P2Y 12 receptor antagonists may have pleiotropic benefits. Little is known, however, about the expression of P2Y 12 receptors in coronary atherosclerotic plaques. We investigated the expression of P2Y 12 receptor in coronary atherectomy tissues retrieved from patients with acute myocardial infarction (AMI) or stable angina pectoris (SAP). Tissue specimens were collected from 35 patients with AMI and 19 with SAP who underwent directional coronary atherectomy. Specimens were analyzed immunohistochemically using antibodies specific to P2Y 12 receptor and to markers of endothelial cells, macrophages, and smooth muscle cells. The 2 groups had similar baseline clinical characteristics. Plaque types were more likely to be cellular in the AMI group. The proportion of areas immunopositive for α-smooth muscle actin was smaller but those positive for CD31 and CD68 were larger in the AMI than in the SAP group. In addition, the relative area immunopositive for P2Y 12 receptor was significantly larger for AMI than SAP (1.1 ± 0.9% vs 0.5 ± 0.4%, respectively, p <0.001). P2Y 12 receptor positivity coincided with areas positive for CD31 and α-smooth muscle actin. In conclusion, P2Y 12 receptor is present in coronary atherosclerotic plaques and is increased in culprit plaques of patients with AMI. P2Y 12 receptor may play a role in plaque destabilization.
P2Y 12 receptors are expressed on endothelial cells, vascular smooth muscle cells, and platelets. Clopidogrel can decrease markers of vascular inflammation and improve endothelial function. Recent results from the Platelet Inhibition and Patient Outcomes (PLATO) trial showed that the cardiovascular benefits of ticagrelor outweighed those of clopidogrel despite being associated with more spontaneous bleeding events. The vascular benefits of ticagrelor could not be explained by its faster and more potent platelet inhibition alone, suggesting that ticagrelor has platelet-independent pleiotropic effects coupled with P2Y 12 receptor antagonism. To date, however, little is known about the expression of P2Y 12 receptors in coronary atherosclerotic plaques and its relation to plaque instability. We therefore compared the expression of P2Y 12 receptors in coronary atherectomy tissues retrieved from patients with acute myocardial infarction (AMI) to those from patients with stable angina pectoris (SAP).
Methods
Atherectomy specimens were obtained from a biobank at our institution, which had collected tissues from 35 consecutive patients with AMI and 19 with SAP, defined as typical exertional angina with no change in symptoms within 1 month before the procedure. Patients were suitable for directional coronary atherectomy if they had a significant stenotic lesion with a large plaque burden but lacked heavy thrombi in a nontortuous epicardial coronary artery >3 mm in diameter. Each specimen corresponded to the de novo lesion responsible for the clinical presentation in a single patient. Directional coronary atherectomy was performed using a Flexi-Cut catheter (Abbott Laboratories/Guidant Vascular Interventions, Santa Clara, California) under intravascular ultrasound guidance. The study protocol was approved by our institutional review committee, and all patients provided written informed consent.
Tissue specimens were fixed in formalin and embedded in donor paraffin blocks. Tissue microarrays were produced by re-embedding tissues from these paraffin blocks into arrays on recipient paraffin blocks. Sections from the master block were cut using a microtome, mounted on a microscope slide, and used for subsequent staining.
Samples were stained with hematoxylin and eosin to determine cellularity and general morphologic features. The area of each plaque was measured using a microscopic image analysis system (Motic Images Advanced 3.2, Motic, Xiamen, China). Plaques were classified as atheromatous (i.e., with necrotic cores and cholesterol clefts but without connective tissue matrix) or fibrocellular and graded as sparsely cellular (<30 spindle cells per high-power field), moderately cellular (30 to 100 spindle cells per field), or hypercellular (≥100 spindle cells per field). All slides were graded by 2 pathologists (C.-S.P. and I.H.) blinded to patients’ clinical status. Any discrepancies between their findings were resolved by consensus.
Sections of each tissue specimen were stained with rabbit polyclonal antibodies against P2Y 12 receptor (1:100; Novus Biologicals, Littleton, Colorado) and monoclonal antibodies against α-smooth muscle actin (1:200; mouse antihuman macrophage antibody clone 1A4; DAKO, Carpinteria, California), CD31 (1:100; mouse antihuman CD31 [platelet endothelial cell adhesion molecule-1] antibody clone 1A10; Leica, Newcastle, United Kingdom), and CD68 (1:200; mouse antihuman macrophage antibody clone KP-1; DAKO) using an Envision-plus immunostaining kit and 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole as the chromogen as described by the manufacturer (DAKO). Briefly, samples were incubated with primary antibodies for 1 hour, washed 2 times for 5 minutes each with Tris buffered saline/Tween-20, incubated with secondary antibodies conjugated with horseradish peroxidase–labeled polymer (DAKO) for 1 hour, and washed again. As negative controls, adjacent sections were stained with species- and isotype-matched irrelevant antibodies including normal rabbit immunoglobulin G (Abcam, Cambridge, United Kingdom). A sample of platelets was used as a positive control for anti-P2Y 12 receptor antibodies. Cell types positive for P2Y 12 receptor were identified by immunostaining of serial sections with anti-P2Y 12 receptor antibodies. The immunopositive area was calculated as the ratio of positively stained regions to total plaque area.
For immunofluorescent staining, fixed sections were hydrated in phosphate buffered saline (PBS) for 10 minutes at room temperature, incubated with DakoCytomation Protein Block (DakoCytomation, Carpinteria, California) for 5 minutes at room temperature, and washed 3 times in PBS/Tween-20. Sections were subsequently incubated with mouse monoclonal antibody to human CD31 (Leica), CD68 (DakoCytomation), or α-smooth muscle actin (DakoCytomation) or rabbit polyclonal antibody to P2Y 12 receptor for 60 minutes at room temperature. After 3 additional washes in PBS/Tween-20, sections were incubated with fluorescein isothiocyanate–conjugated antirabbit immunoglobulin G or allophycocyanin-conjugated antimouse immunoglobulin G for 60 minutes at room temperature and washed 3 times with PBS/Tween-20. Coverslips were mounted onto glass slides using DAKO fluorescent mounting medium (DakoCytomation). Fluorescein isothiocyanate was excited using an argon laser at 488 nm and allophycocyanin was excited by a helium–neon laser at 633 nm. Detector slits were configured to minimize any crosstalk between channels. Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with a 40× objective (model NA 0.75) and a Zoom 1-4 × and processed using Leica TCS-NT/SP software (version LCS) and Adobe Photoshop 7.0 (San Jose, California).
Continuous variables are expressed as mean ± SD or median with interquartile range, whereas categorical variables are expressed as frequency. Continuous variables were compared using Student’s t test or Mann–Whitney U test, and categorical variables were analyzed using chi-square test. Linear regression analysis was used to correlate areas positive for P2Y 12 receptor with those positive for markers for endothelial cells, macrophages, and smooth muscle markers. Statistical significance was defined as a 2-sided p value <0.05.
Results
Baseline clinical characteristics were similar between the 2 groups except for their lipid profiles and medications ( Table 1 ). Median time from symptom onset to reperfusion was 5.5 hours (interquartile range 2.5 to 7.5) for ST-segment elevation AMI (n = 27) and 35.5 hours (interquartile range 24 to 90) for non–ST-segment elevation AMI (n = 8). At the time of directional coronary atherectomy, larger percentages of patients in the SAP group were taking calcium channel blockers and statins than those in the AMI group.
| Characteristics | AMI | SAP | P Value |
|---|---|---|---|
| (n = 35) | (n = 19) | ||
| Age (years) | 56.4 ± 11.2 | 61.0 ± 6.9 | 0.107 |
| Men/women | 29/6 | 12/7 | 0.181 |
| Current smoker | 16 (45.7%) | 6 (31.6%) | 0.391 |
| Diabetes mellitus | 7 (20%) | 7 (36.8%) | 0.206 |
| Hypertension | 17 (48.6%) | 11 (57.9%) | 0.577 |
| Total cholesterol (mg/dl) | 192.9 ± 44.6 | 153.0 ± 30.1 | 0.001 |
| Triglycerides (mg/dl) | 179.4 ± 84.2 | 110.5 ± 45.5 | 0.002 |
| High-density lipoprotein cholesterol (mg/dl) | 34.8 ± 7.9 | 43.0 ± 14.4 | 0.031 |
| High-sensitivity C-reactive protein (mg/L) | 3.3 ± 3.3 | 1.7 ± 1.8 | 0.059 |
| Multivessel coronary disease | 17 (48.6%) | 7 (36.8%) | 0.567 |
| Target coronary artery | 0.795 | ||
| Left anterior descending | 19 (54.3%) | 12 (63.2%) | |
| Left circumflex | 3 (8.6%) | 1 (5.3%) | |
| Right | 13 (37.1%) | 6 (31.6%) | |
| Medications at time of directional coronary atherectomy | |||
| Aspirin | 35 (100%) | 19 (100%) | 1.0 |
| Clopidogrel | 35 (100%) | 19 (100%) | 1.0 |
| Angiotensin-converting enzyme inhibitor/angiotensin receptor blocker | 2 (5.7%) | 2 (10.5%) | 0.607 |
| β Blockers | 7 (20%) | 9 (4.7%) | 0.060 |
| Calcium channel antagonists | 5 (14.3%) | 11 (57.9%) | 0.001 |
| Statins | 11 (31.4%) | 13 (68.4%) | 0.012 |
Total plaque area was smaller, whereas atheroma area was larger, in the AMI than in the SAP group ( Table 2 ). Plaque types were more likely to be cellular in the AMI group. Thrombi were significantly more common in the AMI than in the SAP group (68.6% vs 1.1%, p <0.001). Relative plaque area immunopositive for α-smooth muscle actin was significantly smaller in the AMI group than in the SAP group, whereas areas immunopositive for CD31 and CD68 were significantly larger in the AMI group ( Table 2 ). In addition, relative area immunopositive for P2Y 12 receptor was significantly larger in plaques from patients with AMI than from those with SAP (1.1 ± 0.9% vs 0.5 ± 0.4%, p <0.001). In multivariate analysis, areas immunopositive for P2Y 12 receptor (r = 0.54) were significantly correlated with those positive for CD31 (beta 0.30, p = 0.022) and CD68 (beta 0.36, p = 0.007).
| Variables | AMI | SAP | P Value |
|---|---|---|---|
| (n = 35) | (n = 19) | ||
| Histology | |||
| Atheroma | 48.7 ± 26.1 | 31.1 ± 30.3 | 0.030 |
| Fibrocellular area | |||
| Sparsely cellular | 35.5 ± 23.5 | 51.6 ± 30.4 | 0.035 |
| Moderately cellular | 7.6 ± 9.0 | 15.7 ± 24.6 | 0.179 |
| Hypercellular | 2.5 ± 5.1 | 0.5 ± 1.9 | 0.041 |
| Thrombus | 5.2 ± 16.2 | 0.0 ± 0.1 | <0.001 |
| Calcium | 0.5 ± 1.9 | 1.0 ± 3.0 | 0.509 |
| Total plaque area (mm 2 ) | 704.4 ± 344.0 | 496.5 ± 314.9 | 0.034 |
| Immunohistochemistry | |||
| α-Smooth muscle actin | 2.9 ± 2.7 | 12.3 ± 14.4 | 0.011 |
| CD31 | 1.1 ± 1.6 | 0.2 ± 0.2 | 0.001 |
| CD68 | 15.5 ± 13.6 | 7.0 ± 14.6 | 0.038 |
| P2Y 12 | 1.1 ± 0.9 | 0.5 ± 0.4 | <0.001 |
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