Cardiovascular Revascularization Medicine
Volume 13, Issue 1 , Pages 11-19, January 2012

The role of oxidized phospholipids, lipoprotein (a) and biomarkers of oxidized lipoproteins in chronically occluded coronary arteries in sudden cardiac death and following successful percutaneous revascularization☆

  • Paul Fefer

      Affiliations

    • Schulich Heart Center, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5
    • Current address: Sheba Medical Center, Tel Aviv University, Tel Aviv.
    • ,
  • Sotirios Tsimikas

      Affiliations

    • Vascular Medicine Program, University of California, San Diego, USA
    • ,
  • Amit Segev

      Affiliations

    • St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    • Current address: Sheba Medical Center, Tel Aviv University, Tel Aviv.
    • ,
  • John Sparkes

      Affiliations

    • Schulich Heart Center, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5
    • ,
  • Fumiyuki Otsuma

      Affiliations

    • CVPath Institute, Gaithersburg, MD, USA
    • ,
  • Frank Kolodgie

      Affiliations

    • CVPath Institute, Gaithersburg, MD, USA
    • ,
  • Renu Virmani

      Affiliations

    • CVPath Institute, Gaithersburg, MD, USA
    • ,
  • Joseph Juliano

      Affiliations

    • Vascular Medicine Program, University of California, San Diego, USA
    • ,
  • Thierry Charron

      Affiliations

    • St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    • Current address: Hôpital du Sacré-Coeur de Montréal, Montréal, Québec.
    • ,
  • Bradley H. Strauss

      Affiliations

    • Schulich Heart Center, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5
    • Corresponding Author InformationCorresponding author. Schulich Heart Center, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada M4N 3M5. Tel.: +1 416 480 6066; fax: +1 416 480 4745.

Received 17 April 2011; received in revised form 5 August 2011; accepted 15 August 2011. published online 14 November 2011.

Article Outline

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.

Keywords: Chronic total occlusion, Oxidized phospholipids, Oxidized LDL, Angioplasty

 

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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 [1], [2], [3], [4]. In particular, unstable plaques appear to be preferentially enriched in OxLDL and lipoprotein (a) [Lp(a)] [5], [6], [7], [8], and OxLDL and oxidized phospholipids on apolipoprotein B-100 particles in plasma (OxPL/apoB) have been shown to be associated with acute coronary syndromes [5], [9], [10] and endothelial dysfunction [11], [12], [13].

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 [14]. 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 [15]. 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 [16]. Furthermore, we have recently documented significant increase in a variety of oxidized phospholipids in debris captured in both saphenous and carotid distal protection devices [17]. 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 [18], [19], [20], before and serially up to 1 week after PCI of CTO vessels and compared the results to PCI of non-CTO vessels.

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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 [21].

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 [21], [22] and the OxPL and OxLDL biomarker data used in our study for comparison purposes were published previously [16]. 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 [23].

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 [24].

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 [25]. 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 [26].

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 [9], [16]. 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 [9]. 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 [9] 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 [9], [16]. 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.

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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 [18], [27].

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  • Fig. 1. 

    Representative histopathologic images of chronic total occlusion in human coronary arteries. (A–D) Histologic sections of chronic total occlusion at the proximal site of right coronary artery from a 65-year-old woman who died after hip fracture surgery. (A) A low-power image (Movat pentachrome) showing totally occluded artery with organized and recanalized thrombus. (B) A high-power image (hematoxylin-eosin stain) showing neovascular channels. (C) Same section as (A) showing positive staining for oxidized phospholipid epitopes using antibody EO6 in particular around organized thrombus. (D) Same section as (A) showing positive staining for malondialdehyde-like epitopes using human antibody IK17 within the organized thrombus. (E–H) Histologic sections of chronic total occlusion at the proximal site of right coronary artery from a 49-year-old man who died suddenly. (E) A low-power image (Movat pentachrome) showing organized and recanalized thrombus with multiple neovascular channels. (F) A high-power image (hematoxylin-eosin stain) showing large and small neovascular channels within the organized thrombus. (G and H) Same sections as (E) showing positive staining for EO6 (G) and IK17 (H) epitopes within the organized thrombus.

3.2. PCI cohorts 

The baseline clinical, procedural and angiographic characteristics of the PCI groups are shown in Table 1. Patients with PCI for CTO lesions had significantly increased prevalence of prior MI, were more likely to have a procedure involving the right coronary artery, and less likely to have a procedure involving the left anterior descending artery compared to those with PCI for non-CTO lesions. Background medication data for patients with CTO include aspirin in 31 (100%), angiotensin-converting enzyme blockers in 17 (59%), and statins in 26 (90%). Lesion length was 9.29±3.29 mm, and lesion diameter was 2.95±0.54 in control patients, whereas stent diameter was 2.98±0.38 mm and stent length was 48.4±27.3 mm in CTO patients.

Table 1. Baseline characteristics
CTO (n=33)Controls (n=156)P diff
Age (mean±S.D.)61±1156±10.62
Males, n (%)24 (73)120 (77).65
Diabetes mellitus n (%)11 (33)28 (18).06
Hypertension, n (%)21 (64)69 (44).06
Smoking, n (%)7 (22)61 (39).07
Previous myocardial infarction, n (%)16 (48)41 (26).02⁎
Coronary artery undergoing PCI, n (%)
Left anterior descending artery9 (27)90 (58)<.002**
Left circumflex artery4 (11)34 (22).24
Right coronary artery20 (62)31 (20)<.0001**
Stent diameter (mm)2.98±0.382.95±0.54.95

⁎Significant; ⁎⁎highly significant.

3.3. Acute (≤24 h) changes in OxPL/apoB and Lp(a) levels 

The acute (≤24 h) changes in OxPL/apoB and Lp(a) were very different in the CTO group compared with the non-CTO controls. Following PCI of a non-CTO vessel there was a 35% mean percent increase in OxPL/apoB (P<.001) (Fig. 2) and a 68% mean percent increase in Lp(a) levels (P <.0001) after PCI compared with before PCI (Fig. 3). In contrast, following PCI of CTO vessels, there was no significant increase in OxPL/apoB and an 11% increase in Lp(a) levels (P =.031) (Fig. 2, Fig. 3). The mean percent increases for non-CTO vessels were also significantly greater than in CTO vessels (P<.0001), as shown in Fig. 2, Fig. 3. While the levels of OxPL/apoB and Lp(a) in the non-CTO group demonstrated a swift return towards baseline by 6 h post-PCI, in CTO vessels, levels of OxPL/apoB and Lp(a) continued to rise, achieving statistical significance compared with non-CTO levels by 6 hours post-PCI. In CTO vessels, levels of OxPL/apoB increased 15% above baseline (P =.009) and levels of Lp(a) increased 13% above baseline (P = .044) by 6 h.

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  • Fig. 2. 

    Percent change in OxPL/apoB in CTO after angioplasty of CTO and non-CTO coronary vessels. Note early rise and fall in OxPL/apoB in non-CTO vessels compared with a gradual and persistent rise in CTO vessels. P values are for comparison of CTO and non-CTO vessels at each time point.

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  • Fig. 3. 

    Percent change in Lp(a) in CTO after angioplasty of CTO and non-CTO coronary vessels. Note early rise and fall in Lp(a) in non-CTO vessels compared with a very mild rise in CTO vessels. P values are for comparison of CTO and non-CTO vessels at each time point.

3.4. Delayed (1–7 days) changes in OxPL/apoB and Lp(a) levels 

While post-procedure Day 1–7 plasma levels of OxPL/apoB and Lp(a) remained close to baseline following PCI in the non-CTO control group, the plasma levels of OxPL/apoB and Lp(a) in the CTO group continued to rise out to Day 7 post PCI (Fig. 2, Fig. 3). In the CTO group, Day 7 mean percent levels of OxPL/apoB were significantly higher than immediate post-PCI levels (P = .0017) and trended higher for Lp(a) at this time point (P = .077). OxPL/apoB mean percent levels between non-CTOs and successful CTO patients were significantly different immediately after PCI, at 3 days and at 7 days (P<.0001, P = .0002 and P<.0001, respectively). Differences in Lp(a) between non-CTOs and successful CTO patients were only significant immediately after PCI (P<.0001).

3.5. Acute (≤24 h) changes in autoantibodies to MDA-LDL and apoB-IC 

In both CTO and non-CTO patients, the mean percentage change in MDA-LDL IgG decreased significantly immediately post PCI and returned to baseline by 6 h post PCI in both groups. MDA-LDL IgM also decreased immediately post PCI but was significant only for the larger control group. At 24 h, MDA-LDL IgM and IgG were non-significantly lower in the CTO group compared with non-CTOs. There were no significant differences between CTO patients and controls at any time point (Fig. 4A).

Levels of apo-B immune complexes (IC) in the non-CTO group remained close to baseline throughout the first 24 h, as did the apo-B IC IgG in the CTO group. However, apo-B IgM rose non-significantly by 6 h post PCI in the CTO group and remained elevated over the first 24-h period. There was a significant difference between CTO and non-CTO patients at 6 and at 24 h for IC IgM (P = .0045 and P=.036, respectively) (Fig. 5A).

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  • Fig. 5. 

    Percent change in IC IgG (A) and IgM (B) after angioplasty of CTO and non-CTO coronary vessels. Significant differences are noted for IC-IgG at one week post angioplasty and at all time points post angioplasty for IC-IgM.

3.6. Delayed (1-7 days) changes in autoantibodies and apoB-IC 

In both CTO and non-CTO patients, the mean percent change in MDA-LDL IgG and MDA-LDL IgM returned to baseline by 3 days post-PCI and significantly increased by 7 days in all groups except for MDA-LDL IgM in CTO patients. There were no significant differences between CTO and non-CTO patients at any time points with regard to MDA-LDL IgG and IgM (Fig. 4A and Fig4B).

Apo-B IC IgG and IgM both rose significantly over post PCI Days 3–7 in the CTO group. This rise was significantly above any rise observed in the non-CTO group. The increase in IC IgM in the CTO group was maintained from 6 hours post PCI, whereas the IC IgG rise became significant only at 3 days post PCI (Fig. 5A and 5B).

3.7. Acute and delayed changes in hsCRP levels 

As shown in Fig. 6, levels of hsCRP rose immediately post-PCI and peaked at around 3 days post PCI. Absolute levels of hsCRP rose about fourfold from 2 to 8 mg/L. No differences were noted either in absolute levels or in percent change in the post-procedural CRP levels between CTO vessels and non-CTO vessels.

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4. Discussion 

This study demonstrates two novel findings regarding coronary CTOs: (1) immunohistological sections demonstrate the strong presence of both OxPL and MDA-like epitopes in clinically relevant areas of the plague including in organized thrombus, neovessels and necrotic core. This suggests that they may be involved pathophysiologically as has been shown in non-CTO vessels [5], [6], [7], [17], [18], [26]; (2) differences in temporal changes in plasma oxidative biomarkers between CTO and non-CTO vessels may reflect both different pathophysiology and vessel architecture as well as acute vessel response to PCI and sub-acute remodeling over the first week.

In the immunohistology CTO study, CTOs evaluated contained both significant plaque burden and evidence of organized thrombus and neovascularization. Immunohistologically, OxPL and MDA-like epitopes were strongly present around these areas, although they did not completely colocalize, which is consistent with generation of such chemically distinct epitopes at different stages of the plaques [18], [27]. These findings suggest the temporal sequence of thrombotic occlusion of an oxidation-rich plaque and subsequent thrombotic occlusion, remodeling and neovascularization, as has been suggested in prior studies. [28]. Older CTOs are associated with increasing content of hard fibrous tissue and calcium [15].

In the PCI of CTO study, we demonstrate a number of differences in the responses to PCI of CTO vessels: (1) the acute rise (immediately post) and rapid fall (by 6 h post) in OxPL/apoB and Lp(a) was not seen following PCI of CTO vessels; (2) instead, a gradual rise in the levels of OxPL/apoB and Lp(a) was observed up to 7 days post PCI of CTO vessels, whereas levels remained at baseline for patients undergoing PCI of non-CTO vessels; (3) the short-term (≤7 days) immune response as assessed by IgM and IgG antibodies to MDA-LDL and IC differed between the groups, whereas IC levels rose significantly more in CTO patients compared with controls, levels of IgM and IgG antibodies to MDA-LDL rose both in CTO patients and in controls, with no differences noted between groups. Furthermore, showing specificity for oxidized lipoproteins rather than inflammation in general, no significant changes was noted in hsCRP levels between CTO and non-CTO vessels. We will address each of these points separately.

4.1. Acute (≤24 h) changes in OxPL/apoB and Lp(a) levels 

The acute rise in levels of OxPL/apoB and Lp(a) was modest following PCI of CTO vessels. This is in sharp distinction to the 35% and 68% acute rise in levels of OxPL/apoB and Lp(a) respectively, following PCI of non-CTO vessels. Previous studies from our group have shown that the OxPL/apoB rises abruptly after acute coronary events [9] and immediately after PCI [16]—situations in which the release of OxPL (or OxLDL, or both) from the vessel wall might be postulated. If we assume that the rise in OxLDL levels following PCI is indeed due to disruption and embolization of plaque content, clearly, PCI of CTO vessels did not induce such release of oxidized phospholipids.

4.2. Delayed (1–7 days) changes in OxPL/apoB and Lp(a) levels 

While the rise of OxPL/apoB and Lp(a) levels after PCI to CTO vessels was minimal in the first 24 h, levels continued to rise from 24 h to 7 days after PCI. This is in contrast to the levels of OxPL/apoB and Lp(a) after PCI of non-CTO vessels which demonstrated a sharp rise and fall within 6 h but remained stable at baseline values for the duration of the first week after PCI. This second phase of a progressive rise in these markers, may be related to the renewal of antegrade flow into a previously occluded artery, exposing a large area of atherosclerosis which was previously relatively isolated from the systemic circulation. Increased shear forces may induce metabolic activity within these plaques, leading to increased oxidative stress and formation of lipid peroxides that could oxidize phospholipids in the vessel wall or/and plasma. Lp(a) levels have been shown to correlate closely with OxPL/apoB levels [9]. There is support to the hypothesis that Lp(a) preferentially binds to OxLDL in the plasma [29], [30], in effect acting as a “sink” for such OxLDL in vivo, providing a mechanism for their transport and degradation [16]. One proposed mechanism for up-regulation of Lp(a) levels is through up-regulation of liver apo(a) synthesis by pro-inflammatory cytokines (e.g., IL-6) [16].

4.3. Immune response 

Following PCI of non-CTO vessels, there is an acute drop in autoantibodies to MDA-LDL and a simultaneous rise in apoB-IC levels. This is thought to represent release of oxidized lipoproteins that bind to preformed antibodies (MDA-LDL) and the formation of IC [16]. A very similar immune response was seen following PCI of CTO vessels, albeit a more robust response as evidenced by the greater levels of IC observed over time.

4.4. Changes in hsCRP Levels 

The observed changes in hsCRP after PCI are consistent with previous studies [31], [32], although the absolute magnitude of rise may have been slightly greater than these, possibly due to the fact that most patients in our study were not being treated with a statin at the time of PCI. Interestingly, although biomarkers of OxLDL rose significantly more after successful PCI of CTO vessels compared with non-CTO vessels, no differences were noted in the CRP response between these two groups. Thus, it would seem that despite higher total exposure to OxLDL in CTO patients, the inflammatory reaction as assessed by CRP did not differ between groups, suggesting that the changes in OxPL and biomarkers of OxLDL are perhaps more specifically associated with changes in the vessel wall rather than due to generalized inflammation.

4.5. Limitations 

First, as expected, there are significant differences in baseline characteristics between patients with and without CTO. While such differences are inherent in the study design, it is possible that some of the variability in biomarkers is related to these baseline differences. Second, the use of historical controls is a weakness but is used instructively as a comparison for hypothesis generating, rather than as a definitive test of differences in CTO vs. non-CTO responses to PCI. The majority of historical controls had balloon angioplasty without stent implantation, whereas all CTO patients had stents implanted. However, in the original study, no significant differences in markers of OxPL were found between control patients undergoing balloon angioplasty or stent implantation; thus, the effect of stenting versus balloon angioplasty is likely limited. Also, while 90% of the CTO patients were treated with statins, few of the control group were receiving such therapy, which may affect some of these changes. However, we have shown that stains and low fat diets tend to increase OxPL/apoB and Lp(a), which may reflect a flux of OxPL from the vessel wall as suggested in animal studies [20], [33] or up-regulation of Lp(a), and its OxPL content, by statins. However, this would favor a higher acute increase in OxPL/apoB in the CTO group, not a slower one as noted. Finally, a comparison with patients undergoing unsuccessful PCI to CTO was not made, nor do we have available any advanced pre-procedural plaque imaging such as intravascular ultrasound or optical coherence tomography imaging. However, we recently demonstrated in non-CTO vessels that there is strong immunostaining for OxPL and Lp(a) in human atherosclerotic plaques as they progress from intima xanthomas, to thin cap fibroatheromas to plaque rupture [7].

4.6. Clinical significance 

Relatively little data are available evaluating biomarkers in patients with CTO undergoing PCI. Since both Lp(a) and OxPL are associated with plaque progression, impaired fibrinolysis and inhibition of platelet aggregation [20], [34], they may contribute to the increased incidence and prevalence of CTO and may also impact acute success and reocclusion and, ultimately, adverse cardiovascular events observed in patients with CTO. This study may provide a rationale to study changes in such biomarkers in larger clinical and pathological studies of CTO, both in coronary and peripheral vessels.

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Acknowledgment 

The Fondation Leducq (ST) funded this study.

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☆ Disclosure: Dr Tsimikas is named as co-inventor of patents and patent applications related to oxidation-specific antibodies, has equity interest in Atherotope, and is a consultant to Quest Diagnostics. The other authors declare no conflicts of interest.

PII: S1553-8389(11)00501-X

doi:10.1016/j.carrev.2011.08.001

Cardiovascular Revascularization Medicine
Volume 13, Issue 1 , Pages 11-19, January 2012

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