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Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.

ABSTRACT: BACKGROUND:In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo. METHODS AND RESULTS:To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold-polyvmer-iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3-4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10nM lysophosphatidylcholine (LPC), for 3days, cell clusters exhibited translucent extensions/rods 60-80?m wide and 200-250?m long. When 0.5?g/?l Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590-650nm light, these extensions emitted intrinsic fluorescence at >667nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518nm, the ?max for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminoglycans than cells treated with LPC and Schnurri-3. CONCLUSIONS:In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3. GENERAL SIGNIFICANCE:The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.

SUBMITTER: Castro-Chavez F

PROVIDER: S-EPMC4383529 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.

Castro-Chavez Fernando F Vickers Kasey C KC Lee Jae Sam JS Tung Ching-Hsuan CH Morrisett Joel D JD

Biochimica et biophysica acta 20130314 6

<h4>Background</h4>In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for represent ...[more]

PMID: 23500015

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Project description:Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFR? markers. Sca-1(+) (Sca-1(+)/PDGFR?(+) and Sca-1(+)/PDGFR?(-)) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1(-) (Sca-1(-)/PDGFR?(+) and Sca-1(-)/PDGFR?(-)) progenitor cells. Among Sca-1(+) progenitor populations, Sca-1(+)/PDGFR?(-) cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1(+)/PDGFR?(+) cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor ? (PPAR?) agonist, Sca-1(+)/PDGFR?(-) cells preferentially differentiated into osteoclast-like cells. Sca-1(+) progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1(+) calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1(+) progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1(+)/PDGFR?(-) cells treated with a PPAR? agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1(+)/PDGFR?(-) cells into Apoe(-/-) mice increased the severity of calcified atherosclerotic plaques. However, Sca-1(+)/PDGFR?(-) cells in which PPAR? was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1(+)/PDGFR?(-) cells mainly expressed osteocalcin; however, activation of PPAR? triggered receptor activator for nuclear factor-?B (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPAR? activation may be an option to reverse calcification.

Dataset Information

Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.

Publications

Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.

Similar Datasets

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