Annexin-mediated matrix vesicle calcification in vascular smooth muscle cells

Abstract

In bone, osteoblasts and chondrocytes synthesize matrix vesicles (MVs) that interact with collagen to initiate calcification. MVs have been identified in human calcified arteries but are poorly characterized. The objective of this study is to determine the role of annexins and fetuin-A in MV formation and activity during calcification in bovine vascular smooth muscle cells (BVSMCs). BVSMCs were treated with control or calcification (high phosphorus) media, and cellular MVs were isolated by collagenase digestion and secreted MVs were isolated from cultured media by ultracentrifugation. The results showed that alkaline phosphatase (ALP) activity was significantly increased in MVs from calcified BVSMCs compared with noncalcified BVSMCs, as was annexin II and VI content and (45)Ca uptake. We also determined that MVs from calcifying BVSMCs could mineralize type I collagen but not type II collagen in the absence of cells in a dose- and time-dependent manner. Blockade of annexin calcium channel activity by K201 significantly decreased ALP activity and reduced the ability of the MVs to subsequently calcify on collagen, whether the K201 was added during or after MV formation. Furthermore, cellular MVs had significantly increased ability to calcify on collagen compared with secreted MVs, likely because of their increased ALP activity and annexin II content but low fetuin-A content. In conclusion, our results suggest that mineralization in VSMCs requires both active MVs and an interaction of the MVs with type I collagen, and both steps require annexin activity.

FIG. 1

ALP activity, annexin II and VI content, and ⁴⁵Ca uptake by MVs from calcified and noncalcified BVSMCs. MVs were isolated from calcified and noncalcified (control) BVSMCs by collagenase digestion. (A) MV ALP activity was measured and normalized by total MV protein. (B) Annexin II (top) and annexin VI (bottom) content in MVs was determined by Western blot analysis and band density was quantified. (C) Equal amounts of MVs (10 μg of protein) were incubated in 100 μl reaction media with 1 × 10⁶ cpm ⁴⁵Ca at 37°C for 5 h, and uptake was determined by scintillation counting. The results show that MVs from calcified BVSMCs had increased ALP activity, increased annexin II and VI content, and enhanced ability to take up calcium. Data are shown as mean ± SD from three to four separate experiments. *p < 0.001, calcified vs. control.

FIG. 2

Time course of collagen calcification of MVs from control or calcified BVSMCs in MV-collagen calcification assay. Glass coverslips were coated with type I collagen (10 μg/cm²), and MVs (10 μg) isolated from calcified and noncalcified (control) BVSMCs were added in the presence of calcification media (DMEM with 15% FBS and 10 mM β-glycerophosphate) and incubated at 37°C for 1, 2, or 3 days to yield an acellular MV-collagen culture. The calcium content was determined by HCL extraction. The results show MVs from calcified BVSMCs calcify type I collagen in a time-dependent manner, but MVs from control BVSMC fail to calcify. Data are shown as mean ± SD from three separate experiments. p < 0.0001, calcified vs. control; same day, ^# p < 0.0002, day 3 vs. day 2, control or calcified.

FIG. 3

MV calcification on type I collagen in MV-collagen calcification assay. Glass coverslips were coated with type I collagen (10 μg/cm²). Increasing concentrations (5, 7.5, 10, and 20 μg) of MVs isolated from calcified BVSMCs were added in the presence of calcification media (DMEM with 15% FBS and 10 mM β-glycerophosphate) and incubated at 37°C for 72 h to yield an acellular MV-collagen culture. The calcium content was determined by HCL extraction. The results show a dose-dependent increase in the amount of calcification with the addition of increasing concentrations of MVs added. Data are shown as mean ± SD from three separate experiments. *p < 0.005 compared with 5 μg MVs; ^# p < 0.05 compared with 7.5 μg MVs.

FIG. 4

Effect of extracellular matrix in MV-collagen calcification assay. Glass coverslips were coated with various amounts of type I or type II collagen (0, 10, 50 μg/cm²), and MVs were added in the presence of calcification media (DMEM with 15% FBS and 10 mM β-glycerophosphate) and incubated at 37°C for 72 h to yield an acellular MV-collagen culture. The calcium content was determined by HCL extraction. The results show a dose-dependent increase in MV calcification with type I collagen, whereas MVs fail to calcify type II collagen. Data are shown as mean ± SD from three separate experiments. *p < 0.0001, coating vs. noncoating control; ⁺ p < 0.005, 50 vs. 10 μg/cm²; ^# p < 0.0001, type I vs. type II collagen coated, 50 or 10 μg/cm².

FIG. 5

K201 inhibits the ability of MV to calcify on type I collagen. To determine the role of annexins in MV activity, BVSMCs were incubated in DMEM in calcification media (calcified, with 10 mM β-glycerophosphate) or noncalcifying media (control, no β-glycerophosphate) for 7 days in the presence or absence of K201, a broad inhibitor of annexin calcium channel activity and MVs were isolated. (A) Equal amounts of MVs (10 μg of protein) were incubated in 100 μl reaction media with 1 × 10⁶ cpm ⁴⁵Ca at 37°C for 5 h and determined by scintillation counting. (B) MVs were placed on type I collagen-coated coverslips in culture dishes and incubated with calcification media (10 mM β-glycerophosphate) for 3 days, and the calcification was examined by HCL extraction. The results show that blockade of annexin calcium channel activity by K201 during MV synthesis significantly decreases ALP activity and decreases the ability of the MV to subsequently calcify collagen in MVs isolated from calcified BVSMCs. Data are shown as mean ± SD from four separate experiments. *p < 0.01, calcified vs. control; same treatment; ^# p < 0.001, K201 vs. no K201, control or calcified.

FIG. 6

Characterization of secreted (media) vs. cellular (collagenase-released) MVs in BVSMCs during calcification. BVSMCs were incubated in DMEM in calcification media (with 10 mM β-glycerophosphate) or control media (no β-glycerophosphate) for 7 days. Secreted MVs were isolated from media in cultured cells by ultracentrifugation, and cellular MVs were isolated from collagenase digestion of these cultured cells from the same experiments. (A) Secreted and cellular MV ALP activity was determined and normalized by total MV proteins. (B) Annexin II and (C) fetuin-A content in secreted and cellular MVs was determined by Western blot and quantified. The results show that there was an significant increase in ALP activity and annexin II content but a decrease in fetuin-A content in cellular MVs compared with secreted MVs. Data are shown as mean ± SD from four separate experiments. Also shown are representative Western blot from the experiments. *p < 0.005, calcified vs. control, secreted, or cellular MVs; ^# p < 0.001, cellular MVs vs. secreted MVs, control, or calcified.

FIG. 7

Calcification of secreted vs. cellular MVs in M-collagen calcification assay. BVSMCs were incubated in DMEM in calcification media (with 10 mM β-glycerophosphate) or control media (no β-glycerophosphate) for 7 days. Secreted MVs were isolated from media in cultured cells by ultracentrifugation and cellular MVs were isolated from collagenase digestion of these cultured cells followed by centrifugation from the same experiments. MVs (10 μg) were added to type I collagen-coated coverslips in the presence of calcification media (DMEM with 15% FBS and 10 mM β-glycerophosphate) and incubated for 3 days. The calcium content was determined by HCL extraction. The results show that cellular MVs isolated from calcified BVSMCs significantly increases calcification of collagen but secreted MVs do not calcify. Data are shown as mean ± SD from four separate experiments. *p < 0.0001, calcified vs. control, cellular, or secreted; ^# p < 0.0001, cellular vs. secreted MVs, control, or calcified.