Calpain-1 Mediated Disorder of Pyrophosphate Metabolism Contributes to Vascular Calcification Induced by oxLDL

Abstract

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.

Fig 1. Calpain activity increased in the calcified aorta of rats and RVSMCs, which was inhibited by CAI and calpain-1 siRNA respectively.

Rats were treated with high dose of vitamin D₂ (Control) with or without high cholesterol diet (HCD) in the presence or absence of CAI. RVSMCs were treated with or without oxLDL (25-100mg/L) in the presence or absence of calpain-1 siRNA. The parameters were measured as described in Materials and Methods. A: Calpain activity in aortic tissue of rats; B: Calpain activity in RVSMCs. Results were expressed as mean±S.D. n = 8 for A and n = 4 for B. *: P<0.05 was considered statistically significant.

Fig 2. CAI reduced vascular calcification in rats.

Rats were treated with high dose of vitamin D₂ (Control) with or without high cholesterol diet (HCD) in the presence or absence of CAI. The parameters were measured as described in Materials and Methods. A: Calcium deposition in aorta arch in brown yellow color (A1: representative photograph, black arrow indicates the calcium deposition; A2: statistical results); B: Calcium content in aortic tissue; C: DHE in red color indicating superoxide anion (C1: representative photograph; C2: statistical results); D: Serum content of total cholesterol (TC); E: oxLDL content in serum. Results were expressed as mean±S.D. n = 8. *: P<0.05 was considered statistically significant.

Fig 3. CAI corrected the imbalance in the degradation and synthesis of PPi in rats.

Rats were treated with high dose of vitamin D₂ (Control) with or without high cholesterol diet (HCD) in the presence or absence of CAI. The parameters were measured as described in Materials and Methods. A: PPi content in serum; B: ATP content in serum; C: ATP content in aortic tissue; D: ALP activity in serum; E: ALP activity in aortic tissue; F: ALP mRNA expression in aortic tissue; G: ALP protein expression in aortic tissue (upper: representative photograph; lower: statistical results); H: Enpp1 mRNA expression in aortic tissue; I: Ank mRNA expression in aortic tissue. Results were expressed as mean±S.D. n = 8 for A, B, C, D and E; n = 4 for F, G and I. *: P<0.05 was considered statistically significant.

Fig 4. CAI increased the activity of ATP synthase and protein expression of ATP5D in aortic tissue of rats.

Rats were treated with high dose of vitamin D₂ (Control) with or without high cholesterol diet (HCD) in the presence or absence of CAI. The parameters were measured as described in Materials and Methods. A: ATP synthase activity; B: ATP5D protein expression (upper: representative photograph; lower: statistical results). Results were expressed as mean±S.D. n = 8 for A; n = 4 for B. *: P<0.05 was considered statistically significant.

Fig 5. Suppression of calpain-1 inhibited RVSMCs calcification induced by oxLDL.

RVSMCs were treated and the parameters were measured as described in Materials and Methods. A: Protein expression of calpain-1 in RVSMCs transfected with siRNA against calpain-1 (upper: representative photograph; lower: statistical results); B: Protein expression of calpain-2 in RVSMCs transfected with siRNA against calpain-2 (upper: representative photograph; lower: statistical results); C: Calpain actitity in RVSMCs transfected with siRNA against calpain-1 and calpain-2 respectively; D: Calcium deposition in RVSMCs in red and black color (D1: representative photograph; D2: statistical results); E: Calcium content in RVSMCs. Results were expressed as mean±S.D. n = 4. *: P<0.05 was considered statistically significant

Fig 6. Suppression of calpain-1 corrected the imbalance of PPi metabolism in RVSMCs treated with oxLDL.

RVSMCs were treated with or without oxLDL in the presence or absence of calpain-1 siRNA. The parameters were measured as described in Materials and Methods. A: PPi content in medium; B: ATP content in medium; C: ATP content in RVSMCs; D: ALP activity in medium; E: ALP activity in RVSMCs; F: ALP mRNA expression in RVSMCs; G: ALP protein expression in RVSMCs (upper: representative photograph; lower: statistical results); H: Enpp1 mRNA expression in RVSMCs; I: Ank mRNA expression RVSMCs. Results were expressed as mean±S.D. n = 4. *: P<0.05 was considered statistically significant.

Fig 7. Suppression of calpain-1 increased the activity of ATP synthase and protein expression of ATP5D in RVSMCs treated with oxLDL.

RVSMCs were treated with or without oxLDL in the presence or absence of calpain-1 siRNA. The parameters were measured as described in Materials and Methods. A: ATP synthase activity; B: ATP5D protein expression (upper: representative photograph; lower: statistical results). Results were expressed as mean±S.D. n = 4. *: P<0.05 was considered statistically significant.

Fig 8. Inhibition of ROS in mitochondria reduced calcification of RVSMCs and regulated the expression of ALP.

In one experiment, RVSMCs were treated with or without oxLDL in the presence or absence of calpain-1 siRNA. In the other experiments, RVSMCs were treated with or without oxLDL in the presence or absence of TEMPO. The parameters were measured as described in Materials and Methods. A: Mito-SOX in red color indicating ROS production in mitochondria of RVSMCs (A1: representative photograph; A2: statistical results); B: Calcium deposition in RVSMCs in red and black color (B1: representative photograph; B2: statistical results); C: PPi content in medium; D: ALP activity in medium; E: ALP activity in RVSMCs; F: ALP mRNA expression in RVSMCs; G: ALP protein expression in RVSMCs (upper: representative photograph; lower: statistical results). Results were expressed as mean±S.D. n = 4. *: P<0.05 was considered statistically significant.