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 mono-layers 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 µm wide and 200–250 µm long. When 0.5 µg/µl 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 λmax for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminogly-cans 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.

File Compression and Expansion of the Genetic Code by the Use of the Yin/Yang Directions to Find its Sphered Cube

“…The optimization of the 3-D cell culture methodology for atherosclerosis required more than two years; however, reference [42], not only added the LPC that was successfully demonstrated by us [39], while using my methodologies and materials with no credits, but also ruined our experimental model by adding two extra, non-necessary and nonphysiological reagents: β-glycerophosphate (7.5 mM) and ascorbic acid (50 μg/ml), being these additions reflected in their work by the retention of hydroxyapatite within the VSMCs of their 3-D cluster (seen at the end of their third composite illustration), instead of being normally secreted, as it was shown in all of our Figures [38].…”

“…Our original work [38] demonstrates that a 3-D perspective is highly useful in biomedical research, not only for its 3-D cell cultures, but also for the current study of the genetic code in 3-D as shown here. [45] (also see [44]) Figure 27 shows how both our 384-grid Cube and Sphered Cube representation of the genetic code can be compared to the Pythagorean music series.…”

“…Also, and apart of exploring the 3-D properties of the genetic code, cell cultures in 3-D have been recently used by the author of this article in an attempt to simulate the current conditions of living tissues through the modeling of the origin and attenuation of atherosclerosis [38] in human osteogenic trans-differentiating vascular smooth muscle cells (VSMCs). We saw that Lyso-phosphatidylcholine (LPC) at physiological concentrations (10 nm), instead of the entire Low Density Lipoprotein (LDL) or 'bad cholesterol', seemed to have been the main responsible for the development of calcification in atherosclerosis [39][40][41], being this a defensive response of the arterial muscular cells against the detergentlike effects of the LPC, a mechanism for those cells to protect themselves.…”

Morphometric analysis of calcification and fibrous layer thickness in carotid endarterectomy tissues

“…We have found that the very small calcified particles are almost exclusively round in 2D histology sections (Figure 6C). Earlier studies have suggested that the action of Lipoprotein-associated phospholipase A2 (Lp-PLA2) on oxLDL produce lyso-phosphatidylcholine, an atherogenic agent which can, in turn, induce VSMCs to differentiate into calcifying vascular cells in vitro (9, 10). Our group has shown previously that levels of oxLDL and Lp-PLA2 activities were high in the bifurcation segment of CEA tissues (25), which may give rise to the very small calcified particles observed in this study.…”

“…Over time a subset of VSMCs may transdifferentiate into calcified vascular cells (CVCs), an osteogenic phenotype that contributes to plaque calcification (6–8). In our previous studies, lysophosphatidylcholine, a product of oxidized phosphatidylcholine hydrolysis has been found in carotid endarterectomy (CEA) atheroma and was identified as a candidate for stimulating VSMCs to transdifferentiate into CVCs (9, 10). However, the current understanding of the atherosclerotic calcification process does not account for the observed heterogeneous distribution of intra-plaque calcification (11).…”