Novel Role for Matrix Metalloproteinase 9 in Modulation of Cholesterol Metabolism

Abstract

Background: The development of atherosclerosis is strongly linked to disorders of cholesterol metabolism. Matrix metalloproteinases (MMPs) are dysregulated in patients and animal models with atherosclerosis. Whether systemic MMP activity influences cholesterol metabolism is unknown.

Methods and results: We examined MMP-9-deficient (Mmp9^-/-) mice and found them to have abnormal lipid gene transcriptional responses to dietary cholesterol supplementation. As opposed to Mmp9^+/+ (wild-type) mice, Mmp9^-/- mice failed to decrease the hepatic expression of sterol regulatory element binding protein 2 pathway genes, which control hepatic cholesterol biosynthesis and uptake. Furthermore, Mmp9^-/- mice failed to increase the expression of genes encoding the rate-limiting enzymes in biliary cholesterol excretion (eg, Cyp7a and Cyp27a). In contrast, MMP-9 deficiency did not impair intestinal cholesterol absorption, as shown by the ¹⁴C-cholesterol and ³H-sitostanol absorption assay. Similar to our earlier study on Mmp2^-/- mice, we observed that Mmp9^-/- mice had elevated plasma secreted phospholipase A₂ activity. Pharmacological inhibition of systemic circulating secreted phospholipase A₂ activity (with varespladib) partially normalized the hepatic transcriptional responses to dietary cholesterol in Mmp9^-/- mice. Functional studies with mice deficient in other MMPs suggested an important role for the MMP system, as a whole, in modulation of cholesterol metabolism.

Conclusions: Our results show that MMP-9 modulates cholesterol metabolism, at least in part, through a novel MMP-9-plasma secreted phospholipase A₂ axis that affects the hepatic transcriptional responses to dietary cholesterol. Furthermore, the data suggest that dysregulation of the MMP system can result in metabolic disorder, which could lead to atherosclerosis and coronary heart disease.

Keywords: atherosclerosis; cholesterol; lipid metabolism; liver; matrix metalloproteinase; plasma phospholipase A2.

Figure 1

MMP‐9 deficiency is associated with abnormalities in lipid distribution and excretion. A, Triglyceride and cholesterol levels in lipoprotein fractions of plasma separated by FPLC. Traces correspond to pools of plasma from WT and Mmp9 ^−/− mice (n=4 per genotype). Semiquantitative assessment based on peak heights indicates that Mmp9 ^−/− mice have 2.7‐, 4.3‐, 1.5‐, and 1.2‐fold increases in VLDL triglycerides, VLDL cholesterol, LDL cholesterol, and HDL cholesterol, respectively (compared with WT mice). The same volume was injected onto the FPLC. B, Cholesterol absorption measured by radioactive ¹⁴C‐cholesterol and ³H‐sitostanol absorption assay (n=4 per genotype). C, Bile acid content in mouse stool in response to cholesterol (n=5 for WT with normal chow, n=5 for Mmp9 ^−/− with normal chow, n=4 for WT with cholesterol, n=5 for Mmp9 ^−/− with cholesterol). *P<0.05 vs WT. ^† P<0.05 vs normal chow, t test. FPLC indicates fast‐performance liquid chromatography; HDL, high‐density lipoprotein; LDL, low‐density lipoprotein; MMP, matrix metalloproteinase; VLDL, very low‐density lipoprotein; WT, wild type.

Figure 2

Hepatic transcriptional responses to dietary cholesterol supplementation. Mice were fed regular chow or chow supplemented with cholesterol (0.15%) for up to 2.5 days. Gene expression analysis was conducted at days 0 and 2.5 (n=4 to 5 mice per time point). *P<0.05 vs WT. ^† P<0.05 vs 0 days on cholesterol, 1‐way repeated‐measures ANOVA. Abca1 indicates ATP‐binding cassette sub‐family A member 1; Abcg5/Abcg8, ATP‐binding cassette sub‐family G member 5/8; Cyp27a1, sterol 27 hydroxylase; Cyp7a1, cholesterol 7 alpha hydroxylase; Fasn, fatty acid synthase; Hmgcr, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase; Ldlr, low density lipoprotein receptor; Mmp, matrix metalloproteinase gene; Nr1h3/Nr1h2, liver X receptor α/β; Pcsk9, proprotein convertase subtilisin/kexin type 9; Srebf1, sterol regulatory element binding protein 1; Srebf2, gene for sterol regulatory element binding protein 2; SREBP, sterol regulatory element binding protein; WT, wild type.

Figure 3

Impact of dietary cholesterol on hepatic transcriptional responses. Mice were fed either regular chow or chow supplemented with cholesterol (0.15% or 1.5%) for 2.5 days. Gene expression analysis was conducted at 0 and 2.5 days (n=4 to 5 mice per group [or treatment]). *P<0.05 vs normal chow for each genotype, all pairwise multiple comparisons vs control group (Holm–Sidak method), ANOVA. Cyp27a1 indicates sterol 27 hydroxylase; Cyp7a1, cholesterol 7 alpha hydroxylase; Fasn, fatty acid synthase; Hmgcr, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase; Ldlr, low density lipoprotein receptor; LXR, liver X receptor; Mmp, matrix metalloproteinase gene; Nr1h3/LXR‐α, liver X receptor α; Pcsk9, proprotein convertase subtilisin/kexin type 9; Srebf1, sterol regulatory element binding protein 1; Srebf2, gene for sterol regulatory element binding protein 2; SREBP, sterol regulatory element binding protein; WT, wild type.

Figure 4

Plasma sPLA ₂ activity is elevated by MMP‐9 deficiency. A, sPLA ₂ activity in the plasma, liver, and heart of Mmp9 ^−/− mice (n=4 mice per genotype). *P<0.05 vs WT, t test. B, The elevated plasma PLA ₂ activity in Mmp9 ^−/− mice (compared to WT) was confirmed using 2 unrelated assays: the Cayman sPLA ₂ assay kit (substrate: di‐heptanoyl‐thio‐PC; n=4 per genotype; *P<0.05 vs WT, t test) and the ³H‐oleate E coli membrane assay (data are representative of technical duplicates for a pool of 5 mice per genotype). C, EGTA and varespladib inhibition profiles for the sPLA ₂ from Mmp9 ^−/− plasma vs heart and plasma from Mmp2 ^−/− mice. The analysis was performed in duplicate using samples from pools of 4 mice per genotype using the ³H‐oleate E coli membrane assay. Similar results were obtained using the Cayman assay kit (data not shown). D, Profiling of PLA ₂ activity inhibition demonstrates that the plasma sPLA ₂ that is present in Mmp2 ^−/− and Mmp9 ^−/− mice is the same enzyme or very similar enzymes (pools of plasma: n=5 for Mmp2 ^−/− and n=5 for Mmp9 ^−/−). Data are representative of technical duplicates. For comparison, the activity of cardiac sPLA ₂ from an Mmp2 ^−/− mouse (mouse “E” in figure S3 of Hernandez‐Anzaldo et al2) is presented. *P<0.05 vs Mmp2 ^−/− plasma, t test. The x‐axis values indicate (0) no inhibitor; (1) EDTA, inhibits Ca²⁺‐dependent PLA ₂s; (2) dithiothreitol, sulfhydryl redox agent; (3) MJ33, active site‐directed PLA ₂ inhibitor; (4) KH064, sPLA2 inhibitor; (5) YM 26734, sPLA ₂ (PLAG2A, PLA2G5) inhibitor; (6) arachidonyl trifluoromethyl ketone, cytosolic PLA₂ and iPLA ₂ inhibitor; (7) N‐(p‐amylcinnamoyl) anthranilic acid, PLA ₂ inhibitor; (8) bromoenol lactone, iPLA2 inhibitor; and (9) heparin, inhibits some sPLA ₂s. AACOCF3 indicates arachidonyl trifluoromethyl ketone; ACA, N‐(p‐Amylcinnamoyl) anthranilic acid; BEL, bromoenol lactone; cPLA₂, cytosolic phospholipase A₂; DTT, dithiothreitol; E. coli, Escherichia coli; EDTA, ethylenediaminetetraacetic acid (divalent metal ion chelator); EGTA, ethylene glycol‐bis(?‐aminoethyl ether)‐tetraacetic acid (Ca²⁺ chelator); iPLA₂, calcium‐independent phospholipase A₂; MJ33, 1‐hexadecyl‐3‐trifluoroethylglycero‐sn‐2‐phosphomethanol; Mmp, matrix metalloproteinase gene; MMP, matrix metalloproteinase; MMP‐2, matrix metalloproteinase‐2; MMP‐9, matrix metalloproteinase‐9; PC, phosphatidylcholine; SH, sulfhydryl; sPLA₂, secreted phospholipase A₂; WT, wild type; YM 26734, 1,1?‐[5‐[3,4‐dihydro‐7‐hydroxy‐2‐(4‐hydroxyphenyl)‐2H‐1‐benzopyran‐4‐yl]‐2,4,6‐trihydroxy‐1,3‐phenylene]bis‐1‐dodecanone.

Figure 5

Circulating systemic sPLA ₂ modulates hepatic transcriptional responses to dietary cholesterol supplementation. A, Plasma sPLA ₂ activity ofMmp9 ^−/− mice administered the pan‐sPLA ₂ inhibitor varespladib (10 mg/kg per day) or vehicle for 5 days (n=4 mice per group). *P<0.05 vs WT, t test. ^† P<0.05 vs untreated, t test. B, Study protocol for varespladib treatment prior to cholesterol supplementation. Mice were fed either regular chow or chow supplemented with 0.15% cholesterol for 2.5 days. Varespladib treatment (10 mg/kg per day for 5 days) started 2.5 days prior to commencement of cholesterol supplementation of the diet. C, Hepatic expression of lipid metabolic genes in WT mice administered varespladib (10 mg/kg per day; n=8 WT without varespladib and n=8 WT with varespladib mice, n=4 per time point). *P≤0.05 vs WT without varespladib at day 2.5. ^† P<0.05 vs day 0. All pairwise multiple comparisons vs control group (Holm–Sidak method), ANOVA. D, Hepatic expression of lipid‐metabolic genes in Mmp9 ^−/− mice administered varespladib (10 mg/kg per day; n=8 Mmp9 ^−/− without varespladib and n=8 Mmp9 ^−/− with varespladib, n=4 per time point). *P<0.05 vs Mmp9 ^−/− without varespladib at day 2.5. ^† P<0.05 vs day 0. All pairwise multiple comparisons vs control group (Holm–Sidak method), ANOVA. Abca1 indicates ATP‐binding cassette sub‐family A member 1; Abcg5/Abcg8, ATP‐binding cassette sub‐family G member 5/8; Cyp27a1, sterol 27 hydroxylase; Cyp7a1, cholesterol 7 alpha hydroxylase; Fasn, fatty acid synthase; Hmgcr, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase; Ldlr, low density lipoprotein receptor; LXR, liver X receptor; Mmp, matrix metalloproteinase gene; MMP, matrix metalloproteinase; Nr1h3/Nr1h2, liver X receptor α/β; Pcsk9, proprotein convertase subtilisin/kexin type 9; sPLA₂, secreted phospholipase A₂; Srebf1, sterol regulatory element binding protein 1; Srebf2, gene for sterol regulatory element binding protein 2; SREBP, sterol regulatory element binding protein; WT, wild type.

Figure 6

The MMP system modulates hepatic transcriptional responses to dietary cholesterol. A, Deficiency of MMP‐2, MMP‐7, or MMP‐9 is associated with abnormalities in the hepatic transcriptional responses to dietary cholesterol. Mice were fed either regular chow or chow supplemented with 0.15% cholesterol for 6.5 days. Gene expression analysis was conducted at 0, 2.5, and 6.5 days. Analysis involved 6 WT mice, 8Mmp2 ^−/− mice, 5 Mmp7 ^−/− mice, and 5 Mmp9 ^−/− mice. For clarity, the symbols indicating statistically significant differences were excluded. An expanded version of these analyses is presented in Figure S6. B, Proposed model for regulation of cholesterol homeostasis by systemic MMP activity. MMP deficiencies (due to genetic deletion or functional blockade) can alter the hepatic cholesterol metabolism. The mechanism by which MMPs act may or may not require the release of sPLA ₂ activity from peripheral organs and is governed by tissue inhibitors of metalloproteinase. Cyp27a1 indicates sterol 27 hydroxylase; Cyp7a1, cholesterol 7 alpha hydroxylase; Fasn, fatty acid synthase; Hmgcr, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase; Ldlr, low density lipoprotein receptor; Mmp, matrix metalloproteinase gene; MMP, matrix metalloproteinase; Nr1h3, liver X receptor α; Pcsk9, proprotein convertase subtilisin/kexin type 9; sPLA₂, secreted phospholipase A₂; Srebf1, sterol regulatory element binding protein 1; Srebf2, gene for sterol regulatory element binding protein 2; TIMP, tissue inhibitor of metalloproteinase; WT, wild type.