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Use of a Recombinant Fluorescent MMP‐2 as a Reporter for Tracking the Involvement of This Protease in Target Organ Damage Found in Cardiovascular Diseases
Author(s) -
Prado Alejandro Ferraz,
Azevedo Aline,
Castro Myrella Lessio,
Gerlach Raquel Fernanda
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.lb607
Subject(s) - green fluorescent protein , zymography , western blot , matrix metalloproteinase , recombinant dna , blot , microbiology and biotechnology , in vivo , biology , pathology , chemistry , biochemistry , medicine , gene
Matrix Metalloproteinase 2 (MMP‐2) is elevated in hypertensive heart and vessel remodeling, being also highly expressed in organs that show function change and structural damage due to hypertension and atherosclerosis. Expression of MMP‐2 alone caused structural and functional changes in heart and kidney. It is not known yet which are the consequences of increased circulating MMP‐2 in vivo . To investigate this, we constructed a clone containing the MMP‐2 cDNA encoding MMP‐2's catalytic domain fused to the green fluorescent protein (GFP). Recombinant protein was expressed in bacteria, purified and checked for integrity and activity. Zymography, SDS‐PAGE and western blotting (WB) analyses were used for characterization. The purified protein was injected into ApoE Knockout mice (ApoE−/−) and controls (C57BL6) for four weeks. Whole organs (arteries, brain, heart, and kidneys) were excised to check for the presence of MMP‐2 according to the level of fluorescence using the IVIS spectrum imaging system. Homogenized organs levels of MMP‐2 were checked in a fluorimeter, being those further confirmed by Western Blot against the GFP protein. Results suggest spread of MMP‐2 via circulation to organs that display funtional and structural damage in cardiovascular diseases. This study was supported by The State of Sao Paulo Research Foundation (FAPESP)