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. 2009 Jan;83(2):830-5.
doi: 10.1128/JVI.01363-08. Epub 2008 Oct 22.

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Klas Strååt  1 , Rainier de Klark, Sara Gredmark-Russ, Per Eriksson, Cecilia Söderberg-Nauclér
Affiliations

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Klas Strååt et al. J Virol. 2009 Jan.

Abstract

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.

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Figures

FIG. 1.
FIG. 1.
Quantitative real-time PCR analysis of MMP-2, MMP-7, MMP-9, MMP-12, and MMP-14 (A) and TIMP-1, -2, -3, and -4 (B) mRNA levels in uninfected versus HCMV-infected macrophages (n = 8). The mRNA levels were normalized to β2-microglobulin (β2M) and are presented as percentages of control values (MOI = 1). *, P < 0.05. Experiments were performed 4 days after infection. Error bars indicate standard errors of the means.
FIG. 2.
FIG. 2.
Quantitative real-time PCR analysis of MMP-9 (A) and TIMP-1 (B) mRNA levels in macrophages (n = 3). Macrophages were mock infected or challenged with HCMV, UV-irradiated HCMV inocula, filtered viral inocula from HCMV-infected cultures, or HCMV-infected macrophages treated with Foscavir 2 h after infection. The mRNA levels were normalized to β2-microglobulin (β2M) (MOI = 1). *, P < 0.05. Experiments were performed 4 days after infection. Error bars indicate standard errors of the means.
FIG. 3.
FIG. 3.
(A to C) Quantitative real-time PCR analysis of HCMV pp150 mRNA levels in macrophages from three donors. The macrophages were mock infected or challenged with HCMV, UV-irradiated HCMV inocula, filtered viral inocula from HCMV-infected cultures, or HCMV-infected macrophages treated with Foscavir 2 h after infection. The mRNA levels were normalized to β2-microglobulin (β2M). (D) Nested PCR for detection of the major IE (MIE) gene in the Foscavir-treated samples from three donors; a triplicate for each donor is displayed (MOI = 1). Experiments were performed 4 days after infection. G6PD, glucose-6-phosphate dehydrogenase.
FIG. 4.
FIG. 4.
(A and B) Quantitative real-time PCR analysis of the viral kinetic effect on the expression of MMP-9 (A) and TIMP-1 (B) mRNA levels in HCMV-infected macrophages (MOI = 1). (C and D) Quantitative real-time PCR analysis of MMP-9 (C) and TIMP-1 (D) mRNA levels in macrophages that were uninfected or infected with HCMV at different MOIs. Dose-dependent effects on the expression of MMP-9 and TIMP-1 mRNA levels were observed 4 days after infection. The mRNA levels were normalized to β2-microglobulin (β2M) and presented as percentages of control values. *, P < 0.05. Error bars indicate standard errors of the means.
FIG. 5.
FIG. 5.
(A) Representative Western blot analysis for pro-MMP-9 (black arrow) and active-MMP-9 (gray arrow) protein levels in mock-infected macrophages and macrophages infected with HCMV; β-actin was used as a loading control. (B) Representative Western blot analysis for TIMP-1 protein levels in mock-infected macrophages and macrophages infected with HCMV; β-actin was used as a loading control. (C) A representative zymograph image of mock-infected and HCMV-infected macrophages assayed by gelatin zymography. The black arrow indicates pro-MMP-9, and the gray arrow indicates active-MMP-9. Experiments were performed 4 days after infection at an MOI of 1.
FIG. 6.
FIG. 6.
ELISA measurements of secreted MMP-9 and TIMP-1 in supernatants from noninfected or HCMV-infected macrophages at 4 and 7 days after infection. (A) MMP-9 levels in supernatants 4 days after infection. (B) MMP-9 levels in supernatants 7 days after infection. (C) TIMP-1 levels in supernatants 4 days after infection. (D) TIMP-1 levels in supernatants 7 days after infection. Data are presented as percentages of control values (n = 3). *, P < 0.05. Error bars indicate standard errors of the means.
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