Name: predicting amputation using local circulating mononuclear progenitor cells in angioplasty-treated patients with critical limb ischemia
Uploaded: 2020-09-22T13:00:00
Description: Watch this Scientific Journal Video about Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia at JoVE.com

The authors thank the support of Institutional Program E015 for the project ID 356.2015.

The institutional research ethics committee from Centro M&#233;dico Nacional &#8220;20 de Noviembre&#8221; 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 prevalen...

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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 vascula...

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 amputation2,3.
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 year3,4,5.
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 therapy1,5. 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 recognized8,9.
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&#8217;s ability to proliferate, migrate and show vascular adherence; these cells have become good candidates to reflect endothelial repair in response to ischemia10,11,12. 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 repair7,13,14.
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 PAD15,16.
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.

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			<td>BSA</td>
			<td>Roche</td>
			<td>10735086001</td>
			<td>Bovine Serum Albumin (BSA) as a buffering agent, stabilizer, standard and for blending.</td>
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		<tr>
			<td>Calibration Beads</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-093-607</td>
			<td>MACQuant calibration beads are supplied in aqueous solution containing 0.05% sodium azide. 3.5 ml for up to 100 tests</td>
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			<td>CD133/1 (AC133)-PE</td>
			<td>Milteny Biotec / MACS</td>
			<td>#130-080-801</td>
			<td>Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer</td>
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			<td>CD184 (CXCR4)-PE-VIO770</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-103-798</td>
			<td>Monoclonal, Isotype recombinant human IgG1, conjugated</td>
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		<tr>
			<td>CD309 (VEGFR-2/KDR)-APC</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-093-601</td>
			<td>Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer</td>
		</tr>
		<tr>
			<td>CD34-FITC</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-081-001</td>
			<td>The monoclonal antibody clone AC136 detecs a class III epitope of the CD34</td>
		</tr>
		<tr>
			<td>CD45- VioBlue</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-092-880</td>
			<td>Monoclonal CD45 Antibody, human conjugated</td>
		</tr>
		<tr>
			<td>Conical Tubes</td>
			<td>Thermo SCIENTIFIC</td>
			<td>#339651</td>
			<td>15ml conical centrifuge tubes</td>
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			<td>Cytometry Tubes</td>
			<td>FALCON Corning Brand</td>
			<td>#352052</td>
			<td>5 mL Polystyrene Round-Bottom Tube. 12x75 style. Sterile.</td>
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			<td>EDTA</td>
			<td>BIO-RAD</td>
			<td>#161-0729</td>
			<td>Heavy metals, (as Pb) &#60;10ppm, Fe&#60;0.01%, As&#60;1ppm, Insolubles&#60;0.005%</td>
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			<td>Improved Neubauer</td>
			<td>Without brand</td>
			<td>Without catalog number</td>
			<td>Hemocytometer for cell counting. (range 0.1000mm, 0.0025mm2)</td>
		</tr>
		<tr>
			<td>K2 EDTA Blood Collection Tubes</td>
			<td>BD Vacutainer</td>
			<td>#367863</td>
			<td>Lilac plastic vacutainer tube (K2E) 10.8mg, 6 mL.</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>01-63-12-002-A</td>
			<td>Sterile and checked on the presence of endotoxins. Density: 1.077&#177;0.001g/mL</td>
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			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>#SZBF0920V</td>
			<td>Fixation of biological samples, (powder, 95%)</td>
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			<td>Pipette Transfer 1,3mL</td>
			<td>CRM Globe</td>
			<td>PF1016, PF1015</td>
			<td>The transfer pipette is a tool that facilitates liquid transfer with greater accuracy.</td>
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			<td>Test Tubes</td>
			<td>KIMBLE CHASE</td>
			<td>45060 13100</td>
			<td>Heat-resistant test tubes. SIZE/CAP 13 x 100 mm</td>
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predicting amputation using local circulating mononuclear progenitor cells in angioplasty-treated patients with critical limb ischemia

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.

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 subpopulation...

Watch this Scientific Journal Video about Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia at JoVE.com

Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia

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.

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; ...

Although angioplasty improves blood flow in critical limb ischemia, some patients will still progress to limb amputation. Compared to prognosis based on traditional risk factors and intrinsic vascular features, the number of local MPCs can be more predictive of the clinical outcome, regarding endothelial dysfunction and limb amputation. The number of local MPCs represents a promising indicator of the prognosis of vascular events, such as ischemia heart disease, and lower limb ischemia, with potential application in risk stratification and prevention enforcement.Demonstrating the procedures will be Eduardo Vera-Gomez, Alejandro Hernandez-Patricio, Carolina Aranda-Rodriguez, Juan Ariel Gutierrez-Buendia, Atzin Sua Ruiz-Hernandez, Mario Antonio Tellez-Gonzalez, and Gabrielle Alexandra Dominguez-Perez. PhD's from the laboratories of experimental metabolism and tissue regeneration, as well, will be Gabrielle Hernandez-De Rubin and Oscar Antonio Loman-Zuniga. And these from the vascular surgery department.To determine the pre and post angioplasty FMD, use a vascular linear transducer to measure the diameter of the brachial artery of the patient. Next place, the cuff of a sphygmomanometer above the measurement site in the forearm, and insufflate the cuff 50 millimeters of mercury above the systolic blood pressure for five minutes before deflating. Within 60 seconds of deflating, measure the brachial artery diameter again, and use the equation to estimate the FMD.To evaluate the clinical severity of limb ischemia, according to the Rutherford classification, place an 18 gauge needle into the blood vessel at the selected groin site, and place an introducer over the needle. Advance a flexible guidewire into the introducer and replace the guide with a six French introducer. Using fluoroscopic guidance, inject contrast medium to identify the artery trajectory and blocked vascular sites.Introduce two, 0.014 French navigation guidewires and two, 0.014 French support guidewires into the vessel, and advance the guide wires to the blocked site. Next, introduce one five French catheter, and then one three French catheter, and collect 10 milliliters of blood from the site closest to the vascular obstruction. Place the sample on ice and advance a guidewire again.When the guidewire is in place, introduce an angioplasty balloon catheter, containing an inflatable balloon located at the end of the catheter, and advance the angioplasty balloon catheter to the site of the lesion. Inflate the balloon against the blocking plaque, located at the vascular wall, and verify restoration of the blood flow. 30 minutes after performing the angioplasty, advance a catheter to the site closest to the vascular block and collect 10 milliliters of blood.Then, remove all of the guidewires and provide the appropriate post-operative care. Two weeks after the angioplasty, evaluate the clinical severity of the limb ischemia, the resolving rest pain, lower ischemia, and functional foot preservation, according to the Rutherford classification, and identify those cases requiring a major amputation due to an unfavorable outcome. Within one hour of collection, transfer six milliliters of each collected blood sample into new 15 milliliter tubes, and dilute the samples at a one-to-one ratio in PBS.Add two milliliters of density gradient medium to each of three test tubes, and add three equal volume aliquots of the diluted blood to each tube to no more than three quarters full. Separate the blood cells by density gradient centrifugation, and use a pipette to collect the cells of the interface of the resulting layers. Pull the cells from each sample into a single 15 milliliter tube, and wash each sample six times, with two milliliters of PBS per wash.After the last wash, re-suspend the MPC containing pellets in one milliliter of PBS per sample for counting, and add one times 10 to the six cells per tube to the appropriate number of five milliliter flow cytometry tubes per sample. Pellet the cells by centrifugation, and re-suspend each cell sample in 100 microliters of the antibody cocktail of interest for 10 seconds, before incubating the cells for 20 minutes at four degrees Celsius, protected from light. At the end of the incubation, use isotype-matched control antibodies to set up the appropriate forward and side scatter gates for analysis, and use a tube containing a high number of CD45 and CD34 positive cells to gate the NPCs.To select for double positive immuno phenotypes, create new plots and gate-identify the KDR, CD133 and CD184 positive, CD34 positive, CD45 positive cells, then gate to identify the main MPC subpopulations. The number of MPCs can then be correlated with the baseline FMD value, and post angioplasty delta of FMD, and the proportion of patients requiring major amputation of the lower limb. In this representative analysis, 30 days after lower limb angioplasty, the baseline number of CD45 positive, CD34 positive, KDR positive MPCs, negatively correlated with the FMD.Whereas the change in CD45 positive, CD34 positive, CD133 positive, CD184 positive MPCs, after angioplasty, significantly correlated with FMD improvement. An increased baseline number of MPCs CD45 positive, CD34 positive, KDR positive subpopulations, as well as post angioplasty reduction in the MPC CD45 positive, CD34 positive, CD133 positive, CD184 positive cells, were also observed in patients, who evolved to limb amputation, During the vascular approach, be sure to collect the blood from the site closest to the vascular obstruction.

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