Geometric Control of Cell Life and Death

Presenter's Summary

Most cells in tissues are attached to each other and to the protein network of the extracellular matrix (ECM). Endothelial cells require this attachment for their healthy growth and proliferation. When the endothelial cell-ECM attachment is disrupted, this leads to programmed cell death, or apoptosis. The apoptosis which results from the lack of cell anchorage to the ECM is termed "anoikis".

Cell attachment to the ECM is mediated by cell surface receptors. The most important ones belong to the integrin family. In cultured cells integrin receptors are localized in specialized structures of the cell membrane called "focal adhesions". In addition to ECM-attached integrin receptors, focal adhesions contain a number of cytoskeletal components and various signalling proteins. Cell adhesion is regulated by the integrin receptor-mediated signalling which includes intracellular pathways activated by growth factor receptors,(i.e. protein kinase C, phosphatidyl-inositol 3'-kinase, and the Ras-Raf-mitogen activated kinase pathway) and the focal adhesion kinase (FAK) pathway.

The adhesion-dependent control of apoptosis is thought to be mediated by the integrin receptors. This notion is supported by the following data:

1. The addition of soluble antagonists to integrin alpha v beta 3 in cultured cells induces apoptosis.
2. This process can be prevented by allowing the cells to grow on immobilized antibodies to integrin beta 1.

In the present study Chen et al., set out to determine if it is true that cell shape can control apopoptosis.

They grew human and bovine capillary endothelial cells on ECM-coated micro beads of a range of different sizes, or on ECM-coated adhesive islands, of varying size. The size of these islands was comparable to focal adhesions 3 micro m or 5 micro m, and the distance between them could be varied. This arrangement allowed for a constant area of ECM -cell contact, while varying the extent of cell spreading. Cellular proliferation and apoptosis was determined under the varying growth conditions. (DNA synthesis as the percentage of 5-bromo-deoxyuridine labeled nuclei, and apoptotic index as the percentage of cells exhibiting TUNEL staining).

The key findings can be summarized as follows:

• Capillary endothelial cells cultured in suspension, in the absence of any beads remained small and spherical. After 24 hrs. 60% of the cells were positive for apoptosis.
• When these cells were cultured in suspension, in the presence of fibronectin (FN)-coated beads (25 micron diameter), they adhered and became flattened. After 24 hrs. fewer than 10 % of these cells entered the cell death programme.
  These results confirmed the cell-attachement to the ECM, mediated by the integrin receptors is important for cell survival.
• When these cells were grown on 10-micro m diameter beads, the cells became more rounded, the apoptotic index increased, and was similar to that of the suspended cells.
  These findings confirmed that the extent of cell spreading played a role in cell death or survival.
• When cells were grown on different sized, square shaped FN-coated islands, apoptosis declined, and DNA synthesis increased with increasing island size from 75-3000 micro m squared.
  These results confirmed that increased cell spreading on a homogeneous FN-coated surface lead to cell growth.
• Since in these experiments there was also a concomitant increase in the ECM-cell surface area, the next set of studies were designed to keep the total ECM-cell contact area constant and vary the extent of cell spreading. This was accomplished by growing cells on a microfabricated surface of ECM-coated circular islands on an otherwise nonadhesive surface. The spreading of the cells could be changed by varying the size and the spacing of the small dots while the total area of cell-to ECM attachment was kept constant. Under these conditions growth conditions DNA synthesis increased and apoptosis decreased with increase in cell spreading.
• Using substrates coated with specific antibodies to various integrins, the anti apoptotic effect of spreading was greater with selected integrins (i.e. with alpha v beta3).

Base on these results the authors' concluded that cell shape is regulated by the ECM and cell shape in turn "governs whether individual cells grow or die."

Why is this paper important?

The importance of cell adhesion to endothelial cell survival has been shown before. It has also been demonstrated that loss of cell anchorage results in apoptosis.

What is novel in the paper of Chen et al., is that they demonstrate for the first time that cell geometry has an effect on cell growth and survival. If this process can also be demonstrated in vivo, than local control of cell growth and death may represent a "fundamental mechanism" for regulating normal tissue development.

As suggested by the commentary of Erkki Ruoslahti (Science, 1997, 276:1345-1346)

"A highly speculative, but tantalizing, possibility raised by these and earlier results from the same laboratory is that cell shape might directly control gene regulation".

Abstract of the authors

Human and bovine endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue environment.

Additional references

• A.F. Horwitz: Integrins and Health. Scientific American, 1997, 276: 68-75.
• E.A. Clark and J.S. Brugge: Integrins and Signal Transduction Pathways: The Road Taken. Science, 1995, 268:233-239.
• F.W. Luscinskas and J. Lawler: Integrins as Dynamic Regulators of Vascular Function. FASEB J., 1994, 8:929-938.
• M.A. Swarttz, M.D. Schaller and M.H. Ginsberg: Integrins: Emerging Paradigms of Signal Transduction. Ann.Rev.Cell & Developmental Biol., 1995, 11:549-599.