Journal: Stem Cell Research & Therapy

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

doi: 10.1186/s13287-018-0894-1

Figure Lengend Snippet: 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

Article Snippet: Beta-glycerophosphate (β-GP, G9422) and Alizarin Red S (A5533) were obtained from Sigma.

Techniques: Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Modification, Derivative Assay

Journal: Stem Cell Research & Therapy

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

doi: 10.1186/s13287-018-0894-1

Figure Lengend Snippet: 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

Article Snippet: Beta-glycerophosphate (β-GP, G9422) and Alizarin Red S (A5533) were obtained from Sigma.

Techniques: Incubation, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Modification

Journal: Stem Cell Research & Therapy

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

doi: 10.1186/s13287-018-0894-1

Figure Lengend Snippet: 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

Article Snippet: Beta-glycerophosphate (β-GP, G9422) and Alizarin Red S (A5533) were obtained from Sigma.

Techniques: Cell Culture, Staining, Standard Deviation

Journal: Stem Cell Research & Therapy

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

doi: 10.1186/s13287-018-0894-1

Figure Lengend Snippet: 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

Article Snippet: Beta-glycerophosphate (β-GP, G9422) and Alizarin Red S (A5533) were obtained from Sigma.

Techniques: Incubation, Staining, Modification, Derivative Assay

Journal: Stem Cell Research & Therapy

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

doi: 10.1186/s13287-018-0894-1

Figure Lengend Snippet: 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

Article Snippet: Beta-glycerophosphate (β-GP, G9422) and Alizarin Red S (A5533) were obtained from Sigma.

Techniques: Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Modification, Derivative Assay

Journal: Stem Cell Research & Therapy

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

doi: 10.1186/s13287-018-0894-1

Figure Lengend Snippet: 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

Article Snippet: Beta-glycerophosphate (β-GP, G9422) and Alizarin Red S (A5533) were obtained from Sigma.

Techniques: ALP Assay, Activity Assay, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Modification, Derivative Assay

Journal: Scientific Reports

Article Title: Magnesium prevents vascular calcification in vitro by inhibition of hydroxyapatite crystal formation

doi: 10.1038/s41598-018-20241-3

Figure Lengend Snippet: Crystal or particle clusters formed in BGP cell culture supernatant are morphologically and chemically distinct. Morphology of crystal clusters formed in BGP and in BGP supplemented with Mg 2+ cell culture supernatants, as overview (upper) and focused (lower) for EDX analysis ( a ). The averaged EDX-spectrum and resulting quantification of the crystal cluster isolated from Mg 2+ supplemented culture supernatant reveals reduced Ca (green) and P (purple), as visualized in the elemental map ( c ) compared to crystal clusters found in BGP treated cultures ( b ). In addition, Mg 2+ supplemented crystal clusters showed increased Na (blue) and Cl (orange) compared to BGP crystal clusters. Data are presented as mean of the analyses of ten individual crystal clusters. BGP, β -glycerophosphate; CPS, counts per second; EDX, energy dispersive spectroscopy; (k)eV, (kilo)-electron volt.

Article Snippet: Experimental design For calcification, medium was supplemented with 5% (v/v) FBS and 10 mM β -glycerophosphate (BGP, Merck Millipore, Massachusetts, USA).

Techniques: Cell Culture, Isolation, Spectroscopy

Journal: Scientific Reports

Article Title: Magnesium prevents vascular calcification in vitro by inhibition of hydroxyapatite crystal formation

doi: 10.1038/s41598-018-20241-3

Figure Lengend Snippet: Impact of Mg 2+ on BGP-induced Ca 2+ -apatite formation. X-ray powder diffraction analysis of cell culture supernatants show presence of NaCl (*) and Ca-apatite (arrows) in BGP treated cultures (black line), but not in BGP cultures supplemented with Mg 2+ (gray line). Of note: as the BGP culture supplemented with Mg 2+ contained less precipitates; one measurement represents 21 cultures (6-well format) compared to 7 in the BGP condition in order to reach the detection threshold. BGP, β -glycerophosphate.

Article Snippet: Experimental design For calcification, medium was supplemented with 5% (v/v) FBS and 10 mM β -glycerophosphate (BGP, Merck Millipore, Massachusetts, USA).

Techniques: Cell Culture