TIMP‐1 protein but not secretion is increased by shear stress in the skeletal muscle microvasculature

Abstract

Luminal splitting is a form of angiogenesis caused by increased shear stress. A feature of luminal splitting is a lack of extracellular matrix proteolysis correlated with decreased production of matrix metalloproteinase (MMP)‐2. We hypothesized that an MMP inhibitor, tissue inhibitor of matrix metalloproteinase (TIMP)‐1, is upregulated by shear stress. TIMP‐1 mRNA increased in the EDL of prasozin‐treated rats (p<0.05, n=4) as measured by RT‐PCR. Rat skeletal muscle microvascular endothelial cells were exposed to shear stress (12 dyne/cm2). TIMP‐1 mRNA increased in cells after 2 and 24 hours of shear (p<0.05, n=3), and TIMP‐1 protein increased at 2 and 24 but not 4 hours (p<0.05, n=3) as assessed by Western. TIMP‐1 secretion decreased at 2 hours of shear and returned to control levels at 24 hours (p<0.01, n=3) as measured by reverse zymography of concentrated media. Similarly, MMP‐2 activity increased in media at 2 but not 24 hours of shear (p<0.05, n=7) as assessed by an in vitro MMP‐2 activity assay (Anaspec). 2 hours of shear caused no change in TIMP‐1 surface staining (ns, n=3) as assessed by confocal microscopy, but did cause an increase in intracellular TIMP‐1 staining (p<0.05, n=3). Our results show that intracellular TIMP‐1 protein is increased by shear stress, due to increased production and decreased secretion. The function of intracellular TIMP‐1 is unknown.Funded by the Heart and Stroke Foundation of Canada.Grant Funding SourceHeart and Stroke Foundation of Canada