TIMP3 is reduced in atherosclerotic plaques from subjects with type 2 diabetes and increased by SirT1

Marina Cardellini, Rossella Menghini, Eugenio Martelli, Viviana Casagrande, Arianna Marino, Stefano Rizza, Ottavia Porzio, Alessandro Mauriello, Anna Solini, Arnaldo Ippoliti, Renato Lauro, Franco Folli, Massimo Federici, Marina Cardellini, Rossella Menghini, Eugenio Martelli, Viviana Casagrande, Arianna Marino, Stefano Rizza, Ottavia Porzio, Alessandro Mauriello, Anna Solini, Arnaldo Ippoliti, Renato Lauro, Franco Folli, Massimo Federici

Abstract

Objective: Atherosclerosis is accelerated in subjects with type 2 diabetes by unknown mechanisms. We identified tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of A disintegrin and metalloprotease domain 17 (ADAM17) and other matrix metalloproteinases (MMPs), as a gene modifier for insulin resistance and vascular inflammation in mice. We tested its association with atherosclerosis in subjects with type 2 diabetes and identified Sirtuin 1 (SirT1) as a major regulator of TIMP3 expression.

Research design and methods: We investigated ADAM10, ADAM17, MMP9, TIMP1, TIMP2, TIMP3, and TIMP4 expression levels in human carotid atherosclerotic plaques (n = 60) from subjects with and without diabetes. Human vascular smooth muscle cells exposed to several metabolic stimuli were used to identify regulators of TIMP3 expression. SirT1 small interference RNA, cDNA, and TIMP3 promoter gene reporter were used to study SirT1-dependent regulation of TIMP3.

Results: Here, we show that in human carotid atherosclerotic plaques, TIMP3 was significantly reduced in subjects with type 2 diabetes, leading to ADAM17 and MMP9 overactivity. Reduced expression of TIMP3 was associated in vivo with SirT1 levels. In smooth muscle cells, inhibition of SirT1 activity and levels reduced TIMP3 expression, whereas SirT1 overexpression increased TIMP3 promoter activity.

Conclusions: In atherosclerotic plaques from subjects with type 2 diabetes, the deregulation of ADAM17 and MMP9 activities is related to inadequate expression of TIMP3 via SirT1. Studies in vascular cells confirmed the role of SirT1 in tuning TIMP3 expression.

Figures

FIG. 1.
FIG. 1.
TIMP3 is reduced in atherosclerotic plaques of subjects with type 2 diabetes (DM2). ADAM10, ADAM17, and MMP9 (A) as well as TIMPs (B) expression in NGT (n = 37) and type 2 diabetes (n = 23) subjects; ***P < 0.001 by one-way ANOVA. C: Western blot using extracellular matrix extracts from representative NGT (n = 2) and type 2 diabetic (n = 4) subjects. *P < 0.05 NGT vs. type 2 diabetes by Student's t test. D–J: Immunohistochemistry confirmed that TIMP3 is reduced in type 2 diabetic (n = 8) versus NGT (n = 8) subjects; one representative image is shown for TIMP3, anti–α smooth muscle actin, and CD68 for NGT (D–F; 4× magnification) and type 2 diabetic (G–J; 4× magnification) subjects. K and I: ADAM17 activity measured by a fluorimetric assay (K) and MMP9 activity measured by a fluorimetric assay (I) are increased in type 2 diabetic (n = 23) compared with NGT (n = 37) subjects; ***P < 0.001 by Student's t test for both. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Effects of diabetes on TIMP3 expression in vascular cells. A: TIMP3 expression in CASMC treated with various metabolic stimuli: high glucose (HG) 20 mmol/l; mannitol (Man) 20 mmol/l; insulin (Ins) 10−7 M; LDL 100 μg/ml; oxidized LDL (oxLDL) 100 μg/ml; glycated LDL (glyLDL) 100 μg/ml; LXR agonists (T0901317 [T090] 5 μmol/l; GW3965 [GW] 3 μmol/l; R-hydroxycholesterol [RH] 10 μmol/l; 22-S-hydroxycholesterol [SH] 10 μmol/l); SirT1 inhibitor (Sirtinol) 50 μmol/l. n = 4 for all experiments; *P < 0.05 by Student's t test versus control (CT). B: Sirtinol increased ADAM17 activity in CASMC. n = 4 for all experiments; ***P < 0.001 by Student's t test. C: TIMP3 expression in HUVEC and THP1 treated with Sirtinol and high glucose (20 mmol/l). n = 4 for all experiments; *P < 0.05 by Student's t test versus control. D: SirT1 expression is reduced in CASMC, HUVEC, and THP1 treated with high glucose (20 mmol/l) compared with control. n = 4 for all experiments; *P < 0.05 by Student's t test. E: SirT1 levels are decreased in type 2 diabetic compared with NGT subjects. *P < 0.05 by Student's t test. F: SirT1 correlates with TIMP3 in atherosclerotic plaques from NGT (n = 37) and type 2 diabetic (DM2) (n = 23) subjects.
FIG. 3.
FIG. 3.
Regulation of TIMP3 expression in CASMC. A: SirT1 knockdown decreased TIMP3 expression but not TIMP1, TIMP2, TIMP4, ADAM10, ADAM17, or MMP9 in CASMC. n = 4 for all experiments; ***P < 0.001 by Student's t test versus control. B: SirT1 knockdown decreased TIMP3 expression in HUVEC and THP1. n = 4 for all experiments; ***P < 0.001 by Student's t test. C: SirT1 cDNA overexpression increased Timp3 promoter activity. n = 4 for all experiments; **P < 0.01 by one-way ANOVA. D: SirT1 overexpression increased and prevented loss of TIMP3 expression caused by high glucose (HG; 20 mmol/l) in CASMC, HUVEC, and THP1. n = 4 for all experiments; *P < 0.05 by Student's t test.

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