Jornal Vascular Brasileiro
https://www.jvascbras.org/article/doi/10.1590/jvb.2014.038
Jornal Vascular Brasileiro
Brief Communication

Enzymatic activity analysis of MMP-2 and 9 collected by swab from lower limb venous ulcers

Análise da atividade enzimática de MMP-2 e 9 coletadas por swab em úlcera venosa de membro inferior

Flávio Santos da Silva; Diego Neves Araujo; João Paulo Matos Santos Lima; Adriana Augusto de Rezende; Bento João da Graça Azevedo Abreu; Fernando Augusto Lavezzo Dias

Downloads: 0
Views: 637

Abstract

Metalloproteinases play a role in repair of venous ulcers of the lower limbs. The great majority of studies of metalloproteinase enzyme activity conducted to date have employed material from biopsies of ulcers. We evaluated the viability of using zymography to measure the enzyme activity of metalloproteinases 2 and 9 in samples of venous ulcer exudate collected on swabs. The method chosen for processing the samples proved viable in terms of its ability to provide adequate protein concentrations for analysis. Using zymography, we observed that the parameters that provided the best results for analysis of gelatinolytic activity were 0.125 to 0.5 μg of total protein content in the gels and enzymatic activation time of 19 hours (at 37 °C). Collection of venous ulcer fluid using swabs proved to be a simple, rapid and effective method for obtaining samples for measurement of gelatinolytic activity with a minimum degree of invasivity.

Keywords

venous ulcer, metalloproteinases, gelatinases

Resumo

As metaloproteinases participam do reparo das úlceras venosas de membros inferiores. Até o momento, estudos de atividade enzimática utilizaram, em sua maior parte, biópsia das úlceras. Objetivamos avaliar a viabilidade da mensuração da atividade enzimática de metaloproteinases 2 e 9, extraídas por swab, em amostras de exsudato de úlcera venosa, através de zimografia. O método de processamento da amostra coletada mostrou-se viável, visto que foi possível obter concentração proteica adequada para análise. Através de zimografia, observamos que as quantidades de proteína total das amostras carregadas nos géis entre 0,125 e 0,5 μg, além do tempo de ativação enzimática de 19 horas (a 37 °C), foram parâmetros adequados e de melhor resultado para a análise da atividade gelatinolítica. A coleta através de swab mostrou-se um método simples, rápido e eficaz para a coleta de fluido de úlcera venosa com o objetivo de mensuração da atividade gelatinolítica com grau mínimo de invasividade.

Palavras-chave

úlcera venosa, metaloproteinases, gelatinases

References

de Aguiar ET, Pinto LJ, Figueiredo MA, Savino Neto S. Úlcera de Insuficiência Venosa Crônica: Diretrizes sobre Diagnóstico, Prevenção e Tratamento da Sociedade Brasileira de Angiologia e Cirurgia Vascular (SBACV). J Vasc Bras. 2005;4(^sSupl.2):S195-200.

Lopes CR, Figueiredo M, Ávila AM, Soares LMBM, Dionisio VC. Avaliação das limitações de úlcera venosa em membros inferiores. J Vasc Bras. 2013;12(1):5-9.

de Araujo T, Valencia I, Federman DG, Kirsner RS. Managing the patient with venous ulcers. Ann Intern Med. 2003;138(4):326-34.

Wipke-Tevis DD, Rantz MJ, Mehr DR. Prevalence, incidence, management, and predictors of venous ulcers in the long-term-care population using the MDS. Adv Skin Wound Care. 2000;13(5):218-24.

Benjamin MM, Khalil RA. Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease. EXS. 2012;103:209-79.

Dalton SJ, Mitchell DC, Whiting CV, Tarlton JF. Abnormal extracellular matrix metabolism in chronically ischemic skin: a mechanism for dermal failure in leg ulcers. J Invest Dermatol. 2005;125(2):373-9.

Meyer FJ, Burnand KG, Abisi S, Tekoppele JM, van Els B, Smith A. Effect of collagen turnover and matrix metalloproteinase activity on healing of venous leg ulcers. Br J Surg. 2008;95(3):319-25.

Murphy G, Nagase H. Progress in matrix metalloproteinase research. Mol Aspects Med. 2008;29(5):290-308.

Wysocki AB, Staiano-Coico L, Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol. 1993;101(1):64-8.

Rayment EA, Upton Z, Shooter GK. Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer. Br J Dermatol. 2008;158(5):951-61.

Trengove NJ, Stacey MC, MacAuley S. Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen. 1999;7(6):442-52.

Snoek-van Beurden PA, Von den Hoff JW. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques. 2005;38(1):73-83.

Hu X, Beeton C. Detection of functional matrix metalloproteinases by zymography. J Vis Exp. 2010:e2445.

Beidler SK, Douillet CD, Berndt DF, Keagy BA, Rich PB, Marston WA. Multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy. Wound Repair Regen. 2008;16(5):642-8.

La Rocca G, Pucci-Minafra I, Marrazzo A, Taormina P, Minafra S. Zymographic detection and clinical correlations of MMP-2 and MMP-9 in breast cancer sera. Br J Cancer. 2004;90(7):1414-21.

Schmohl M, Beckert S, Joos TO, Königsrainer A, Schneiderhan-Marra N, Löffler MW. Superficial wound swabbing: a novel method of sampling and processing wound fluid for subsequent immunoassay analysis in diabetic foot ulcerations. Diabetes Care. 2012;35(11):2113-20.

Sociedade Brasileira de Angiologia e Cirurgia Vascular (SBACV)"> Sociedade Brasileira de Angiologia e Cirurgia Vascular (SBACV)">
5ddd81660e882558391da3e9 jvb Articles

J Vasc Bras

Share this page
Page Sections