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

Abstract —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 p47phox‐/‐ 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 p47phox membrane translocation that is followed by an increase in Nox‐1 transcripts. ROS formation was completely abrogated in p47phox‐/‐ VSMCs. cDNA array further revealed an increase of MMP‐2 mRNA in response to mechanical stretch, which was validated by RT‐PCR. Using p47phox‐/‐ 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 p47phox‐/‐ 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. (Circ Res. 2003;92:e80‐ e86.)

[1]  R. Nerem,et al.  Cyclic strain induces an oxidative stress in endothelial cells. , 1997, The American journal of physiology.

[2]  A. Tedgui,et al.  Signal transduction of mechanical stresses in the vascular wall. , 1998, Hypertension.

[3]  Robert M. Nerem,et al.  The Role of Matrix Metalloproteinase-2 in the Remodeling of Cell-Seeded Vascular Constructs Subjected to Cyclic Strain , 2001, Annals of Biomedical Engineering.

[4]  L. Langille,et al.  Remodeling of Developing and Mature Arteries: Endothelium, Smooth Muscle, and Matrix , 1993, Journal of cardiovascular pharmacology.

[5]  A. Naftilan Role of the tissue renin-angiotensin system in vascular remodeling and smooth muscle cell growth. , 1994, Current opinion in nephrology and hypertension.

[6]  C. Meischl,et al.  NADPH oxidase(s): new source(s) of reactive oxygen species in the vascular system? , 2002, Journal of clinical pathology.

[7]  S. Kawashima,et al.  Lysophosphatidylcholine increases the secretion of matrix metalloproteinase 2 through the activation of NADH/NADPH oxidase in cultured aortic endothelial cells. , 2001, Atherosclerosis.

[8]  A. Kugler Matrix metalloproteinases and their inhibitors. , 1999, Anticancer research.

[9]  K. Stuhlmeier,et al.  A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate. , 1992, Journal of immunological methods.

[10]  H. Drexler,et al.  Role of NAD(P)H Oxidase in Angiotensin II–Induced JAK/STAT Signaling and Cytokine Induction , 2000, Circulation research.

[11]  D. Fishman,et al.  Membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation in primary human ovarian epithelial carcinoma cells. , 1996, Invasion & metastasis.

[12]  P. Kaminski,et al.  Stretch Enhances Contraction of Bovine Coronary Arteries via an NAD(P)H Oxidase–Mediated Activation of the Extracellular Signal–Regulated Kinase Mitogen-Activated Protein Kinase Cascade , 2003, Circulation research.

[13]  V. Dzau The role of mechanical and humoral factors in growth regulation of vascular smooth muscle and cardiac myocytes. , 1993, Current opinion in nephrology and hypertension.

[14]  A. Takeshita,et al.  Important role of local angiotensin II activity mediated via type 1 receptor in the pathogenesis of cardiovascular inflammatory changes induced by chronic blockade of nitric oxide synthesis in rats. , 2000, Circulation.

[15]  J. Keiser,et al.  Expression of Membrane-Type Matrix Metalloproteinase in Rabbit Neointimal Tissue and Its Correlation with Matrix-Metalloproteinase-2 Activation , 1998, Journal of Vascular Research.

[16]  M. Klagsbrun,et al.  AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells. , 1999, American journal of physiology. Cell physiology.

[17]  J. O'Connell,et al.  Regulation of Matrix Metalloproteinase Activity a , 1994, Annals of the New York Academy of Sciences.

[18]  G. Owens,et al.  Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells , 1988, The Journal of cell biology.

[19]  E. Schiffrin,et al.  Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries: Regulation by Angiotensin II , 2002, Circulation research.

[20]  A. Bolcato-Bellemin,et al.  Expression of mRNAs Encoding for α and β Integrin Subunits, MMPs, and TIMPs in Stretched Human Periodontal Ligament and Gingival Fibroblasts , 2000 .

[21]  T. Lüscher,et al.  Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor-κB in Human Coronary Smooth Muscle , 1997 .

[22]  Avrum I. Gotlieb,et al.  Wall Tissue Remodeling Regulates Longitudinal Tension in Arteries , 2002, Circulation research.

[23]  S. Holland,et al.  p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice. , 2001, The Journal of clinical investigation.

[24]  Christopher J. O’Callaghan,et al.  Mechanical Strain–Induced Extracellular Matrix Production by Human Vascular Smooth Muscle Cells: Role of TGF-&bgr;1 , 2000, Hypertension.

[25]  S. Holland,et al.  The p47phox mouse knock-out model of chronic granulomatous disease , 1995, The Journal of experimental medicine.

[26]  D. Harrison,et al.  Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. , 1996, The Journal of clinical investigation.

[27]  M. Mochizuki,et al.  Involvement of mechanical stretch in the gelatinolytic activity of the fibrous sclera of chicks, in vitro. , 2002, Japanese journal of ophthalmology.

[28]  S. Holland,et al.  Genetic Demonstration of p47phox-Dependent Superoxide Anion Production in Murine Vascular Smooth Muscle Cells , 2001, Circulation.

[29]  Z. Galis,et al.  This Review Is Part of a Thematic Series on Matrix Metalloproteinases, Which Includes the following Articles: Matrix Metalloproteinase Inhibition after Myocardial Infarction: a New Approach to Prevent Heart Failure? Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: the Good, the Ba , 2022 .

[30]  Hua Cai,et al.  Role of p47phox in Vascular Oxidative Stress and Hypertension Caused by Angiotensin II , 2002, Hypertension.

[31]  T. Acott,et al.  Effects of mechanical stretching on trabecular matrix metalloproteinases. , 2001, Investigative ophthalmology & visual science.

[32]  R W Alexander,et al.  Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. , 1994, Circulation research.