Mechanism of inhibition of matrix metalloproteinase-2 expression by doxycycline in human aortic smooth muscle cells Jian Liu, MD, Wanfen Xiong, PhD,

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Mechanism of inhibition of matrix metalloproteinase-2 expression by doxycycline in human aortic smooth muscle cells Jian Liu, MD, Wanfen Xiong, PhD, Lisa Baca-Regen, PharmD, Hideaki Nagase, MD, B.Timothy Baxter, MD Journal of Vascular Surgery Volume 38, Issue 6, Pages 1376-1383 (December 2003) DOI: 10.1016/S0741-5214(03)01022-X

Fig 1 Effect of doxycycline on matrix metalloproteinase-2 (MMP-2) production from cultured aortic smooth muscle cells (SMCs). Aortic SMCs cultured in either serum-free M-199 (Control) or macrophage or lymphocyte co-culture medium (Stimulated) were treated with doxycycline at indicated concentrations for 24 hours. MMP-2 concentration in cell culture medium at indicated doses of doxycycline were determined with gelatin zymography (A) and Western blot analysis (B). C, MMP-2 band intensity was determined with quantitative densitometry. Quantitative values in arbitrary integrator units represent mean obtained from three independent experiments. D, Inhibition of MMP-2 production from SMCs treated without (Control) or with (Stimulated) co-cultured macrophage or lymphocyte media in response to 5 μg/mL of doxycycline. Gelatin zymography (lower panel) represents SMCs from three separate plates. *P < .05 compared with control SMCs; # P < .05 compared with medium-treated SMCs without doxycycline. Journal of Vascular Surgery 2003 38, 1376-1383DOI: (10.1016/S0741-5214(03)01022-X)

Fig 2 Effect of doxycycline on proliferation of cultured aortic smooth muscles (SMCs). SMCs were treated with indicated dose of doxycycline in serum-free M-199 medium for 24 hours. Cell numbers were determined by measuring conversion of tetrazolium salt into blur formazan in viable cells. Values are mean ± SE of triplicate measurements. OD, Optic density. Journal of Vascular Surgery 2003 38, 1376-1383DOI: (10.1016/S0741-5214(03)01022-X)

Fig 3 Effect of doxycycline on steady-state level and stability of matrix metalloproteinase-2 (MMP-2) mRNA from smooth muscle cells. A, Aortic SMCs were treated with doxycycline at concentrations of 5 and 10 μg/mL for 24 hours. Total RNA was extracted from the cells, and MMP-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined with Northern blot analysis. B, Confluent cultures of human SMCs were incubated in presence or absence of 20 μg/mL of doxycycline. After 24 hours, actinomycin D (5 μg/mL) was added (0 h), and cultures were further incubated for indicated times. Each value represents results from three separate plates. Total RNA was extracted, dot blotted onto Nytran membrane, and probed with [32P]deoxycitidine triphosphate–labeled MMP-2 complementary DNA fragment. Dot blots were quantified with densitometry, and data were semilogarithmic plotted as mean ± SE (n = 5). Best-fit curves were generated and used to estimated messenger RNA half-life. Journal of Vascular Surgery 2003 38, 1376-1383DOI: (10.1016/S0741-5214(03)01022-X)

Fig 4 Doxycycline-inhibited matrix metalloproteinase (MMP)–9 and MMP-2 expression from cultured human abdominal aortic aneurysm (AAA) tissue. Human AAA tissue was cultured in serum-free M-199 medium and treated without or with doxycycline at 2.5 to 40 μg/mL for 10 days. Media were collected, and MMP-2 and MMP-9 concentration was examined with gelatin zymography and Western blot analysis. A, Representative gelatin zymography shows both MMP-2 and MMP-9 concentrations in media of AAA tissue culture treated at indicated dose of doxycycline. MMP-9 (B) and MMP-2 (C) activity was quantified with densitometry. Values in arbitrary integrator units were reported. D, Western blot analysis of MMP-2 concentration in culture media of AAA tissue treated with indicated concentration of doxycycline. Journal of Vascular Surgery 2003 38, 1376-1383DOI: (10.1016/S0741-5214(03)01022-X)

Fig 5 Effect of therapeutic dose of doxycycline on matrix metalloproteinase-2 (MMP-2) expression from cultured AAA tissue. Explanted AAA tissue was treated with 5 μg/mL of doxycycline for 10 days. MMP-2 concentration in tissue culture medium was determined with gelatin zymography, and quantified with densitometry. Values represent mean ± SE of three different patient samples run in triplicate. *P < .05; **P < .01. Gelatin zymography (lower panel) represents a patient sample run in triplicate. Journal of Vascular Surgery 2003 38, 1376-1383DOI: (10.1016/S0741-5214(03)01022-X)