fragment generated with phage-display library technology that binds to a unique epitope present on both MDA-LDL and Cu-OxLDL .
2.5
Immunohistochemistry
Formalin-fixed paraffin sections (5 μm) were incubated overnight at 4°C with primary antibody E06 at 1:400 dilution. The detection of primary antibody was achieved using the biotinylated link antibody LSAB2 System-HRP DAB kit (Dako, Carpenteria, CA, USA) with appropriate secondary antibodies directed to mouse IgM. Histologic sections for antibody staining against IK17 were initially incubated overnight with non-immune goat anti-human IgG (GAH, Vector, BA-3000) at a dilution of 1:100 in 2% goat serum to reduce non-specific background staining. For IK17 immunostaining, IK17 was diluted 1:600 in 2% goat serum and incubated for one hour at room temperature (RT). Primary labeling was then visualized using an alkaline phosphatase-labeled goat anti-human secondary antibody (dilution 1:200, Sigma A3813) for 1 h at RT and visualized with Vector Red (Vector SK-5100). The degree of E06 and IK17 positivity was assessed qualitatively.
2.6
Determination of OxPL/apoB, OxLDL autoantibody titers, apolipoprotein B-100 immune complexes, Lp(a) and high-sensitivity C-reactive protein (hsCRP) levels
Chemiluminescence enzyme-linked immunosorbent assay was used to measure OxPL and OxLDL markers as previously described in detail . OxPL/apoB is a measure of the content of OxPL per apoB-100 particle (OxPL/apoB) using the murine monoclonal antibody E06, which specifically binds to the phosphorylcholine head group of oxidized, but not native, phospholipids. A 1:50 dilution of plasma in phosphate-buffered saline is added to microtiter wells coated with the monoclonal antibody MB47, which specifically binds apoB-100 particles. Under these conditions, a saturating amount of apoB-100 is added to each well and consequently an equal number of apoB-100 particles are captured in each well for all assays. The content of OxPL per apoB-100 is then determined with biotinylated E06 as previously described . Plasma titers of immunoglobulin G (IgG) and immunoglobulin M (IgM) malondialdehyde (MDA-LDL) (1:200 plasma dilution) autoantibodies and IgG and IgM apoB-IC previously were measured as previously described Lp(a), which has previously been shown to preferentially bind OxPL compared to other lipoproteins, was also measured at each time point as previously described . hsCRP levels were assessed at four time points (before PCI, after PCI and at 3 and 7 days).
2.7
Statistical analysis
Assay results were expressed as percent changes in markers relative to the baseline levels for each subject. This approach was taken mainly to provide assay measures that could be compared between CTO and non-CTO subjects as this assay’s evolving methodology was expressed in relative light units rather than a standard curve and absolute levels between studies cannot be easily compared. However, the assay is internally validated and high and low standards are used on microtiter well plates to minimize variability. Changes were analyzed in SAS 9.0 using a generalized linear model (mixed procedure) with an unstructured covariance. Two sets of analysis were performed. In the first analysis of variance (ANOVA), CTO and non-CTO data were analyzed separately; percentage changes in markers post-PCI were compared over time. In a second ANOVA, percent changes in markers of CTO and non-CTO vessels were compared at each time point (post PCI, 6 h, 24 h, 3 days and 7 days). The probability of each comparison was estimated by calculating a Fisher probability for the comparison. The p values for comparisons between CTO and non-CTO vessels at each time point are the ones shown in our figures. A p value of less than .05 was considered statistically significant.
3
Results
3.1
Immunohistochemistry of CTOs of subjects with sudden cardiac death
Sufficient clinical information was not available for the eight subjects with sudden cardiac death. Histologic sections of chronic total occlusion and immunohistochemistry at the proximal sites of the right coronary artery from a 65-year-old woman and 49-year-old man with sudden death are shown in Fig. 1 . Both examples show evidence of organized and recanalized thrombus, with both small and large neovessels. E06 (brown stain) and IK17 (red stain) epitopes are abundant in the lesions and particularly around the organized thrombus. There is also significant E06 and particularly IK17 staining in the necrotic areas of the plaque (i.e., from 9 to 6 o’clock clockwise in Patient 1 and 4 to 9 o’clock clockwise in Patient 2). Interestingly, there is some, but not complete, co-localization, which reflects the different epitopes each antibody detects. This suggests that they are generated either at different stages of the lesion as it occludes and/or different stages of oxidation within the lesion. These chemical modifications of lipids and proteins in atheromata representing oxidation-specific epitopes are generated at different stages, depending on stage of oxidation .
