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Morphogenesis of Vascular Smooth Muscle in Atherosclerosis and Cell Culture

Russell Ross, Beverly Kariya

10.1002/cphy.cp020203

Source: Supplement 7: Handbook of Physiology, The Cardiovascular System, Vascular Smooth Muscle

Originally published: 1980

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Smooth Muscle Cells
1.1 Morphology and Growth Characteristics
1.2 Problems With Studies of Diploid Cells—Variability in Response
1.3 Senescence in Culture
1.4 Growth Requirements in Culture
1.5 Platelet Factors and Growth Control
1.6 Connective Tissue Formation by Smooth Muscle Cells in Culture
2 Endothelium
3 Fibroblasts
4 Summary
5 Appendix
5.1 Surgical Procedures for Dissection of Thoracic Aorta
5.2 Preparation of Primary Cultures and Maintenance of Secondary Cultures
5.3 Preparation of Pooled Monkey Serum for Stock Cultures and Dose‐Response Curves
5.4 Preparation of Monkey Blood Serum and Monkey Plasma Derived Plasma Serum for Growth Curves
5.5 Growth Curves
Figure 1. Figure 1.

Electron micrograph of a typical en face view of an aortic smooth muscle cell from the primate Macaca nemestrina in culture. Most of the cytoplasm of this cell is abundant in thin and intermediate filaments with relatively few areas of the cytoplasm lacking filaments, a characteristic of quiescent smooth muscle cells in culture. In several regions (arrows) there are strings of vesicles representing surface vesicles (caveolae). × 6,700.
Figure 2. Figure 2.

A: light micrograph showing the characteristic appearance of cultured confluent aortic smooth muscle cells demonstrating the characteristic hills (arrow) and valleys that smooth muscle cells form and their more irregular shape. × 370. B: light micrograph showing confluent dermal fibroblasts, both from Macaca nemestrina, with their characteristic spindle shape and concentric pattern of growth. × 370.
Figure 3. Figure 3.

Electron micrograph of a transverse section through part of one of the hills that is characteristic of arterial smooth muscle cells in culture. Between the layers of the smooth muscle cells are several extracellular structures, most of which are microfibrils (110–Å diam). Myofilaments sectioned both transversely and longitudinally are also apparent in these cells. × 60,000.
Figure 4. Figure 4.

Electron micrograph of a transverse section of two primate aortic smooth muscle cells at the site of a gap junction between the two cells. There is a distance of approximately 20 Å between the outer leaflets of the two unit membranes of the cells at this junctional site. Cytoplasm of each cell contains numerous transverse sections of myofilaments of two sizes, approximately 50‐Å and 100‐Å diam. Cells are bordered by a filamentous extracellular material reminiscent of the appearance of basement membrane. × 108,000.
Figure 5. Figure 5.

A: graph representing growth curves of primate thoracic aorta smooth muscle cells (M42 Smooth Muscle). B: graph representing growth curves of primate dermal fibroblasts (M42 Skin Fibroblasts). Both smooth muscle cells and dermal fibroblasts were from the same donor in the same transfer (T4) generation under identical culture conditions. Both cell types responded to whole‐blood serum (either human or monkey) and to human plasma serum to which platelet factor had been readded. The smooth muscle cells, however, grew logarithmically for 9 days or longer after exposure to whole‐blood serum or to platelet factors, whereas the dermal fibroblasts grew logarithmically for only 2–3 days before becoming quiescent under the same conditions.

From Rutherford and Ross 105
Figure 6. Figure 6.

Eight growth curves representing the response of aortic smooth muscle cells in the second passage (T2) from eight different age‐ and sex‐matched Macaca nemestrina to the same pool of homologous serum. Monkey no. 52 (M52) grew as well in 1% serum as did monkey no. 50 (M50) in 5% serum. The cells from monkey no. 52 grew equally well in 2.5–20% serum. These eight growth curves demonstrate the marked variability from donor animal to donor animal.
Figure 7. Figure 7.

Three growth curves demonstrating the response of aortic smooth muscle cells from the same donor during senescence in culture. At the third passage (T3) there is a linear dose response to increasing concentrations of serum. At T5 5%, 10%, and 20% serum approach each other in terms of the final density reached by the cells. At T8 the cells responded poorly to increasing concentrations of serum, and the differences between 0.5% and 10% are small. At T8 the cells in 20% serum reached a final density equivalent to that reached in 2.5% serum at T3 and T5; this behavior demonstrated a marked decrease in the ability to respond to increasing concentrations of the serum with increasing age in culture.


Figure 1.

Electron micrograph of a typical en face view of an aortic smooth muscle cell from the primate Macaca nemestrina in culture. Most of the cytoplasm of this cell is abundant in thin and intermediate filaments with relatively few areas of the cytoplasm lacking filaments, a characteristic of quiescent smooth muscle cells in culture. In several regions (arrows) there are strings of vesicles representing surface vesicles (caveolae). × 6,700.



Figure 2.

A: light micrograph showing the characteristic appearance of cultured confluent aortic smooth muscle cells demonstrating the characteristic hills (arrow) and valleys that smooth muscle cells form and their more irregular shape. × 370. B: light micrograph showing confluent dermal fibroblasts, both from Macaca nemestrina, with their characteristic spindle shape and concentric pattern of growth. × 370.



Figure 3.

Electron micrograph of a transverse section through part of one of the hills that is characteristic of arterial smooth muscle cells in culture. Between the layers of the smooth muscle cells are several extracellular structures, most of which are microfibrils (110–Å diam). Myofilaments sectioned both transversely and longitudinally are also apparent in these cells. × 60,000.



Figure 4.

Electron micrograph of a transverse section of two primate aortic smooth muscle cells at the site of a gap junction between the two cells. There is a distance of approximately 20 Å between the outer leaflets of the two unit membranes of the cells at this junctional site. Cytoplasm of each cell contains numerous transverse sections of myofilaments of two sizes, approximately 50‐Å and 100‐Å diam. Cells are bordered by a filamentous extracellular material reminiscent of the appearance of basement membrane. × 108,000.



Figure 5.

A: graph representing growth curves of primate thoracic aorta smooth muscle cells (M42 Smooth Muscle). B: graph representing growth curves of primate dermal fibroblasts (M42 Skin Fibroblasts). Both smooth muscle cells and dermal fibroblasts were from the same donor in the same transfer (T4) generation under identical culture conditions. Both cell types responded to whole‐blood serum (either human or monkey) and to human plasma serum to which platelet factor had been readded. The smooth muscle cells, however, grew logarithmically for 9 days or longer after exposure to whole‐blood serum or to platelet factors, whereas the dermal fibroblasts grew logarithmically for only 2–3 days before becoming quiescent under the same conditions.

From Rutherford and Ross 105


Figure 6.

Eight growth curves representing the response of aortic smooth muscle cells in the second passage (T2) from eight different age‐ and sex‐matched Macaca nemestrina to the same pool of homologous serum. Monkey no. 52 (M52) grew as well in 1% serum as did monkey no. 50 (M50) in 5% serum. The cells from monkey no. 52 grew equally well in 2.5–20% serum. These eight growth curves demonstrate the marked variability from donor animal to donor animal.



Figure 7.

Three growth curves demonstrating the response of aortic smooth muscle cells from the same donor during senescence in culture. At the third passage (T3) there is a linear dose response to increasing concentrations of serum. At T5 5%, 10%, and 20% serum approach each other in terms of the final density reached by the cells. At T8 the cells responded poorly to increasing concentrations of serum, and the differences between 0.5% and 10% are small. At T8 the cells in 20% serum reached a final density equivalent to that reached in 2.5% serum at T3 and T5; this behavior demonstrated a marked decrease in the ability to respond to increasing concentrations of the serum with increasing age in culture.

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Article Title: Morphogenesis of Vascular Smooth Muscle in Atherosclerosis and Cell Culture
 

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Russell Ross, Beverly Kariya. Morphogenesis of Vascular Smooth Muscle in Atherosclerosis and Cell Culture. Compr Physiol 2011, Supplement 7: Handbook of Physiology, The Cardiovascular System, Vascular Smooth Muscle: 69-91. First published in print 1980. doi: 10.1002/cphy.cp020203