Un abonnement à JoVE est nécessaire pour voir ce contenu. Connectez-vous ou commencez votre essai gratuit.

Summary

Lower limb amputation may occur even after angioplasty of obstructed vessels in Critical Limb Ischemia (CLI). Mononuclear Progenitor Cells (MPCs) reflect vascular repair. The present protocol describes the quantification of MPCs from circulation close to angioplasty, and its relationship with endothelial dysfunction and prediction of lower limb amputation.

Abstract

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; however, some patients irreversibly progress to limb amputation. The extent of vascular damage and the mechanisms of vascular repair are factors affecting post-angioplasty outcome. Mononuclear Progenitor Cells (MPCs) are reactive to vascular damage and repair, with the ability to reflect vascular diseases. The present protocol describes quantification of MPCs obtained from blood circulation from vessel close to the angioplasty site, as well as its relationship with endothelial dysfunction and its predictive ability for limb amputation in the next 30 days after angioplasty in patients with CLI.

Introduction

Peripheral Arterial Disease (PAD) is characterized by a chronic and progressive vascular obstruction with limitation of blood supply¹. At a global scale, PAD of the lower limbs affects around 10% of the elderly population, while up to 7% of such cases are submitted to limb amputation^2,3.

Critical Limb Ischemia (CLI) represents the most serious presentation of PAD¹. Patients usually experience pain at rest, ulcers, or gangrene attributable to occluded arteries; while clinical prognosis is unfavorable and marked by a 30% risk of limb amputation and mortality during 1 year^3,4,5.

Angioplasty is a minimally invasive endovascular procedure that can restore blood flow to the lower limb in patients with CLI; however, some patients will inevitably require major limb amputation, even after angioplasty therapy^1,5. Early identification of unfavorable outcomes after angioplasty is quite valuable, due to the possibility of therapy enforcement.

Traditional risk factors may provide a limited predictive ability for major limb amputation in patients with CLI undergoing angioplasty⁶. Pathophysiology-oriented biomarkers represent novel methods with potential clinical applications, which may result specifically useful in diseases related to vascular injury⁷. Nowadays, the participation of cellular populations owning endothelial repair properties, at the site of the atherosclerotic plaque, has been increasingly recognized^8,9.

Mononuclear Progenitor Cells (MPCs) are derived from the bone marrow and own structural and functional characteristics of stem cells with vascular regenerative abilities. Due to MPC’s ability to proliferate, migrate and show vascular adherence; these cells have become good candidates to reflect endothelial repair in response to ischemia^10,11,12. In addition, continuous interest in mechanisms underlying vascular injury has motivated exploring the prognostic role of local occurring biomarkers, since they are considered to reflect vascular damage and repair^7,13,14.

The purpose of the present study is to describe how to determine the amount of MPCs that circulate close to the vascular obstruction in patients with CLI undergoing angioplasty; and how to evaluate the relation between MPCs with indicators of endothelial dysfunction and limb amputation.

Compared to the prognosis based on comorbidities and intrinsic vascular features, the amount of local MPCs show specific ability to predict clinical outcome regarding endothelial dysfunction and limb amputation. Consistently, some studies have described the prognostic role of similar biomarkers during the evaluation of patients with PAD^15,16.

Based on previous results⁷, the method described here may be useful for an early identification of population at risk of adverse vascular outcomes in several clinical settings, such as lower limb and coronary ischemia, stroke, vasculitis, venous thrombosis and others involving vascular injury and repair.

Protocol

The institutional research ethics committee from Centro Médico Nacional “20 de Noviembre” ISSSTE approved this prospective protocol, all enrolled patients provided written informed consent.

1. Evaluation of vascular block of lower limb, blood sampling and balloon angioplasty

NOTE: The study sample used for this experiment comprised of 20 diabetic patients, aged 68 years old and 10 out of 20 were males. Half of the sample were smokers and most prevalent co-morbidities were type 2 diabetes mellitus, systemic arterial hypertension and/or dyslipidemia. The sample was intended to be standardized for age-, sex- and co-morbidities. Possible bias due to clinical-demographic influence on the relation between MPCs and CLI could not be ruled out.

1. Evaluate clinical severity of limb ischemia according to the Rutherford classification¹³ (see Supplementary Table 1).
2. Perform lower limb angiography, blood sampling and balloon angioplasty.
   1. Use anticoagulant and anesthetic drugs before surgery.
   2. Place an 18 G needle into the blood vessel at the groin site selected.
   3. Place an introducer and advance a flexible guide wire. Then, the initial guide is further changed for a 6 Fr introducer.
   4. Use periodic injection of contrast media or CO₂ under fluoroscopic guide to identify artery trajectory and vascular blocked sites (Figure 1).
      NOTE: Use contrast media 40 cc, diluted 1:1 in 0.9% saline, or CO₂ at 10 to 20 mL per shot at a pressure of 12 psi.
   5. Introduce two 0.014 Fr navigation guide wires and two 0.014 Fr support guide wires into the vessel and advance them up to the blocked site.
      NOTE: Two 0.014 Fr guide wires (260 cm long) are used for navigation and two 0.014 Fr guide wires (260 cm long) are used for support, during a single procedure.
   6. Introduce 5 Fr and 3 Fr catheters sequentially and collect 10 mL of blood from the closest site to the vascular obstruction. Maintain blood samples on ice.
      NOTE: Catheters sizes may be 5 Fr and 3 Fr, and they are changed during the procedure.
   7. Advance a guide wire again. Then, introduce an angioplasty balloon catheter, which contains an inflatable balloon located at the end of the catheter. Advance the angioplasty balloon catheter and place the balloon right at the site of lesion. Perform the angioplasty by inflating the balloon against the blocking plaque located at the vascular wall. Verify blood flow restoration.
      NOTE: A stent may be placed in the blocked area to help keep the artery open after the procedure.
   8. Introduce a catheter and advance up to the closest site to the vascular block. Collect 10 mL of blood at 30 min time interval after angioplasty. Maintain blood samples on ice.
      NOTE: Collection of blood samples before and after the angioplasty is recommended to further evaluate the influence of the angioplasty on the number of MPCs.
   9. Remove all the wires under fluoroscopic guidance.
   10. Provide post-operative care procedures, including anti-coagulation therapy using enoxaparin at 1 mg/kg subcutaneous every 12 h, aspirin 100 mg, statin, and analgesia. Compress at the site of vascular puncture during 24 h.

2. Quantification of circulating mononuclear progenitor cells (MPCs) (Figure 2)

1. To a fresh 15 mL conical tube, transfer 6 mL of the collected blood and dilute 1:1 (v/v) with PBS.
   NOTE: Process the blood within 1 h from collection.
2. Prepare for density gradient separation, by adding 2 mL of the density gradient medium to 3 test tubes each. Then, add 3 equal volume aliquots of the diluted blood into each test tube.
   NOTE: Do not exceed three-fourths of the test tube maximal capacity.
3. Centrifuge at 1,800 x g for 30 min at 4 °C. Collect the interface layer present as a ring using a pipette, and transfer into a new tube. Add 2 mL of PBS and spin at 1,800 x g for 6 min at 4 °C. Save the pellet as this will contain MPCs.
4. Wash the pellet containing MPCs with PBS by centrifugation as described in step 2.3. Use fresh PBS for each wash and spin at 1,800 x g for 2 min at 4 °C. Repeat the process for 6 times.
5. After the last wash, use 1 mL of PBS to resuspend the cell pellet. Dilute 20 µL of the cell suspension with 20 µL of 0.4% trypan blue, 1:1 (v/v). Use 10 µL of this cell suspension for cell counting using hemocytometer and a light microscope.
6. Aliquot 1 x 10⁶ MPCs in previously labeled 5 mL flow cytometry tubes.
   NOTE: Prepare corresponding isotype-matched control antibodies.
7. Centrifuge the tubes at 1,800 x g for 6 min at 4 °C. Aspirate and discard the supernatant.
8. Dilute primary antibody in 100 µL of antibody incubation solution [1x PBS, pH 7.4, EDTA 2 mM, BSA 0.05%] and add to the tube. Resuspend for 10 s and incubate for 20 min at 4 °C, in the dark.
   NOTE: Final concentrations of primary antibodies used in the present protocol were CD45 1:50, CD34 1:20, KDR 1:50, CD184 1:20, CD133 1:50. Protocol may be stopped at this step by fixing lymphocytes in 4% paraformaldehyde in PBS and storing samples up to 24 h at 4 °C.
9. Centrifuge at 1,800 x g for 2 min at 4 °C and discard the supernatant. Resuspend in 500 µL of 1x PBS, pH 7.4, EDTA 2 mM.
10. Perform flow cytometry analysis.
    1. Set up the background with isotype-matched control antibodies. Then, at the FSC/SSC plot select lymphocytes spread, trying to exclude cellular debris, residual granulocytes, and other particles. Such distribution is considered as 100%.
       NOTE: Lymphocytes usually spread in the lower-left region of the plot.
    2. Use a gate with common immunophenotype containing high number of cells CD45⁺ and CD34⁺. Then, select for double positive immunophenotypes using gate which previously identified CD45⁺, CD34⁺, and adding either KDR (VEGFR-2)⁺, CD133⁺ or CD184⁺. Identify MPCs subpopulations by their specific cell surface markers. Report as the percentage of gated events.
11. Identify main subpopulations of MPCs. In the present study the immunophenotypes analyzed were CD45⁺CD34⁺CD133⁺; CD45⁺CD34⁺CD184⁺; CD45⁺CD34⁺CD133⁺CD184⁺; CD45⁺CD34⁺KDR⁺; CD45⁺CD34⁺KDR⁺CD133⁺ and CD45⁺CD34⁺KDR⁺CD184^7,17.
    NOTE: These cell surface markers were used for the study: 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. Relation of MPCs with modification of endothelial function and hemodynamic test (FMD)

1. Determine flow-mediated dilation (FMD), pre- and post-angioplasty.
   1. Use a vascular linear transducer to measure the diameter of the brachial artery.
   2. Place the cuff of the sphygmomanometer above the measurement site in the forearm and insufflate at 50 mmHg above the systolic blood pressure for 5 min and deflate.
   3. Determine again the diameter of the brachial artery within the next 60 s. Use the equation below to estimate FMD.
      NOTE: Calculate the degree of dilatation using the equation (%) = (maximum diameter after transient ischemia – basal diameter) × 100 / basal diameter.
2. Correlate the number of MPCs with the baseline FMD value and post-angioplasty delta of FMD.

4. Prognostic ability of MPCs for limb amputation

1. Schedule periodic medical appointments after balloon angioplasty and patient discharge, to evaluate the quality of blood flow to the lower limb.
2. Evaluate clinical severity of limb ischemia at 2 weeks after angioplasty. Evaluate resolving rest pain, lower ischemia, and preservation of a functional foot, according to the Rutherford classification¹³.
3. Compare clinical severity of limb ischemia, at baseline versus follow up. Identify those cases requiring major amputation due to unfavorable outcome.
4. Correlate the number of MPCs with the proportion of patients requiring major amputation of the lower limb.

Representative Results

Blood samples from blocked arteries, at the site addressed for angioplasty, were collected from 20 diabetic patients, aged 68 years old and 10 out of 20 were males. Half of sample population were smokers. Vascular lesions were mainly scored as Rutherford class VI; whereas patients showed a higher prevalence of type 2 Diabetes Mellitus (100%), hypertension (70%) and dyslipidemia (55%).

A 30 days clinical follow up after lower limb angioplasty was carried out. The percentage of MPC subpopulations at baseline or dynamics after angioplasty were correlated (Spearman analysis) with the degree of endothelial dysfunction, as evaluated by FMD; and the baseline number of MPCs were compared between patients undergoing, or not, limb amputation after angioplasty (U-Mann Whitney). The study showed that baseline MPCs subpopulation CD45⁺CD34⁺KDR⁺ negatively correlated with FMD (Figure 3A, left), whereas the change of MPCs CD45⁺CD34⁺CD133⁺CD184⁺ after angioplasty significantly correlated with FMD improvement (Figure 3B, right). Furthermore, increased baseline number of MPCs subpopulation CD45⁺CD34⁺KDR⁺ (Figure 4A,B, left) were observed in those patients who evolved to limb amputation; as well as post-angioplasty reduction of MPCs subpopulation CD45⁺CD34⁺CD133⁺CD184⁺ (Figure 4A,C, right).

Figure 1: Lower limb angiography and blood collection. (A) Vascular trajectory evidenced by contrast media under fluoroscopy. (B) Vascular obstruction before angioplasty. (C) Vascular obstruction after angioplasty. (D) Vascular surgeon uses a catheter to collect blood from the closest location to the vascular obstruction and atheroma plaque, and Lab researcher is ready to obtain the blood sample. Arrows indicate the site of vascular obstructions. Please click here to view a larger version of this figure.

Figure 2: Blood sample preparation and mononuclear progenitor cells (MPCs) determination. (A) Density gradient preparation. (B) Lymphocytes ring separation after blood centrifugation. (C) Collection of the lymphocyte phase. (D) Centrifugation. (E) Pellet formation at the bottom of the test tube. (F) Cell suspension count. (G) Preparation of lymphocytes for flow cytometry. (H) Determination of cell subpopulations by flow cytometry. Please click here to view a larger version of this figure.

Figure 3: Relation of MPCs with hemodynamic indicators. (A) Position of the ultrasound to acquire FMD and representative results. (B) Relationship between baseline %MPCs subpopulations (left, CD45⁺CD34⁺KDR⁺; right, CD45⁺CD34⁺CD133⁺CD184⁺) and baseline FMD values; as well as the relation of (C) %MPCs after angioplasty with FMD improvement after angioplasty. Abbreviatures: MPCs, Mononuclear Progenitor Cells; FMD, Flow Mediated Dilation. Please click here to view a larger version of this figure.

Figure 4: MPCs and prognosis of lower limb amputation after angioplasty. (A) Representative flow cytometry images of MPCs subpopulations. (B) The association of baseline %MPCs subpopulations (left, CD45⁺CD34⁺KDR⁺; right, CD45⁺CD34⁺CD133⁺CD184⁺), or (C) %MPCs after angioplasty, with lower limb amputation after angioplasty, during a 30-days follow up. Abbreviatures: MPCs, Mononuclear Progenitor Cells. Please click here to view a larger version of this figure.

Supplementary File 1: Rutherford’s classification of severity of limb ischemia. Please click here to download this file.

Discussion

Blood collection at the precise site of the vascular block may show technical difficulties; therefore, we performed blood collection in the proximity to vascular block. Likewise, the amount of MPCs close to the vascular plaque seems to be highly dynamic and may originate variations before and after angioplasty. According to our observations, it is recommended to evaluate baseline- and 30min-post-angioplasty changes in the number of MPCs, since they may reflect several pathophysiological processes occurring within vascular damage and repair.

Blood sample processing for MPCs determination is recommended to be performed within the first 3 h; therefore, adequate organization plan, and even a previous simulation practice, may be established between angiology-vascular surgery team and lab researchers. MPCs isolation should be carefully performed, particularly deposing blood sample into density gradient and washes of the pellet containing MPCs. Our group use to transfer cells into cytometry tube, add primary antibodies, fix, and store cells overnight at 4 °C; due to time-administration convenience, and flow cytometry reading would be performed the day after.

Regarding the role of circulating MPCs as a useful clinical biomarker of vascular damage and repair, important efforts have been reported to standardize immunophenotypes between progenitor cells¹⁷. A comprehensive characterization should include subpopulations of circulating progenitor cells participating in the different clinical scenarios within vascular diseases. Using the methods described here, we found that post-angioplasty reduction of MPCs subpopulation CD45⁺CD34⁺CD133⁺CD184⁺ is predictive for major amputation. This finding supports the notion that inflammatory response during vascular injury or angioplasty stimulate homing signals for MPCs, promoting local tissue repair^18,19.

Likewise, the observation is consistent with reported effect of reduced number of CD45⁺CD34⁺CD133⁺ and CD45⁺CD34⁺CD133⁺184⁺ subpopulations of MPCs as a predictor of adverse cardiovascular outcomes after coronary angioplasty⁷, which may be explained by the increased ability of less differentiated phenotypes of endothelial progenitors to adhere to extracellular matrix, to proliferate and to respond to vasculogenic stimulus¹⁸.

Furthermore, we observed that an increased number of CD45⁺CD34⁺KDR⁺ subpopulation of MPCs after angioplasty was related with limb amputation although they have been considered to contribute to vascular repair. This controversy may be explained due to: 1) variations in the study design, since other studies have compared the number of CD45⁺CD34⁺KDR⁺ MPCs between patients with CLI and healthy controls¹⁹; 2) variations in the methods for blood sampling, including the site and the time regarding the angioplasty; and 3) the type of artery blocked and vascularized.

Prognostic characterization of novel biomarkers, based on mechanisms responsible for vascular repair in several diseases, has received significant attention in translational research. This is the first description of a method to explore the role of MPCs, locally determined at a site close to the vascular block, in the prognosis of limb amputation after angioplasty in cases with CLI. One limitation is the lack of MPCs determination at more time points after angioplasty. However, we believe that our findings enrich the field of vascular research by characterizing the translational role of MPCs during vascular damage, repair, and prognostic potential for major amputation in patients with CLI. Particularly, we consider that the method described here may be useful in the prediction of adverse vascular outcomes in clinical settings involving a vascular injury and repair mechanisms, such as lower limb and coronary ischemia, stroke, vasculitis, and/or venous thrombosis.

Materials

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