Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.
Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.
Coronary angioplasty and stenting represent a salvage procedure for patients with coronary artery disease (CAD). However, major adverse cardiovascular events (MACEs), including cardiovascular death, myocardial infarction, coronary restenosis, and episodes of angina or decompensate heart failure, may occur months after coronary intervention, prompting unscheduled visits to the hospital. MACEs are common worldwide and their morbi-mortality is high1.
Coronary ischemic injury induces early vascular response and reparative mechanisms involving mobilization of MPCs due to their differentiation ability and/or angio-reparative potential, as well as the production of soluble molecules like intercellular adhesion molecules (ICAMs), matrix metalloproteinases (MMPs), and reactive oxygen species, reflecting cell adhesion, tissue remodeling, and oxidative stress. Although intrinsic vascular features like plaque characteristics and coronary artery complexity have been used to predict MACEs, some studies have suggested that biomarkers related to the mechanisms of injury and repair occurring in the coronary endothelium could be very useful for the early identification and prognosis of cardiovascular events in patients with CAD submitted to coronary angioplasty2,3,4,5.
Continuous interest in understanding the mechanisms underlying CAD injury and repair has motivated investigators to study intracoronary circulating biomarkers, because coronary sampling more closely reflects vascular damage and repair6. However, characterization of coronary biomarkers in human studies has been scarce7,8,9. Therefore, the purpose of the present study was to describe a method to determine the amount of coronary circulating MPCs and soluble molecules, reflecting both vascular injury and repair, and to show whether these biomarkers are associated with MACEs and the clinical prognosis of CAD patients that underwent coronary angioplasty. This method is based on the use of vascular-related, circulating MPCs and soluble molecules obtained by sampling locations closest to the vessel damage. It may also be useful for clinical studies for lower limb ischemia, stroke, vasculitis, venous thrombosis, and other injuries involving vascular injury and repair.
This protocol meets the institutional guidelines from the human research Ethics Committee.
1. Coronary Angiography, Ultrasound, and Blood Sampling
2. Determination of Circulating MPCs (Figure 2)
3. Determination of Plasma Soluble Biomarkers
Coronary, venous sinus, and peripheral blood were collected from 52 patients that underwent coronary angiography (Figure 1) and showed a high prevalence of hypertension and dyslipidemia. At the clinical follow-up, 11 (21.1%) MACEs occurred 6 months after coronary angiography: death (n = 1), angina requiring hospital attendance (n = 6), myocardial infarction (n = 2), and/or evidence of heart failure (n = 4).
The baseline coronary concentration of most MPCs was significantly lower in patients who developed MACEs (Figure 4), with a larger decrease in MPC subpopulations CD34+CD133+ and CD45+CD34+CD133+CD184+. Likewise, patients who developed MACEs had an increased baseline in coronary amounts of sICAM-1 and lower MMP-9 (Table 1).
Coronary MPCs (subpopulations CD45+CD34+CD133+ and CD45+CD34+CD133+CD184+) and sICAM-1 (dichotomized by their median values) demonstrated prognostic ability for MACE-free survival (Figure 5).
We further characterized the dynamics of soluble biomarkers under different conditions, because there is very little information regarding coronary blood determination. The expression of tumor necrosis factor alpha (TNFα) showed variations according to the time of measurement (pre- or post-angioplasty) and the location of coronary sampling based on a comparison of different lumen areas at same coronary artery using intravascular ultrasound (Figure 6).
Figure 1: Coronary angiography and blood collection. The image shows heart catheterization using a radial approach, performed under a fluoroscopy guide in the hemodynamics room. Cardiology experts evaluate the coronary vessels during angiography and collect coronary blood from the closest location to the atheroma plaque and/or sinus blood through a heart catheter just before balloon angioplasty. Please click here to view a larger version of this figure.
Figure 2: Blood sample preparation and MPCs determination by flow cytometry. (A) Density gradient after blood centrifugation (blue arrow = lymphocyte band). (B) Collection of the lymphocyte phase. (C) Washes with 1x PBS. (D) Centrifugation. (E) Pellet formation at the bottom of the test tube. (F) Neubauer cell suspension load. (G) Lymphocyte cell count using light microscopy. (H) Determination of cell subpopulations by flow cytometry. Please click here to view a larger version of this figure.
Figure 3: Immunoassays to determine blood soluble mediators. Upper row: Enzyme-linked immunosorbent assay (ELISA). The image shows how information from the map samples (notebook) was transferred to the software to start the readings after sample preparation, antibody incubation, and washes. It also shows yellow color development, either in the standard wells (left columns in the plaque) or in the test samples (right columns in the plaque). Lower row: Immuno-magnetic multiplexing assay. After sample preparation, magnetic bead-antibody incubation, and washes, the sample information was transferred to the appropriate immuno-magnetic multiplexing assay system reader software, and a typical standard curve is shown in the screen. Please click here to view a larger version of this figure.
Figure 4: Coronary circulating mononuclear progenitor cells (MPCs). The figure shows baseline %MPCs subpopulations. (A) Representative readings from flow cytometry. (B) Quantification of %MPCs subpopulations with flow cytometry, plotted according to the presentation of MACEs (*) = significant difference, with p < 0.05. This figure has been modified from Suárez-Cuenca et al.10. Please click here to view a larger version of this figure.
Figure 5: Coronary circulating cellular (MPCs), soluble biomarkers and prognosis. The figure shows baseline coronary blood amounts of (A) %MPCs subpopulations determined by flow cytometry and (B) plasma concentration of sICAM-1 determined by ELISA, both plotted according to the presentation of MACEs during the 6 month follow-up. The blue line indicates the number of individuals with risk values for each biomarker, such as lower %MPCs or higher sICAM-1. sICAM-1 = soluble intercellular adhesion molecule 1. This figure has been modified from Suárez-Cuenca, et al.10. Please click here to view a larger version of this figure.
Figure 6: Conditions determining variability of coronary circulating soluble biomarkers. The figure shows changes in the intracoronary concentration of tumor necrosis factor alpha (TNFα), according to the time of measurement (A: Pre-angioplasty or B: Post-angioplasty) as well as the location of coronary sampling (comparison between two coronary lumen diameters at a 3.5 mm cutoff, measured by intravascular ultrasound). (*) = p < 0.05 difference of biomarkers obtained pre- vs. post-angioplasty, and difference of sampling at locations of coronary lumen diameters ≤3.5 mm vs. >3.5 mm. This figure has been modified from Suárez-Cuenca et al.11. Please click here to view a larger version of this figure.
Table 1: Baseline blood soluble biomarkers. (*) indicates p < 0.05 difference biomarkers from coronary blood vs. peripheral circulation. (**) indicates p < 0.05, without MACEs vs. with MACEs; one-tailed independent T-test. Abbreviations: sICAM-1 = soluble intercellular adhesion molecule 1; IL-1β = interleukin 1 beta; MMP-9 = matrix metalloproteinase 9.
Blood collection from the affected coronary artery may be difficult. Sometimes, the coronary artery is barely accessible. In this case, sampling from the venous sinus may be an alternative. We performed validation tests comparing circulating biomarkers in coronary artery vs. venous sinus, with no significant differences. However, the performance of circulating biomarkers was validated only for coronary sampling. Therefore, the performance of biomarkers obtained from the venous sinus remains to be explored.
It is best to process the samples for MPCs within the first 3 h after blood collection. Therefore, good communication should be established between the cardiology team and the lab researchers. During MPCs isolation, care should be taken when depositing blood samples during density gradient preparation when washing the MPCs pellet. Finally, for convenience, we always transfer cells into a cytometry tube, add the primary antibodies, fix and store the cells overnight at 4 °C, and perform the flow cytometry reading the day after. Regarding the biomarker role of circulating MPCs, important efforts have been taken to standardize the most clinically useful immunophenotypes between progenitor cells12, but one limitation of the study may be the fact that specific subpopulations of circulating progenitor cells have not been fully characterized for all clinical scenarios within CAD or other vascular diseases. Therefore, different circulating progenitor cell subpopulations should be explored in each study.
During the determination of soluble markers some general recommendations for ELISA and multiplexing assays include the use of a multichannel pipette, depositing solutions at the bottom of each well without touching the side walls, and avoiding the drying out of the wells during the assay. Always check the sample distribution in the plate, particularly for the multiplexing assay, to avoid precipitation of the magnetic beads by constant vortexing. Also, make sure to insert the bottom plate into the hand-held magnetic plate washer to maintain the magnetic beads inside the wells, otherwise the samples will be lost during the washes.
We found that coronary circulating MPCs, mainly those from hematopoietic origin, as well as sICAM-1 and MMP-9, were outstanding biomarkers for prediction and prognosis of MACEs. This is consistent with the notion that inflammatory response and/or vascular damage mediators stimulate homing signals for MPC mobilization and recruitment, promoting local tissue repair4. Accordingly, we found variations in these biomarkers in several settings. Changes in relation to angioplasty and/or location of coronary sampling may be explained by the effect of the impact over the atheroma plaque during angioplasty, the size of the plaque, and the release of soluble mediators sequestered within the plaque into the coronary flow11. Increased IL-1β has been consistently involved in the development of the plaque and clinical complications13.
To our knowledge, this is the first study prospectively evaluating the role of coronary circulating MPCs and soluble mediators of vascular injury and repair as prognostic biomarkers in a population with CAD submitted to coronary angioplasty, including characterization of changes related to angioplasty, location of coronary sampling, and comparison of coronary vs. peripheral sampling. We think that the method can be easily established in any hospital carrying out coronary angiography. However, one limitation is that we applied this methodology mainly in patients with chronic stable angina released from an emergency room department.
Current traditional methods used for MACEs prediction or prognosis in CAD have moderate predictive ability. There has been an increasing amount of interest in finding novel biomarkers based on the pathophysiology mechanisms responsible for repair and regeneration occurring after CAD and angioplasty. Such biomarkers have shown similar or better predictive performance compared with traditional methods3,4,5,14,15. Thus, we think that the role of coronary circulating MPCs and soluble mediators in predicting the risk for MACEs will be explored further in future prospective studies.
The authors have nothing to disclose.
The authors thank the support of Institutional Program E015; and Fondo Sectorial FOSSIS-CONACYT, SALUD-2014-1-233947.
BSA | Roche | 10735086001 | Bovine Serum Albumin (BSA) as a buffering agent, stabilizer, standard and for blending. |
Calibration Beads | Miltenyi Biotec / MACS | #130-093-607 | MACQuant calibration beads are supplied in aqueous solution containing 0.05% sodium azide. 3.5 ml for up to 100 tests |
CD133/1 (AC133)-PE | Milteny Biotec / MACS | #130-080-801 | Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer |
CD184 (CXCR4)-PE-VIO770 | Miltenyi Biotec / MACS | #130-103-798 | Monoclonal, Isotype recombinant human IgG1, conjugated |
CD309 (VEGFR-2/KDR)-APC | Miltenyi Biotec / MACS | #130-093-601 | Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer |
CD34-FITC | Miltenyi Biotec / MACS | #130-081-001 | The monoclonal antibody clone AC136 detecs a class III epitope of the CD34 |
CD45- VioBlue | Miltenyi Biotec / MACS | #130-092-880 | Monoclonal CD45 Antibody, human conjugated |
Conical Tubes | Thermo SCIENTIFIC | #339651 | 15ml conical centrifuge tubes |
Cytometry Tubes | FALCON Corning Brand | #352052 | 5 mL Polystyrene Round-Bottom Tube. 12×75 style. Sterile. |
EDTA | BIO-RAD | #161-0729 | Heavy metals, (as Pb) <10ppm, Fe <0.01%, As <1ppm, Insolubles <0.005% |
Improved Neubauer | Without brand | Without catalog number | Hemocytometer for cell counting. (range 0.1000mm, 0.0025mm2) |
K2 EDTA Blood Collection Tubes | BD Vacutainer | #367863 | Lilac plastic vacutainer tube (K2E) 10.8mg, 6 mL. |
Lymphoprep | Stemcell Technologies | 01-63-12-002-A | Sterile and checked on the presence of endotoxins. Density: 1.077±0.001g/mL |
Paraformaldehyde | SIGMA-ALDRICH | #SZBF0920V | Fixation of biological samples, (powder, 95%) |
Pipette Transfer 1,3mL | CRM Globe | PF1016, PF1015 | The transfer pipette is a tool that facilitates liquid transfer with greater accuracy. |
Test Tubes | KIMBLE CHASE | 45060 13100 | Heat-resistant test tubes. SIZE/CAP 13 x 100 mm |