Periostin Augments Vascular Smooth Muscle Cell Calcification via β-Catenin Signaling

Abstract

Medial vascular calcification is common in chronic kidney disease (CKD) and is closely linked to hyperphosphatemia. Vascular smooth muscle cells (VSMCs) can take up pro-calcific properties and actively augment vascular calcification. Various pro-inflammatory mediators are able to promote VSMC calcification. In this study, we investigated the effects and mechanisms of periostin, a matricellular signaling protein, in calcifying human VSMCs and human serum samples. As a result, periostin induced the mRNA expression of pro-calcific markers in VSMCs. Furthermore, periostin augmented the effects of β-glycerophosphate on the expression of pro-calcific markers and aggravated the calcification of VSMCs. A periostin treatment was associated with an increased β-catenin abundance as well as the expression of target genes. The pro-calcific effects of periostin were ameliorated by WNT/β-catenin pathway inhibitors. Moreover, a co-treatment with an integrin αvβ3-blocking antibody blunted the pro-calcific effects of periostin. The silencing of periostin reduced the effects of β-glycerophosphate on the expression of pro-calcific markers and the calcification of VSMCs. Elevated serum periostin levels were observed in hemodialysis patients compared with healthy controls. These observations identified periostin as an augmentative factor in VSMC calcification. The pro-calcific effects of periostin involve integrin αvβ3 and the activation of the WNT/β-catenin pathway. Thus, the inhibition of periostin may be beneficial to reduce the burden of vascular calcification in CKD patients.

Keywords: chronic kidney disease; periostin; phosphate; vascular calcification; vascular smooth muscle cells; β-catenin.

Figure 1

Periostin aggravates phosphate-induced calcification of HAoSMCs. (A) Alizarin Red staining in HAoSMCs treated with a control (CTR) or calcification medium (Calc.) without or with additional treatment with recombinant human periostin (POSTN). Calcified areas: red staining. (B) Calcium content in HAoSMCs treated with a control (CTR) or calcification medium (Calc.) without or with additional treatment with recombinant human periostin (POSTN). ** p < 0.01; *** p < 0.001 (significant difference versus CTR group); † p < 0.05 (significant difference versus Calc. group).

Figure 2

Periostin increases osteogenic signaling and augments osteoinduction promoted by phosphate in HAoSMCs. (A–C) Relative mRNA expression of MSX2 (A), CBFA1 (B) and ALPL (C) in HAoSMCs treated with control (CTR) or β-glycerophosphate (Pi) without or with additional treatment with recombinant human periostin (POSTN). (D) Normalized ALP activity in HAoSMCs treated with control (CTR) or β-glycerophosphate (Pi) without or with additional treatment with recombinant human periostin (POSTN). ** p < 0.01; *** p < 0.001 (significant difference versus CTR group); † p < 0.05; †† p < 0.01 (significant difference versus Pi group).

Figure 3

Periostin increases WNT/β-catenin signaling in HAoSMCs. (A,B) Relative mRNA expression of WNT7A (A) and WNT3A (B) in HAoSMCs treated with control (CTR) or recombinant human periostin (POSTN). (C) Representative Western blots and normalized β-catenin protein expression in HAoSMCs treated with control (CTR) or recombinant human periostin (POSTN). (D,E) Relative mRNA expression of MMP2 (D) and PIT1 (E) in HAoSMCs treated with control (CTR) or recombinant human periostin (POSTN). ** p < 0.01; *** p < 0.001 (significant difference versus CTR group).

Figure 4

Inhibition of the WNT/β-catenin pathway suppresses periostin-induced osteogenic signaling in HAoSMCs. (A–E) Relative mRNA expression of MMP2 (A), PIT1 (B), MSX2 (C), CBFA1 (D) and ALPL (E) in HAoSMCs treated with control (CTR) or recombinant human periostin (POSTN) without or with additional treatment with the WNT/β-catenin pathway inhibitors LGK974 (LGK), XAV939 (XAV) or PRI-724 (PRI). * p < 0.05; ** p < 0.01 (significant difference versus CTR group); † p < 0.05; †† p < 0.01; ††† p < 0.001 (significant difference versus POSTN group).

Figure 5

Treatment with integrin αvβ3 antibody blunts periostin-induced WNT/β-catenin and osteogenic signaling in HAoSMCs. (A–G) Relative mRNA expression of WNT7A (A), WNT3A (B), MMP2 (C), PIT1 (D), MSX2 (E), CBFA1 (F) and ALPL (G) in HAoSMCs treated with control (CTR) or recombinant human periostin (POSTN) and with mouse IgG as control or integrin αvβ3 antibody (αvβ3Ab). * p < 0.05; ** p < 0.01; *** p < 0.001 (significant difference versus CTR group); † p < 0.05; †† p < 0.01; ††† p < 0.001 (significant difference versus POSTN group).

Figure 6

Silencing of periostin ameliorates phosphate-induced WNT/β-catenin and osteogenic signaling in HAoSMCs. (A–H) Relative mRNA expression of POSTN (A), WNT7A (B), WNT3A (C), MMP2 (D), PIT1 (E), MSX2 (F), CBFA1 (G) and ALPL (H) in HAoSMCs transfected with negative control siRNA (N.si) or POSTN siRNA (POSTNsi) and treated with control or β-glycerophosphate (Pi). (I) Normalized ALP activity in HAoSMCs transfected with negative control siRNA (N.si) or POSTN siRNA (POSTNsi) and treated with control or β-glycerophosphate (Pi). * p < 0.05; ** p < 0.01; *** p < 0.001 (significant difference versus N.si group); † p < 0.05; †† p < 0.01; ††† p < 0.001 (significant difference versus N.si + Pi group).

Figure 7

Silencing of periostin reduces phosphate-induced calcification of HAoSMCs. (A) Alizarin Red staining in HAoSMCs transfected with negative control siRNA (N.si) or POSTN siRNA (POSTNsi) and treated with control (CTR) or calcification medium (Calc.). Calcified areas: red staining. (B) Calcium content in HAoSMCs transfected with negative control siRNA (N.si) or POSTN siRNA (POSTNsi) and treated with control (CTR) or calcification medium (Calc.). * p < 0.05 (significant difference versus N.si group); †† p < 0.01 (significant difference versus N.si + Calc. group).

Figure 8

Serum periostin levels are increased in hemodialysis patients. (A) Serum periostin (POSTN) levels in healthy volunteers (CTR), patients with known CKD (CKD) and hemodialysis patients (Dialysis). * p < 0.05 (significant difference versus CTR group). (B) Correlation between serum periostin (POSTN) concentrations and serum calcification propensity measured as calciprotein particle maturation time (T50). The p-value is indicated in the figure.