Conditioned medium from bone marrow-derived mesenchymal stem cells inhibits vascular calcification through blockade of the BMP2-Smad1/5/8 signaling pathway

Abstract

Background: Arterial calcification is associated with cardiovascular disease as a complication of advanced atherosclerosis and is a significant contributor to cardiovascular morbidity and mortality. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important role in arterial calcification and is characterized by cellular necrosis, inflammation, and lipoprotein and phospholipid complexes, especially in atherosclerotic calcification. The conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) is well known as a rich source of autologous cytokines and is universally used for tissue regeneration in current clinical medicine. Here, we demonstrate that MSC-CM inhibits beta-glycerophosphate (β-GP)-induced vascular calcification through blockade of the bone morphogenetic protein-2 (BMP2)-Smad1/5/8 signaling pathway.

Methods: VSMC calcification was induced by β-GP followed by treatment with MSC-CM. Mineral deposition was assessed by Alizarin Red S staining. Intracellular calcium content was determined colorimetrically by the o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured by the para-nitrophenyl phosphate method. Expression of BMP2, BMPR1A, BMPR1B, BMPR2, msh homeobox 2 (Msx2), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC), representative osteoblastic markers, was assessed using real-time polymerase chain reaction analysis while the protein expression of BMP2, Runx2, and phosphorylated Smad1/5/8 was detected by western blot analysis.

Results: Our data demonstrated that MSC-CM inhibits osteoblastic differentiation and mineralization of VSMCs as evidenced by decreased calcium content, ALP activity, and decreased expression of BMP-2, Runx2, Msx2, and OC. MSC-CM suppressed the expression of phosphorylated Smad1/5/8 and the β-GP-induced translocation from the cytoplasm to the nucleus. Further study demonstrated that human recombinant BMP-2 overcame the suppression of VSMC calcification by MSC-CM.

Conclusion: MSC-CM may act as a novel therapy for VSMC calcification by mediating the BMP2-Smad1/5/8 signaling pathway.

Keywords: Atherosclerosis; BMP2–Smad1/5/8 signaling; Conditioned medium from bone marrow-derived mesenchymal stem cells; Vascular calcification.

Fig. 1

β-GP induced calcium deposition in VSMCs in a dose-dependent manner. VSMCs cultured in normal medium or in calcification medium with 10, 20 and 40 mmol/l of β-GP for 14 days. a Alizarin Red staining of microscopic views (×100). b Formic acid 10% used to elute Alizarin Red dye and quantification of Alizarin Red staining measured with a microplate reader and normalized to total protein content. c Calcium content shown as mean ± standard deviation of three independent experiments conducted in duplicate. β-GP beta-glycerophosphate, OD optical density

Fig. 2

MSC-CM inhibits β-GP-induced calcium deposition in VSMCs. Confluent cells incubated with normal medium or calcification medium with or without MSC-CM for 14 days. Calcification induced by addition of β-GP (10 mM). a Representative Alizarin Red staining of microscopic views (×100, upper) and plates (lower). b Quantification of Alizarin Red staining. c Calcium contents measured and normalized by protein content of cell lysates. β-GP beta-glycerophosphate, DMEM Dulbecco’s Modified Eagle’s Medium, DMEM^β-GP DMEM medium containing 10 mM β-GP, MSC-CM conditioned medium from bone marrow-derived mesenchymal stem cells, MSC-CM^β-GP MSC-CM with 10 mM β-GP, NC DMEM medium, OD optical density

Fig. 3

MSC-CM abolished β-GP-induced ALP activity and expression of specific osteogenic markers in VSMCs. Confluent VSMCs incubated with different media in different groups. a ALP activity measured after 3 days by ALP kit, normalized to cellular protein contents. Levels of Msx2 (b), Runx2 (c) and Osteocalcin (d) mRNA determined after 7 days by rt-PCR. e Representative immunoblot of Runx2. f Runx2 protein quantified by densitometry. β-GP beta-glycerophosphate, DMEM Dulbecco’s Modified Eagle’s Medium, DMEM^β-GP DMEM medium containing 10 mM β-GP, MSC-CM conditioned medium from bone marrow-derived mesenchymal stem cells, MSC-CM^β-GP MSC-CM with 10 mM β-GP, Msx2 msh homeobox 2, NC DMEM medium, Runx2 Runt-related transcription factor 2

Fig. 4

MSC-CM blocks BMP2–Smad1/5/8 signaling pathways. Confluent VSMCs incubated with different media in different groups for 7 days. a Effect of MSC-CM on BMP-2 mRNA expression. b Representative immunoblot of BMP2. c BMP2 protein quantified by densitometry. Levels of (d) BMPR1a, (e) BMPR1b, and (f) BMPR2 mRNA determined by rt-PCR. g Phosphorylation of Smad1/5/8 in whole cells. h Phosphorylation of Smad1/5/8 in nucleus and cytoplasm. β-GP beta-glycerophosphate, BMP-2 bone morphogenetic protein-2, DMEM Dulbecco’s Modified Eagle’s Medium, DMEM^β-GP DMEM medium containing 10 mM β-GP, MSC-CM conditioned medium from bone marrow-derived mesenchymal stem cells, MSC-CM^β-GP MSC-CM with 10 mM β-GP, NC DMEM medium

Fig. 5

BMP2 overcomes inhibited effect of MSC-CM on β-GP-induced VSMC calcification. a Microscopic views (×100) of Alizarin Red staining. b Quantification of Alizarin Red staining. c Calcium contents measured and normalized by protein content of cell lysates. Expression of Msx2 (d), Runx2 (e), and osteocalcin (f) mRNA assessed by rt-PCR. g Cell lysates analyzed by western blot analysis with anti-Runx2 and p-Smad1/5/8. β-GP beta-glycerophosphate, DMEM Dulbecco’s Modified Eagle’s Medium, DMEM^β-GP DMEM medium containing 10 mM β-GP, MSC-CM conditioned medium from bone marrow-derived mesenchymal stem cells, MSC-CM^β-GP MSC-CM with 10 mM β-GP, Msx2 msh homeobox 2, NC DMEM medium, OD optical density, Runx2 Runt-related transcription factor 2

Fig. 6

Noggin, BMP2 antagonist, suppresses VSMC calcification by inhibiting Smad1/5/8 signaling. VSMCs pretreated with Noggin (100 ng/ml) or vehicle for 2 h followed by incubation with 10 mM β-GP. a Alizarin Red staining (×100). b Quantification of Alizarin Red staining. c Calcium contents measured and normalized by protein content of cell lysates. mRNA expression of (d) BMP2, (e) Msx2, (f) Runx2, and (g) osteocalcin analyzed by rt-PCR. h Protein expression of BMP2 and phospho-Smad1/5/8 performed by western blot analysis. β-GP beta-glycerophosphate, DMEM Dulbecco’s Modified Eagle’s Medium, DMEM^β-GP DMEM medium containing 10 mM β-GP, DMEM^Noggin+β-GP DMEM medium containing 10 mM β-GP and 100 ng/ml Noggin, Msx2 msh homeobox 2, NC DMEM medium, OD optical density, Runx2 Runt-related transcription factor 2