Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Summary

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.

Abstract

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.

Introduction

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 angioplasty^2,3,4,5.

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 scarce^7,8,9. 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.

Protocol

This protocol meets the institutional guidelines from the human research Ethics Committee.

1. Coronary Angiography, Ultrasound, and Blood Sampling

1. Request baseline clinical and demographic information before coronary intervention. Collect the individual's data: age, sex, current smoking status, body mass index (BMI), high blood pressure, dyslipidemia, diabetes mellitus, medications, and the indication for current coronary angiography.
2. Perform coronary angiography through heart catheterization using a radial approach. This procedure should be performed under a fluoroscopy guide in the hemodynamics room by expert cardiologists.
   NOTE: Identify evaluable vessels. For the present study, evaluable vessels were defined as arteries with sections larger than 1.5 mm and lumen stenosis of more than 50%.
3. Advance the intravascular ultrasound catheter to the region of interest and record images. Use the appropriate software to locate and measure the smallest luminal area.
4. Use a coronary catheter to collect 10 mL of blood from the closest location to the plaque.
5. After patient discharge, schedule periodical medical evaluations to follow up study endpoints. If telephone contact is not possible or a physician visit is delayed for longer than 2 months, request an authorized person (previously designed) to verify the study endpoints.
   NOTE: Consider any of the following a MACE: 1) cardiovascular death, 2) new myocardial infarction, 3) unstable angina prompting an unscheduled medical visit within 24 h, 4) stent restenosis as demonstrated by coronary angiography, 5) episodes of decompensated heart failure requiring clinical attention.

2. Determination of Circulating MPCs (Figure 2)

1. Process the blood within 1 h from collection. Transfer 6 mL of the collected blood to a 15 mL conical tube and dilute 1:1 (v/v) with 1x phosphate buffered saline (PBS), pH = 7.4.
2. Add 2 mL of density gradient medium to three test tubes. Carefully transfer three equal volume aliquots of diluted blood into each test tube containing the density gradient medium.
   NOTE: The total volume of density gradient medium and diluted blood should not exceed three-fourths of the test tube maximal capacity.
3. Centrifuge at 1,800 x g, 4 °C for 30 min. Transfer the band at the interface between the layers into a new tube. Add 2 mL of PBS and centrifuge at 1,800 x g, 4 °C for 6 min. The pellet will contain the MPCs.
4. Wash the pellet several times. Aspirate off the previous solution and gently resuspend the cell pellet in fresh PBS. For subsequent washes, centrifuge at 1,800 x g, 4 °C for 2 min. Repeat the process 6x.
5. Resuspend the cell pellet in 1 mL of PBS. Mix 20 µL of the cell suspension with 0.4% trypan blue, diluted 1:1 (v/v). Apply a drop to a hemocytometer and count the unstained cells under a light microscope.
6. Proceed to MPCs determination. Label 5 mL flow cytometry tubes and aliquot out 1 x 10⁶ cells per tube. Prepare the corresponding isotype-matched control antibodies. Centrifuge at 1,800 x g, 4 °C for 6 min and discard the supernatant.
7. Add the primary antibody diluted in 100 µL of an antibody incubation solution consisting of 1x PBS (pH = 7.4), 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% bovine serum albumin (BSA). Resuspend for 10 s and incubate for 20 min at 4 °C, light-protected. The protocol may be paused at this step by fixing the lymphocytes in 4% paraformaldehyde in PBS and storing samples up to 24 h at 4 °C.
   NOTE: The final concentrations of the primary antibodies used in the present protocol were CD45 1:50, CD34 1:20, KDR 1:50, CD184 1:20, CD133 1:50.
8. Centrifuge at 1,800 x g, 4 °C for 2 min and discard the supernatant. Resuspend in 500 µL of 1x PBS (pH = 7.4), 2 mM EDTA.
9. Perform flow cytometry analysis. Use isotype-matched control antibodies to set up the background staining. Then, select lymphocytes spread at the FSC/SSC plot, trying to exclude residual granulocytes, cellular debris, and other particles, which are usually located in the lower, left-distributed in the plot. Such distribution is considered as 100%.
10. Use a gate containing a high number of cells with common immunophenotype CD45⁺ and CD34⁺. For double positive immunophenotypes, use a gate previously identifying CD45⁺, CD34⁺, with the addition of either KDR (VEGFR-2)⁺, CD133⁺, or CD184⁺. Identify the MPC subpopulations by their specific cell surface markers. Report as the percentage of gated events.
11. Identify the main subpopulations of MPCs. In the present study the main immunophenotypes were CD45⁺CD34⁺CD133⁺, CD45⁺CD34⁺CD184⁺, CD45⁺CD34⁺CD133⁺CD184⁺, CD45⁺CD34⁺KDR⁺, CD45⁺CD34⁺KDR⁺CD133⁺, and CD45⁺CD34⁺KDR⁺CD184.
    NOTE: The cell surface markers used were CD45 (lymphocytes), CD34 (endothelial and/or vascular cells), KDR (VEGFR-2, membrane marker of endothelial cells), CD133 (endothelial progenitor cells), and CD184 (hematopoietic stem cells and endothelial cells).

3. Determination of Plasma Soluble Biomarkers

1. Use an enzyme-linked immunosorbent assay (ELISA) to determine the concentration of SICAM-1 and MMP-9 (Figure 3, upper row).
   1. Centrifuge the blood samples at 3,000 x g, room temperature for 5 min and collect the plasma.
   2. Label the standards, sample tubes, and control tubes. Equilibrate the pre-coated wells in the assay plate by washing 2x with the washing buffer provided in the ELISA kit.
   3. Transfer the standards, samples, and controls to the wells. Seal and incubate at 37 °C for 90 min.
      NOTE: Do not let the wells dry completely.
   4. Discard the contents and add the biotin-detection antibody. Seal and incubate at 37 °C for 60 min.
   5. Discard the contents and wash 3x. Seal the plaque and incubate sequentially with streptavidine working solution followed by tetramethylbenzidine substrate at 37 °C for 30 min, light-protected. Wash 3x between incubations. When the color develops, add the stop solution, and read the optical density absorbance in a microplate ELISA reader.
2. Use an immuno-magnetic multiplexing assay to determine the concentration of tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β) (Figure 3, lower row).
   1. Label the standards, sample tubes, and control tubes.
   2. Vortex the magnetic beads vials for 30 s. Transfer the bead suspension to appropriately sized tubes, and then to the wells in the multiplexing assay plate. Periodic vortexing avoids precipitation of the beads.
   3. Securely insert the hand-held magnetic plate washer. Wait 2 min for the beads to accumulate on the bottom of each well and quickly invert both the hand-held magnetic plate washer and plate assembly, over a sink or waste container. Remember to use the hand-held magnetic plate washer to maintain the beads inside the wells.
   4. Add 150 µL of wash buffer into each well and wait 30 s to allow the beads to accumulate on the bottom. Discard the contents as in step 3.2.3. Then, add 25 µL of universal assay buffer (provided in the kit) followed by 25 µL of prepared standards, samples, and controls.
   5. Seal the plate and incubate for least 60 min at room temperature, light-protected, with constant shaking at 500 rpm. Alternatively, incubate overnight at 4 °C, light-protected, with constant shaking at 500 rpm if possible.
   6. Wash 2x by adding 150 µL of wash buffer and wait 30 s. Discard the contents by inserting the hand-held magnetic plate washer. Wait 2 min and invert over a sink or waste container.
   7. Incubate sequentially with 25 µL of detection antibody mixture followed by 25 µL of streptavidin-PE solution at room temperature for 30 min, sealed and light-protected, with constant shaking at 500 rpm. Wash 2x between incubations, as described in step 3.2.6.
   8. Obtain the readings. Add 120 µL of reading buffer. Seal the plate and incubate 5 min at room temperature, light-protected, with constant shaking at 500 rpm. Run the reading on a multiplexing assay reader. Adjust the reading parameters according to each analyte.

Representative Results

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.

Discussion

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 methods^3,4,5,14,15. 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.

Materials

Name	Company	Catalog Number	Comments
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. 12x75 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