We previously reported that
oxidized low density lipoprotein (
oxLDL) accelerated the calcification in aorta of rats and rat vascular smooth muscle cells (RVSMCs). However, the molecular mechanism underlying the acceleration remains poorly understood. The present study aimed to investigate the role of calpain-1, Ca2+-sensitive intracellular
cysteine proteases, in the
vascular calcification of rats treated with both high dose of
vitamin D2 and high
cholesterol diet. The results showed that
calpain activity significantly increased in calcified aortic tissue of rats and RVSMCs treated with
oxLDL. Specific
calpain inhibitor I (CAI, 0.5mg/kg, intraperitoneal) inhibited the
vascular calcification in rats with
hypercholesterolemia accompanied by the increase in the level of extracellular inorganic
pyrophosphate (PPi), the endogenous inhibitor of
vascular calcification. In addition, CAI increased the content of
adenosine triphosphate (
ATP), decreased the activity,
mRNA and
protein expression of
alkaline phosphatase (ALP) and reduced the production of
superoxide anion in calcified aortic tissue. CAI also increased the activity of
ATP synthase as well as
protein expression of ATP5D, δ subunit of
ATP synthase. In the in vitro study, suppression of calpain-1 using
siRNA assay inhibited the
calcium deposition, increased the levels of PPi and
ATP, improved the activity of
ATP synthase as well as
protein expression of ATP5D in RVSMCs treated with
oxLDL. Calpain-1 suppression also decreased the activity,
mRNA and
protein expression of ALP and reduced the mitochondrial ROS (Mito-ROS) production in RVSMCs. However, mito-
TEMPO, the mitochondria-targeted ROS scavenger, reduced the
calcium deposition, increased the PPi in culture medium, decreased the activity,
mRNA and
protein expression of ALP in RVSMCs treated with
oxLDL. Taken together, the results suggested that calpain-1 activation plays critical role in
vascular calcification caused by
oxLDL, which might be mediated by PPi metabolism disorder. The results also implied that Mito-ROS might contribute to the PPi metabolism disorder through regulation of the activity and expression of ALP.