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

Forfatterne takker støtten fra Institutional Program E015 for projekt-id 356.2015.

Den institutionelle forskningsetiske komité fra Centro Médico Nacional "20 de Noviembre" ISSSTE godkendte denne potentielle protokol, alle indskrevne patienter gav skriftligt informeret samtykke.
1. Vurdering af vaskulær blok af underekstremiteter, blodprøvetagning og ballon angioplastik
BEMÆRK: Den undersøgelsesprøve, der blev anvendt til dette eksperiment, bestod af 20 diabetikere i alderen 68 år og 10 ud af 20 var mænd. Halvdelen af prøven var rygere, og ...

<ol>
	<li>Serrano-Hernando, F. J., Mart&#237;n-Conejero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+artery+disease:+Pathophysiology,+diagnosis+and+treatment.">Peripheral artery disease: Pathophysiology, diagnosis and treatment.</a> Revista Espa&#241;ola de Cardiolog&#237;a. 60 (9), 969-982 (2007).</li><li>Agarwal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+re-admissions+among+patients+with+critical+limb+ischemia.">Burden of re-admissions among patients with critical limb ischemia.</a> Journal of the American College of Cardiology. 69 (15), 1897-1908 (2017).</li><li>Kolte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty-day+re-admissions+after+endovascular+or+surgical+therapy+for+critical+limb+ischemia:+Analysis+of+the+2013+to+2014+nationwide+re-admissions+databases.">Thirty-day re-admissions after endovascular or surgical therapy for critical limb ischemia: Analysis of the 2013 to 2014 nationwide re-admissions databases.</a> Circulation. 136 (2), 167-176 (2017).</li><li>Rowlands, T. E., Donnelly, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+therapy+for+intermittent+claudication.">Medical therapy for intermittent claudication.</a> European Journal of Vascular and Endovascular Surgery. 34, 314-321 (2007).</li><li>Cronewett, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Acute limb ischemia and lower extremity chronic arterial disease: Rutherford's vascular surgery (8th ed.). , (2014).</li><li>Dick, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+or+endovascular+revascularization+in+patients+with+critical+limb+ischemia:+influence+of+diabetes+mellitus+on+clinical+outcome.">Surgical or endovascular revascularization in patients with critical limb ischemia: influence of diabetes mellitus on clinical outcome.</a> Journal of Vascular Surgery. 45 (4), 751-761 (2007).</li><li>Su&#225;rez-Cuenca, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+circulating+mononuclear+progenitor+cells+and+soluble+biomarkers+in+the+cardiovascular+prognosis+after+coronary+angioplasty.">Coronary circulating mononuclear progenitor cells and soluble biomarkers in the cardiovascular prognosis after coronary angioplasty.</a> Journal of Cellular and Molecular Medicine. 23 (7), 4844-4849 (2019).</li><li>Franz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+autologous+bone+marrow+mononuclear+cell+implantation+therapy+as+a+limb+salvage+procedure+in+patients+with+severe+peripheral+arterial+disease.">Use of autologous bone marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease.</a> Journal of Vascular Surgery. 50 (6), 1378-1390 (2009).</li><li>Benoit, E., O'Donnell, T. F., Patel, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+autologous+cell+therapy+in+critical+limb+ischemia:+A+systematic+review.">Safety and efficacy of autologous cell therapy in critical limb ischemia: A systematic review.</a> Cellular Transplantation. 22 (3), 545-562 (2013).</li><li>Hill, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+endothelial+progenitor+cells,+vascular+function,+and+cardiovascular+risk.">Circulating endothelial progenitor cells, vascular function, and cardiovascular risk.</a> New England Journal of Medicine. 348 (7), 593-600 (2003).</li><li>Schmidt-Lucke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+number+of+circulating+endothelial+progenitor+cells+predicts+future+cardiovascular+events:+proof+of+concept+for+the+clinical+importance+of+endogenous+vascular+repair.">Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair.</a> Circulation. 111 (22), 2981-2987 (2005).</li><li>Smadja, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+endothelial+progenitor+cells+in+bone+marrow+are+a+biomarker+of+cell+therapy+success+in+patients+with+critical+limb+ischemia.">Early endothelial progenitor cells in bone marrow are a biomarker of cell therapy success in patients with critical limb ischemia.</a> Cytotherapy. 14 (2), 232-239 (2012).</li><li>Kremastinos, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracoronary+cyclic-GMP+and+cyclic-AMP+during+percutaneous+transluminal+coronary+angioplasty.">Intracoronary cyclic-GMP and cyclic-AMP during percutaneous transluminal coronary angioplasty.</a> International Journal of Cardiology. 53 (3), 227-232 (1996).</li><li>Truong, Q. A., Januzzi, J. L., Szymonifka, J., Thai, W. E., Wai, B., Lavender, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+sinus+biomarker+sampling+compared+to+peripheral+venous+blood+for+predicting+outcomes+in+patients+with+severe+heart+failure+undergoing+cardiac+resynchronization+therapy:+the+BIOCRT+study.">Coronary sinus biomarker sampling compared to peripheral venous blood for predicting outcomes in patients with severe heart failure undergoing cardiac resynchronization therapy: the BIOCRT study.</a> Heart Rhythm. 11 (12), 2167-2175 (2014).</li><li>Ding, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis+and+inflammatory+markers+and+long-term+risk+of+peripheral+artery+disease:+The+ARIC+study.">Fibrosis and inflammatory markers and long-term risk of peripheral artery disease: The ARIC study.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 40 (9), 2322-2331 (2020).</li><li>Potier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+copeptin+and+risk+of+lower-extremity+amputation+in+Type+1+and+Type+2+diabetes.">Plasma copeptin and risk of lower-extremity amputation in Type 1 and Type 2 diabetes.</a> Diabetes Care. 40 (12), 2290-2297 (2019).</li><li>Schmidt-Lucke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+endothelial+progenitor+cells+using+the+modified+ISHAGE+protocol.">Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol.</a> PLoS One. 5 (1), 13790 (2010).</li><li>Marboeuf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+triggers+colony+forming+endothelial+cell+mobilization+after+angioplasty+in+chronic+lower+limb+ischemia.">Inflammation triggers colony forming endothelial cell mobilization after angioplasty in chronic lower limb ischemia.</a> Journal of Thrombosis and Haemostasis. 6 (1), 195-197 (2008).</li><li>Regueiro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+endothelial+progenitor+cells+in+acute+cardiovascular+events+in+the+PROCELL+study:+Time-course+after+acute+myocardial+infarction+and+stroke.">Mobilization of endothelial progenitor cells in acute cardiovascular events in the PROCELL study: Time-course after acute myocardial infarction and stroke.</a> Journal of Molecular and Cellular Cardiology. 80, 146-155 (2015).</li></ol>

Blodopsamling på det præcise sted for vaskulær blok kan vise tekniske vanskeligheder; derfor udførte vi blodopsamling i nærheden af vaskulær blok. Ligeledes mængden af MPCs tæt på vaskulære plak synes at være meget dynamisk og kan stamme variationer før og efter angioplastik. Ifølge vores observationer anbefales det at evaluere baseline- og 30min-post-angioplastik ændringer i antallet af MPC'er, da de kan afspejle flere patofysiologiske processer, der forekommer inden for vaskulær skade og reparation.
...

Perifer arteriel sygdom (PAD) er kendetegnet ved en kronisk og progressiv vaskulær obstruktion med begrænsning afblodforsyningen 1. På globalt plan påvirker PAD af underekstremiteterne omkring 10% af den ældre befolkning, mens op til 7% af sådanne tilfælde underkastes lemmer amputation2,3.
Critical Limb Ischemia (CLI) repræsenterer den mest alvorlige præsentation af PAD1. Patienter oplever normalt smerter i hvile, sår, eller koldbrand kan henføres til okkluderede arterier; mens klinisk prognose er ugunstig og præget af en 30% risiko for amputation af lemmer og dødelighed i løbet af 1 år3,4,5.
Angioplastik er en minimalt invasiv endovaskulær procedure, der kan genoprette blodgennemstrømningen til underekstremiteterne hos patienter med CLI; nogle patienter vil dog uundgåeligt kræve amputation af større lemmer, selv efter angioplastikbehandling1,5. Tidlig identifikation af ugunstige resultater efter angioplastik er ret værdifuld på grund af muligheden for behandlingshåndhævelse.
Traditionelle risikofaktorer kan give en begrænset prædiktiv evne til amputation af større lemmer hos patienter med CLI, der gennemgår angioplastik6. Patofysiologiorienterede biomarkører repræsenterer nye metoder med potentielle kliniske anvendelser, hvilket kan resultere specifikt nyttigt i sygdomme relateret til vaskulær skade7. I dag er deltagelsen af cellulære befolkninger, der ejer endotelreparationsegenskaber, på stedet for den aterosklerotiske plak, i stigende grad blevet anerkendt8,9.
Mononukleare stamceller (MPC' er) er afledt af knoglemarven og egne strukturelle og funktionelle egenskaber ved stamceller med vaskulære regenerative evner. På grund af MPC's evne til at formere sig, migrere og vise vaskulær tilslutning; disse celler er blevet gode kandidater til at afspejle endotel reparation som reaktion på iskæmi10,11,12. Derudover har kontinuerlig interesse for mekanismer, der ligger til grund for vaskulær skade, motiveret udforskning af den prognostiske rolle hos lokale forekommende biomarkører, da de anses for at afspejle vaskulær skade og reparation7,13,14.
Formålet med denne undersøgelse er at beskrive, hvordan man bestemmer mængden af MPC'er, der cirkulerer tæt på den vaskulære obstruktion hos patienter med CLI, der gennemgår angioplastik; og hvordan man vurderer forholdet mellem MPCs med indikatorer for endotel dysfunktion og lemmer amputation.
Sammenlignet med prognosen baseret på comorbiditeter og iboende vaskulære træk viser mængden af lokale MPCs specifik evne til at forudsige klinisk resultat med hensyn til endotel dysfunktion og lemmer amputation. Konsekvent, nogle undersøgelser har beskrevet den prognostiske rolle lignende biomarkører under evalueringen af patienter med PAD15,16.
Baseret på tidligere resultater7, den metode, der er beskrevet her, kan være nyttig til en tidlig identifikation af befolkning med risiko for negative vaskulære resultater i flere kliniske indstillinger, såsom underekstremitet og koronarisk iskæmi, slagtilfælde, vaskulitis, venøs trombose og andre, der involverer vaskulær skade og reparation.

<table>
	<tbody>
		<tr>
			<td>BSA</td>
			<td>Roche</td>
			<td>10735086001</td>
			<td>Bovine Serum Albumin (BSA) as a buffering agent, stabilizer, standard and for blending.</td>
		</tr>
		<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>CD184 (CXCR4)-PE-VIO770</td>
			<td>Miltenyi Biotec / MACS</td>
			<td>#130-103-798</td>
			<td>Monoclonal, Isotype recombinant human IgG1, conjugated</td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Cytometry Tubes</td>
			<td>FALCON Corning Brand</td>
			<td>#352052</td>
			<td>5 mL Polystyrene Round-Bottom Tube. 12x75 style. Sterile.</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>#SZBF0920V</td>
			<td>Fixation of biological samples, (powder, 95%)</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Test Tubes</td>
			<td>KIMBLE CHASE</td>
			<td>45060 13100</td>
			<td>Heat-resistant test tubes. SIZE/CAP 13 x 100 mm</td>
		</tr>
	</tbody>
</table>

predicting amputation using local circulating mononuclear progenitor cells in angioplasty-treated patients with critical limb ischemia

Kritisk limbiskæmi (CLI) repræsenterer et fremskredent stadium af den perifere arteriel sygdom. Angioplastik forbedrer blodgennemstrømningen til underekstremiteterne; nogle patienter udvikler sig dog uigenkaldeligt til amputation af lemmer. Omfanget af vaskulære skader og mekanismerne for vaskulær reparation er faktorer, der påvirker resultatet efter angioplastik. Mononukleare stamceller (MPCs) er reaktive over for vaskulære skader og reparationer, med evnen til at afspejle vaskulære sygdomme. Denne protokol beskriver kvantificering af MPC'er opnået fra blodcirkulationen fra kar tæt på angioplastikstedet samt dets forhold til endotel dysfunktion og dets prædiktive evne til lem amputation i de næste 30 dage efter angioplastik hos patienter med CLI.

Blodprøver fra blokerede arterier, på stedet adresseret til angioplastik, blev indsamlet fra 20 diabetikere, i alderen 68 år og 10 ud af 20 var mænd. Halvdelen af stikprøvepopulationen var rygere. Vaskulære læsioner blev hovedsageligt scoret som Rutherford klasse VI; der henviser til, at patienterne viste en højere prævalens af type 2-diabetes Mellitus (100 %), forhøjet blodtryk (70 %) og dyslipidemi (55%).
En 30 dages klinisk opfølgning efter underekstremitet angioplastik blev udf?...

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

Amputation af underekstremiteterne kan forekomme selv efter angioplastik af blokerede beholdere i Critical Limb Ischemia (CLI). Mononukleare stamceller afspejler vaskulær reparation. Denne protokol beskriver kvantificeringen af MPC'er fra cirkulation tæt på angioplastik, og dens forhold til endotel dysfunktion og forudsigelse af underekstremitet amputation.

Forudsigelse af amputation ved hjælp af lokale cirkulerende mononukleare stamceller i angioplastikbehandlede patienter med kritisk limbiskæmi

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

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