Smooth muscle cell-specific matrix metalloproteinase 3 deletion reduces osteogenic transformation and medial artery calcification

Abstract

Aims: Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification.

Methods and results: We found that MMP-3 was increased in rodent models of medial calcification as well as in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D3, we found that MMP-3 deficiency significantly suppressed medial calcification in the aorta. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is an important factor in this process.

Conclusion: These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

Keywords: Calcification; MMP-3; Osteogenic transformation; Smooth muscle cells.

Graphical Abstract

Figure 1

MMP-3 is increased under calcifying conditions in vitro and in vivo. (A) MMP-3 was the most highly induced MMP in a model of medial calcification induced by high doses of vitamin D₃ (Vit D₃). 10-week-old male C57BL/6J mice were SC injected with 5 × 10⁵IU/kg for three consecutive days, and the aortas were harvested after 7 days. n = 3 mice. (B and C). Western blotting data showing increased MMP-3 expression in the aortas of VitD₃-injected mice. n = 3 mice. (D and E). The expression of MMP-3 was increased in the calcified arteries of VitD₃-injected rats. Male SD rats were subjected to SC injection with 3 × 10⁵IU/kg Vit D₃ for three consecutive days, and the aortas were harvested after 11 days. (D) Immunohistochemistry staining of MMP-3. Von Kossa staining shows the deposition of calcium phosphate. Scale bar, 100 μm. E, qPCR results. n = 3 rats. (F) Alizarin Red S staining for calcium deposits showed that calcification occurred in calcifying SMCs in vitro. Confluent rat aortic SMCs were cultured in a calcification medium containing 3.5 mM phosphate (Pi) and 3 mM calcium (Ca) for 7 days. (G and H) MMP-3 mRNA and protein levels were increased in response to Pi/Ca in rat aortic SMCs. n = 3 independent experiments. (I) MMP-3 activity was increased in response to Pi/Ca in rat aortic SMCs. The activity of MMP-3 was examined using an MMP-3 Activity Assay Kit. n = 3 independent experiments. (J–L) MMP-3 mRNA and protein levels were also increased in calcifying human aortic SMCs. Confluent human aortic SMCs were cultured in a calcification medium containing 3.2 mM Pi for 6 days. qPCR and western blotting results were normalized using GAPDH. n = 3 independent experiments. Data were analyzed by t-test. Data were analyzed by t-test. Values are mean ± SD. *P < 0.05, P < 0.01, ***P < 0.001.

Figure 2

MMP-3 is highly expressed in calcified arteries from patients with arterial disease. Human tibial arteries were fixed and decalcified, and paraffin sections were prepared. (A) Immunohistochemistry staining showed that MMP-3 and osteogenic markers RUNX2 and BMP2 were increased but SM-α-actin was decreased in calcified arteries. Scale bar, 200 μm. (B–E) The quantitative data. Data were analyzed by t-test. Values are mean ± standard error (SE). n = 4 specimens. *P < 0.05, P < 0.01, ***P < 0.001. L, lumen. Outlined area: calcified area.

Figure 3

MMP-3 mediates SMC calcification and osteogenic transformation. (A) Knockdown of MMP-3 inhibited calcification in rat aortic SMCs. Rat aortic SMCs were transfected with 100 nM control siRNA or MMP-3 siRNA for 2 days and then cultured in a calcification medium containing 3.5 mM Pi/3 mM Ca for 7 days. Calcium content was examined by calcium assay. (B) The small-molecule MMP-3 inhibitor dose-dependently suppressed Pi/Ca-induced SMC calcification. Confluent rat aortic SMCs were treated with various concentrations of MMP-3 inhibitor and then cultured in 3.5 mM Pi/3 mM Ca calcification medium for 7 days. The culture media and inhibitor were replaced every other day. (C) Activated MMP-3 promoted Pi/Ca-mediated SMC calcification. Confluent rat aortic SMCs were pre-treated with indicated doses of human recombinant MMP-3 protein and then cultured in 3.5 mM Pi/3 mM Ca calcification medium for 7 days. (D–G) Inhibition of MMP-3 suppressed SMC osteogenic transformation in rat aortic SMCs. The osteogenic markers Osterix (Sp7) and ALP (TNSALP) and SMC markers SM-MHC (MYH11) and SM22α (TAGLN) were determined by qPCR and normalized with GAPDH. (H) Knockdown of MMP-3 significantly inhibited calcification in human aortic SMCs. Human aortic SMCs were transfected with 100 nM control siRNA or MMP-3 siRNA for 2 days and then cultured in a calcification medium containing 3.2 mM Pi for 6 days. (I) MMP-3 inhibitor dose-dependently suppressed Pi-induced calcification in human aortic SMCs. (J–L) MMP-3 inhibitor suppressed osteogenic transformation in human aortic SMCs. Confluent human aortic SMCs were pre-treated with MMP-3 inhibitor and then cultured in 3.02 mM Pi calcification medium for 6 days. (M) Deletion of MMP-3 blocked Pi-mediated mouse aortic calcification. Mouse aortic SMCs isolated from MMP-3-WT and MMP-3-KO mice were cultured in a calcification medium containing 3.5 mM Pi for 12 days. The culture media and inhibitor were replaced every other day. Data were analyzed by one-way ANOVA or two-way ANOVA with multiple comparisons. One-way ANOVA analysis was corrected with a post hoc test. Values are mean ± SD. n = 3–4 independent experiments *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Global deficiency of MMP-3 reduces medial calcification ex vivo and in vivo. (A) Deletion of MMP-3 inhibited aortic calcification ex vivo. Aortas were isolated from MMP-3-WT and MMP-3-KO mice and then cut into aortic rings, followed by incubation in a calcification culture medium containing 2.6 mM Pi for 12 days. The calcification medium was changed every 2–3 days. n = 3–6 mice. (B and C). Global deletion of MMP-3 reduced medial calcification in vivo. MMP-3-WT and MMP-3-KO mice were subjected to the injection with 5 × 10⁵IU/kg VitD₃ for 3 consecutive days. 7 days later, the aortas were harvested. (B) calcium assay results. (C) Von Kossa staining results. Vehicle, n = 5; VitD₃, n = 15. Scale bar, 100 μm. (D–H) Deletion of MMP-3 inhibited osteogenic transformation in vivo. Immunohistochemistry staining and qPCR were performed for osteogenic markers RUNX2 (RUNX2) and BMP2 (BMP2), and SMC markers SMMHC (MYH11) and SM22α (TAGLN). The results were normalized with GAPDH. n = 3 mice. Scale bar, 100 μm. Data were analyzed by two-way ANOVA with multiple comparisons. Values are mean ± SE. **P < 0.01, ***P < 0.001.

Figure 5

SMC-specific MMP-3 knockout mouse generation. (A) MMP-3 conditional knockout targeting strategy. The exon 2-4 of MMP-3 gene were selected as the knockout regions and the targeting vector was synthesized. CRISPR/Cas9 technology was used for MMP-3 conditional knockout mouse production. (B) The data from agarose gel electrophoresis showed that four MMP-3 floxed F1 mice including two males and two females were identified using the specific primers. (C and E) Validation of MMP-3 floxed mice. The results from qPCR and Western blotting showed that the adenovirus coding Cre robustly decreased MMP-3 expression compared with LacZ controls in SMCs. Mouse aortic SMCs were isolated from MMP-3 floxed mice using the enzymatic method. Three to five passage SMCs were used for this study. The cultured SMCs were infected with 100 multiplicity of infection adenovirus coding LacZ (Adeno-LacZ) or Cre (Adeno-Cre) for 4 days. (C) qPCR results; (D) Representative Western blotting images; (E) Quantitative data for Western blotting. n = 3 independent experiments. (F) SMC-specific MMP-3-KO mouse was generated. MMP-3 floxed mice were first bred with SMC-specific Cre mice SMMHC-CreER^T2 to produce SMMHC^CreERT2/MMP-3 flox^+/+ mice. SMMHC^CreERT2/MMP3 flox^+/+ mice were then injected with oil or tamoxifen (TMX, 75 mg/kg, IP) for five consecutive days, followed by 10 days of rest, to produce wild-type and SMC-specific MMP-3-KO mice. To confirm that MMP-3 was exclusively expressed in SMCs, the aortas were dissected from TMX or Oil-injected SMMHC^CreERT2/MMP3 flox^+/+ mice and then digested with collagenase to remove the adventitial layers. The endothelial cells were also scraped using tiny cotton swabs. The remained medial layers mainly containing SMCs are used for qPCR. qPCR results showed that MMP-3 expression in the medial layers of TMX-injected mice was extremely low compared with Oil-injected mice. n = 4 mice. (G) Immunofluorescence staining showed that MMP-3 expression was largely decreased in the medial layer of the carotid arteries of SMMHC^CreERT2/MMP3 flox^+/+ mice after TMX injection compared with Oil injection. The expression of MMP-3 and SMC marker SM-a-actin were examined by immunofluorescence staining. Scale bar, 40 μm. Data were analyzed by t-test. Values are mean ± SD. **P < 0.01, ***P < 0.001. L, lumen; M, media. A, adventitial.

Figure 6

SMC-specific MMP-3 deletion reduces osteogenic transformation and medial calcification. 7-week-old male SMMHC^CreERT2/MMP-3 flox^+/+ mice were IP injected with 75 mg/kg tamoxifen (TMX) or Oil for five consecutive days and waited for 10 days of rest, followed by subcutaneous injection with 5 × 10⁵IU/kg VitD₃ or vehicle for 3 consecutive days. Aortas were collected after 7 days. (A) Calcium assay showed that deficiency of SMC-specific MMP-3 suppressed VitD₃-induced medial calcification in vivo. (B) Quantitative data from Von Kossa staining showed that SMC-specific MMP-3 deficiency decreased calcium deposits in the abdominal aortas. (C) Representative images of HE, Von Kossa, and VVG staining. Scale bar, 100 μm. (D and E) Deficiency of SMC-specific MMP-3 blocked VitD₃-mediated elastin degradation. The integrity of elastin was examined by VVG staining. The elastin area and elastin break were analyzed. (F) Deletion of SMC-specific MMP-3 inhibited osteogenic transformation in vivo. Representative immunohistochemistry staining images for osteogenic markers RUNX2 and BMP2, and SMC marker SMMHC. Scale bar, 100 μm. (G–J) Quantitative results. Data were analyzed by one-way ANOVA or two-way ANOVA with multiple comparisons. One-way ANOVA analysis was corrected with a post hoc test. Values are mean ± SE. Vehicle, n = 6; VitD₃, n = 16–22. **P < 0.01, ***P < 0.001. L, lumen.