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(Circulation Research. 2003;92:e80.)
© 2003 American Heart Association, Inc.

UltraRapid Communication

Mechanical Stretch Enhances mRNA Expression and Proenzyme Release of Matrix Metalloproteinase-2 (MMP-2) via NAD(P)H Oxidase–Derived Reactive Oxygen Species

Karsten Grote, Inna Flach, Maren Luchtefeld, Elvan Akin, Steven M. Holland, Helmut Drexler, Bernhard Schieffer

From the Department of Cardiology and Angiology (K.G., I.F., M.L., E.A., H.D., B.S.), Medical School Hannover, Germany; Laboratory of Host Defenses (S.M.H.), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.

Correspondence to Bernhard Schieffer, MD, Abteilung Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail Schieffer.Bernhard{at}mh-hannover.de

	Abstract

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Mechanical stretch is a hallmark of arterial hypertension and leads to vessel wall remodeling, which involves matrix metalloproteinases (MMPs). Because mechanical stretch is further capable of inducing reactive oxygen species (ROS) formation via the NAD(P)H oxidase, we assessed whether mechanical stretch enhances MMP expression and activity in a NAD(P)H oxidase–dependent manner. Therefore, vascular smooth muscle cells (VSMCs) isolated from C57BL/6 mice were exposed to cyclic mechanical stretch. The impact of ROS was assessed using VSMCs isolated from p47^phox-/- mice, deficient for a NAD(P)H oxidase subunit responsible for ROS formation. Transcript levels were investigated by cDNA array and confirmed by RT-PCR. ROS formation was determined by DCF fluoroscopy and MMP-2 activity by zymography. Mechanical stretch of wild-type VSMCs resulted in a rapid ROS formation and p47^phox membrane translocation that is followed by an increase in Nox-1 transcripts. ROS formation was completely abrogated in p47^phox-/- VSMCs. cDNA array further revealed an increase of MMP-2 mRNA in response to mechanical stretch, which was validated by RT-PCR. Using p47^phox-/- VSMCs, this increase in MMP-2 mRNA was completely blunted. mRNA expression of tissue inhibitor of MMP-2 TIMP-1 and TIMP-2 and membrane-type 1 MMP was unaffected by mechanical stretch. Gelatinolytic activity of pro-MMP-2 has been increased rapidly in wild-type VSMCs and was completely abolished in p47^phox-/- VSMCs. These results indicate that mechanical stretch induces ROS formation via the NAD(P)H oxidase and thereby enhances MMP-2 mRNA expression and pro-MMP-2 release. These results are consistent with the notion that in arterial hypertension, reactive oxygen species are involved in vascular remodeling via MMP activation. The full text of this article is available online at http://www.circresaha.org.

Key Words: mechanical stretch • reactive oxygen species • NAD(P)H oxidase • p47^phox • matrix metalloproteinases

	Introduction

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Blood vessels are continuously exposed to mechanical forces that, if excessive, ie, in arterial hypertension, lead to adaptive remodeling.¹ The complex process of vascular remodeling involves smooth muscle hypertrophy and hyperplasia, as well as enhanced collagen decomposition and extracellular matrix (ECM) reorganization.² This process involves the enzymatic activity of metalloproteinases (MMPs) within the vessel wall.³ A major role of MMPs is to enable vascular remodeling through the decomposition of the existing ECM scaffold while a new ECM is synthesized and organized. In this regard, mechanical stretch of the vessel wall was reported to enhance MMP activity, although the underlying mechanism remained unclear.^4,5

Regulation of MMPs may occur at multiple levels: either by gene transcription and synthesis of inactive proenzymes, posttranslational activation of proenzymes, or via the interaction of secreted MMPs with their inhibitors named tissue inhibitors of metalloproteinases (TIMPs).⁶ All members of the MMP family are secreted by cells as inactive proenzymes that must be proteolytically processed to become activated. Besides enzymatic activation by other proteases, such as plasmin, or cell-associated membrane-type MMPs (MT-MMPs), oxidative stress may enhance MMP expression and activity in vitro.³ Because mechanical stretch has also been reported to enhance reactive oxygen species (ROS),^7,8 we postulated that mechanical stretch activates the NAD(P)H oxidase, thereby enhancing ROS formation and subsequently leading to the activation of MMPs.

Vascular NAD(P)H oxidases are involved in signal transduction and are structurally related to the phagocytic NAD(P)H oxidases (phox). The enzyme expressed in vascular smooth muscle cells (VSMCs) is a multisubunit complex consisting of membrane-bound (p22^phox, Nox-1/Nox-4, gp91^phox)^9,10 and cytosolic components (p47^phox, Rac-1),⁹ and multiple studies suggested an important role of the p47^phox subunit for ROS production in VSMCs.^11,12 In the present study, we used mice lacking p47^phox (p47^phox-/-)¹³ to investigate the impact of mechanical stretch on MMP-2 found to be transcriptionally enhanced in wild-type VSMCs by microarray analysis via NAD(P)H-dependent ROS formation. We report that under mechanical stretch, ie, in arterial hypertension, ROS may be involved in vascular remodeling via MMP activation.

	Materials and Methods

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Reagents
Cell culture medium (DMEM) was from Biochrom AG, fetal calf serum (FCS) was from Greiner, and penicillin-streptomycin was from Gibco. 2',7'dichlorofluorescin diacetate (DCFH-DA), 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron; 5 mmol/L), and monoclonal -smooth muscle actin antibody (clone 1A4) were from Sigma. Diphenylene iodonium (DPI; 5 µmol/L) and N-acetyl-L-cysteine (L-NAC; 10 mmol/L) were from Calbiochem. Monoclonal p47^phox antibody (H-195) was obtained from Santa Cruz.

Cell Culture
VSMCs were isolated from the aorta of C57BL/6 (wild type, WT) and p47^phox-/- (S.M. Holland) mice by an enzymatic dispersion method.¹⁴ Cells were maintained in DMEM (Biochrom) supplemented with 20% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were growth-arrested in DMEM containing 0.5% FCS for 48 hour before use, experiments were performed under serum-free conditions.

Mechanical Stretch
VSMCs were plated on six-well silicone elastomer plates coated with collagen type I (Bioflex, Flexcell). The cells were exposed to continuous cycles of stretch and relaxation (0.5 Hz) by use of the Flexercell Strain Unit FX-3000 (Flexcell) for the indicated times; a maximum of 15% radial stretch of the membrane was applied.

DCF Fluoroscopy
Reactive oxygen species (ROS) were measured as hydrogen peroxide (H₂O₂) formation generated in equimolar amounts from superoxide anions (^·O₂^-) monitored by fluoroscopy (Fluoroscan, Labsystems) using DCFH-DA (2',7'dichlorofluorescin diacetate) to DCF conversion.¹⁵ VSMCs from WT and p47^phox-/- VSMCs were incubated with DCFH-DA (5 µmol/L), and ROS formation was monitored for the indicated time.

Microarray
VSMCs were subjected to mechanical stretch for 3 hours. Total RNA was isolated using TriFast-Reagent (peqLAB) and compared with unstretched conditions by the use of cDNA microarray specific for mouse cell adhesion and ECM molecules (GEArray kit, SuperArray, Biomol). Total RNA (5 µg) was used as the template for ³²P dCTP–labeled probe synthesis. After hybridization and washing of the microarray, the resulting X-ray film was quantified using a Gel Doc image analysis system (Bio-Rad, Hercules).

RT-PCR
Total RNA from VSMCs subjected to mechanical stretch was isolated using TriFast-Reagent (peqLAB, Inc). Total RNA was reverse transcribed using Superscript reverse transcriptase (RT, Gibco/BRL), oligo(dT) primers, and deoxynucleoside triphosphates. The RT products were amplified using Taq DNA polymerase (Gibco/BRL). PCR was performed for 18 cycles (18S rRNA), for 26 cycles (Nox-1, TIMP-2), for 30 cycles (TIMP-1, MT1-MMP), for 32 cycles (MMP-2), or for 35 cycles (p22^phox, p47^phox-/-, Nox-4), respectively. PCR products were separated by 1% agarose gel electrophoresis and quantified densitometrically using a Gel Doc image analysis system (Bio-Rad, Hercules).

Gelatin Zymography
Supernatants from VSMCs subjected to mechanical stretch were separated by 10% SDS-PAGE supplemented with 1 mg/mL gelatin. Recombinant mouse MMP-2 (Sigma) was used as control. Gelatinolytic activity was quantified densitometrically using a Gel Doc image analysis system (Bio-Rad, Hercules). Gels were renaturated by 2.5% Triton X-100 for 30 minutes, followed by a substrate buffer incubation (50 mmol/L Tris/HCl, pH 7.5, containing 5 mmol/L CaCl₂, 0.02% Brij-35) for 16 hours. Gels were stained with 0.5% Coomassie blue.

p47^phox Membrane Translocation
VSMCs were subjected to mechanical stretch and the membrane fraction was isolated by differential centrifugation. Cells were harvested by scraping with ice-cold phosphate-buffered saline (PBS), spun briefly, and homogenized with mortar and pestle in a sucrose-containing buffer (250 mmol/L sucrose, 10 mmol/L HEPES, protease inhibitor cocktail [Sigma], pH 7.4). Tubes were spun briefly to remove debris and the supernatant was subjected to stepwise centrifugation: first to 10 000g for 10 minutes to isolate the nuclei (discarded) and then to 20 800g for 30 minutes. Pellets containing membrane fragments were resuspended in homogenization buffer and analyzed by Western blot analysis using p47^phox antibodies. For demonstrating equal protein contents membranes were reprobed with -smooth muscle actin antibodies.

Statistical Analysis
All data are given as mean±SEM of at least 3 independent experiments. Differences were evaluated by ANOVA. Statistical significance was defined as P<0.01.

	Results

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Mechanical Stretch Enhances Nox-1 Transcription Levels
Mechanical stretch leads to enhanced transcript levels of the NAD(P)H oxidase subunit Nox-1 in WT VSMCs. Nox-1 mRNA expression increased at 1 hour (1.8±0.4-fold) and was further enhanced up to 24 hours (3.9±0.7-fold) (Figure 1). The mRNA levels of p22^phox, p47^phox, and Nox-4 were not significantly altered by mechanical stretch. The mRNA of the NAD(P)H-oxidase components gp91^phox (Nox-2) were not detectable.

View larger version (33K): [in this window] [in a new window]   Figure 1. Mechanical stretch enhances Nox-1 transcription levels. Murine WT VSMCs were applied to mechanical stretch (0.5 Hz, 15% elongation) for the indicated time, and RNA was subjected to RT-PCR. mRNA expression levels of NAD(P)H oxidase subunits p22phox, p47phox, Nox-1, and Nox-4 were normalized to 18S expression. Data are given as the mean±SEM of 4 independent experiments. *P<0.01 stretched vs unstretched.

Stretch-Dependent ROS Formation Requires p47^phox
Mechanical stretch leads to a rapid ROS formation in WT VSMCs peaking within the first minutes after stretch application and was sustained up to 5 minutes (Figure 2A). Stretch-induced ROS formation was completely abrogated in p47^phox-deficient VSMCs. Activation of the NAD(P)H oxidase requires an assembly of membrane-bound and cytosolic subunits, eg, that of p47^phox. Stretch-induced NAD(P)H oxidase activation was monitored by p47^phox membrane translocation. In stretched WT VSMCs, p47^phox protein levels in the membrane fraction started to increase at 2.5 minutes (122±11%), reached significant levels at 10 minutes (138±13%), and were further enhanced at 20 minutes (154±19%) as compared with unstretched control (Figure 2B).

View larger version (22K): [in this window] [in a new window]   Figure 2. Stretch-dependent ROS formation requires p47phox. A, ROS formation in VSMCs from WT and p47phox-/- mice subjected to mechanical stretch (0.5 Hz, 15% elongation) for the indicated time was determined by fluoroscopy using the DCFH-DA to DCF conversion. Results are given in arbitrary units as the mean±SEM of 4 independent experiments. B, Representative Western blot analysis of p47phox subunit in the membrane fraction obtained by differential centrifugation of WT VSMCs subjected to mechanical stretch. Summary data showing the stretch-induced increase of p47phox membrane translocation. *P<0.01 for stretched vs unstretched; #P<0.01 for p47phox-/- vs WT.

Mechanical Stretch Enhances MMP-2 mRNA Expression via p47^phox
Using a cDNA array chip specific for mouse cell adhesion and extracellular matrix proteins, we identified 5 genes to be significantly upregulated in WT VSMC subjected to mechanical stretch (Table), which was confirmed by RT-PCR (Figure 3). Because reactive oxygen species have been reported to modulate MMP-2 activity,¹⁶ we investigated whether the NAD(P)H oxidase may enhance MMP-2 mRNA expression and activity in response to mechanical stretch. Expression of MMP-2 mRNA in WT VSMCs increased at 3 hours (1.8±0.1-fold) (Figure 3A), peaked at 6 hours (2.0±0.1-fold), and remained elevated up to 24 hours (1.9±0.1-fold). In contrast, mechanical stretch of p47^phox-/- VSMCs had no impact on MMP-2 mRNA expression levels (Figure 3A). Additionally, the flavoprotein inhibitor DPI and the radical scavengers Tiron and L-NAC abolished the stretch-induced increase in MMP-2 transcripts in WT VSMCs at 3 hours of mechanical stretch. Inhibitors alone had no effects (Figure 3B). In contrast, tissue inhibitors of MMP-2, which are TIMP-1, TIMP-2, and membrane-type 1 MMP (MT1-MMP), expression levels remained unchanged in response to mechanical stretch (Figure 3C).

View this table: [in this window] [in a new window]   Table 1. Genes Induced by Mechanical Stretch

View larger version (30K): [in this window] [in a new window]   Figure 3. Mechanical stretch enhances MMP-2 mRNA expression via p47phox. Cultured VSMCs from WT and p47phox-/- mice were subjected to mechanical stretch (0.5 Hz, 15% elongation) for the indicated time, and MMP-2, TIMP-1, TIMP-2, and MT1-MMP mRNA expression levels were determined by RT-PCR. A, Time-dependent expression of MMP-2 mRNA in WT and p47phox-/- VSMCs. Data are given as the mean±SEM of 4 independent experiments. B, Influence of DPI, Tiron, and L-NAC on stretch-induced MMP-2 mRNA expression in WT VSMCs after 3 hours. Data are given as the mean±SEM of 3 independent experiments. C, Expression of TIMP-1, TIMP-2, and MT1-MMP mRNA in WT VSMCs after mechanical stretch. Representative results of 3 independent experiments are shown. *P<0.01 for stretched vs unstretched; #P<0.01 for p47phox-/- vs WT; P<0.01 for stretched with inhibitor vs stretched without inhibitor.

Mechanical Stretch Enhances Proenzyme Release via p47^phox
VSMCs were found to secrete solely the latent form of MMP-2 (pro-MMP-2, 72 kDa) (Figure 4). Moreover, no active form of MMP-2 (68 kDa) was detected by gelatin zymography. However, mechanical stretch leads to a rapid increase of pro-MMP-2 activity reflecting to pro-MMP-2 release observed in VSMCs from WT mice at 1 hour (3.9±0.1-fold; Figure 4), which was sustained at 3 and 6 hours, and further increased at 24 hours (7.0±0.9-fold). However, increase of pro-MMP-2 activity in response to mechanical stretch was completely blunted in p47^phox-/- VSMCs (Figure 4). Similarly, DPI, Tiron, and L-NAC abolished the stretch-induced enhancement in pro-MMP-2 release in WT VSMC at 3 hours of mechanical stretch, whereas the inhibitors alone had no effect (Figure 3B). TIMP-1 and TIMP-2 protein was not detectable in the supernatants by Western blot analysis.

View larger version (29K): [in this window] [in a new window]   Figure 4. Mechanical stretch enhances pro-MMP-2 release via p47phox. Cultured VSMCs from WT and p47phox-/- mice were subjected to mechanical stretch (0.5 Hz, 15% elongation) for the indicated time. Supernatants were tested for MMP-2 activity by gelatin zymography. A, Time-dependent release of pro-MMP-2 in WT and p47phox-/- VSMCs. Data are given as the mean±SEM of 4 independent experiments. PC indicates positive control. B, Influence of DPI, Tiron, and L-NAC on stretch-induced pro-MMP-2 release in WT VSMCs after 3 hours. Data are given as the mean±SEM of 3 independent experiments. *P<0.01 for stretched vs unstretched; #P<0.01 for p47phox-/- vs WT; P<0.01 for stretched with inhibitor vs stretched without inhibitor.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that mechanical stretch not only enhances p47^phox membrane translocation and ROS formation but also enhances the expression of the NAD(P)H oxidase subunit Nox-1. Importantly, stretch-induced ROS formation via the NAD(P)H oxidase enhances MMP-2 mRNA expression and pro-MMP-2 release. These observations are consistent with the notion that mechanical stretch (ie, in arterial hypertension) modulates vascular remodeling via a NAD(P)H oxidase–dependent stimulation of MMP expression and activity.

Maladaptive remodeling of the vessel wall is a characteristic feature of vascular diseases such as arterial hypertension or atherosclerosis and appears to involve vasoactive peptides, ie, angiotensin II (Ang II),¹⁷ but also mechanical stretch.¹⁸ Ang II-AT₁ receptor activation was reported to stimulate superoxide anion formation via the NAD(P)H-oxidase.¹⁹ This mechanism was shown to contribute to vascular inflammation via redox-sensitive signaling mechanisms.²⁰ However, little is known about the mechanism by which mechanical stretch is linked to vascular inflammation and remodeling. It has been reported that superoxide anions, generated by the NAD(P)H oxidase may result in an alteration of vascular function.²¹ Moreover, in the murine apoE^-/- atherosclerosis model, deficiency of the NAD(P)H oxidase subunit p47^phox leads to a significant reduction of total lesion area in the descending aorta compared with apoE^-/- mice.²² Therefore, the NAD(P)H oxidase may be a key enzyme in the regulation of vascular function and remodeling.

The present study demonstrates that mechanical stretch leads to a rapid increase in ROS formation in smooth muscle cells. Moreover, mechanical stretch induces an upregulation of the NAD(P)H oxidase subunit Nox-1. Similarly, we and others were able to demonstrate that mechanical stretch results in a rapid increase in p47phox membrane translocation.²³ We further demonstrate that the stretch-induced ROS formation relies on the NAD(P)H oxidase subunit p47^phox. Based on these results, we postulate that mechanical stretch enhances oxidative stress within the vessel wall via the NAD(P)H oxidase.

We further postulated that this mechanism may be critical for mechanical stretch–induced vascular remodeling. Decomposition of preexisting and de novo synthesis of ECM is a characteristic of arterial hypertension and of plaque development and destabilization. This process is mediated by the enzymatic activity of MMPs. MMP-2 is the predominant MMP secreted by smooth muscle cells and is known to be activated by either ROS^16,24 or mechanical stretch.^25,26 MMPs are regulated at multiple levels, for instance by gene transcription and synthesis of inactive proenzymes. In the present study, we report an upregulation of MMP-2 mRNA by mechanical stretch consistent with previous observations.^27–29 MMP-2 mRNA is upregulated as early as 3 hours after the administration of mechanical stretch and remained elevated for 24 hours. This upregulation is abolished in p47^phox-/- VSMCs, demonstrating the essential role of the NAD(P)H oxidase in the transcriptional regulation of MMP-2.

Similarly, the activity of the latent form of MMP-2 (pro-MMP-2) reflecting the release of the proenzyme is dramatically increased in response to mechanical stretch after 1 hour (>4-fold), remained elevated afterward and increased again at 24 hours by 7-fold. This biphasic activity pattern was completely abolished in p47^phox-/- VSMCs and may be explained by the de novo synthesis of NAD(P)H oxidase components and MMP-2 protein, which may contribute to the second peak of pro-MMP-2 activity. Notably, the rapid increase of pro-MMP-2 activity at 1 hour does not require protein de novo synthesis, because stretch-induced MMP-2 mRNA emerged at 3 hours. Both enhanced mRNA expression and proenzyme release after mechanical stretch were completely blunted by the flavoprotein inhibitor DPI and the radical scavenger Tiron and L-NAC. These findings suggest that stretch-induced ROS formation is a key regulator of vascular MMP-2. Thus, a critical role for ROS generated by the NAD(P)H oxidase in mechanical stretch–induced MMP-2 mRNA expression and pro-MMP-2 release is suggested, although the underlying mechanism needs further investigations.

In this regard, pro-MMP-2 may be activated via the enzymatic removal of its prodomain by MT1-MMP.^30,31 However, further experiments demonstrated that in contrast to MMP-2 mRNA expression in response to mechanical stretch, MT1-MMP mRNA is not enhanced.

Moreover, pro-MMP-2 activation was reported to be modulated via ROS produced by foam cells, which can trigger the activation of latent MMP-2 stored in the vessel wall.¹⁶ Thus, mechanical stretch may lead to pathological maladaptive morphological processes via induction of MMP-2 by superoxide anions derived from the NADP(H) oxidase. TIMPs³² are known to inhibit MMP-2 activity. However, mechanical stretch did not affect TIMP-1 and TIMP-2 mRNA expression in vitro. Moreover, TIMP-1 and TIMP-2 protein were not be detected in supernatants of cells subjected to mechanical stretch. These findings are consistent with the notion that mechanical stretch induces a shift toward a collagenolytic activity in a ROS-dependent manner.

In summary, we show that mechanical stretch elicits MMP expression and activation via NAD(P)H oxidase–derived reactive oxygen species. Because increased mechanical stretch is a hallmark of arterial hypertension, our observations are consistent with the notion that induction of MMPs is a crucial step in vascular remodeling processes of the vessel wall attributed to mechanical forces in hypertension.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich TR02 project B4 and the Leducq Fondation. We thank Tanja Sander and Silke Pretzer for excellent technical assistance.

	Footnotes

 
Original received December 23, 2002; revision received April 28, 2003; accepted May 2, 2003.

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