Effect of Lyso-phosphatidylcholine and Schnurri-3 on Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells to Calcifying Vascular Cells in 3D Culture

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• 作者：Fernando Castro-Chavez^a ; Kasey C. Vickers^a ; Jae Sam Lee^b ; Ching-Hsuan Tung^b ; Joel D. Morrisett^a ; ^{morriset@bcm.edu}
• 关键词：CHA ; calcium hydroxyapatite ; LPC ; lysophosphatidylcholine ; VSMC ; vascular smooth muscle cell ; CVC ; calcifying vascular cell ; HABP-19 ; hydroxyapatite binding peptide containing 19 amino acids ; CEA ; carotid endarterectomy ; Ca/P ; calcium/phosphorus ; FBS ; fet
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2013
• 出版时间：June, 2013
• 年：2013
• 卷：1830
• 期：6
• 页码：3828-3834
• 全文大小：1304 K

文摘

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 10 nM lysophosphatidylcholine (LPC), for 3 days, cell clusters exhibited translucent extensions/rods 60-80 ¦Ìm wide and 200-250 ¦Ìm long. When 0.5 ¦Ìg/¦Ìl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590-650 nm light, these extensions emitted intrinsic fluorescence at > 667 nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518 nm, the ¦Ë_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.