Rho GTPases Signal Isoactin Dependent Cytoskeletal Remodeling

More recently, we have recognized the pivotal role that the Rho GTPases and their downstream signaling effectors play in regulating retinal microvascular shape, motility, and contractile potential [12, 13]. As has been well documented, activation of Rho GTPase in other cells has been shown to cause the bundling of actin filaments into stress fibers, along with the clustering of focal adhesion complex proteins. Like all GTPases, Rho GTPases act as molecular switches, which cycle between an inactive GDP-bound and an active GTP-bound state, and the ratio between these two forms depends on the activity of regulatory factors. GTPase-activating proteins (GAPs) promote the inactive state of the GTPase by increasing the GTPase's intrinsic rate of nucleotide hydrolysis, while guanine nucleotide dissociation inhibitors (GDIs) interfere with both the exchange of GDP for GTP and the hydrolysis of bound GTP. Guanine nucleotide exchange factors (GEFs) promote the active GTP-bound state and tether the GTPases to specific subcellular locations in order to generate an active signal. Biological implications of these findings are wide ranging, and Rho GTPases, together with family members, rac and Cdc42, play key regulatory roles in cell movement, axonal guidance, as well as multicellular morphogenetic processes involving changes in cell polarity and angiogenesis [3].

Two protein families appear to be required for contractility through Rho-induced assembly of stress fibers and focal adhesions: the Rho-associated kinases (ROCK) and the Dia members of the formin family. These include ROCK1 and ROCK2 as well as mDial and mDia2. Activation of ROCK appears to be necessary, but is not sufficient, for stress fiber formation. Inhibition of ROCK using the inhibitor Y-27632 prevents stress fiber formation, whereas a constitutively activated mutant of ROCK1 promotes the formation of stellate actomyosin filaments, which do not resemble stress fibers. Through its downstream phospho-substrates, ROCK can signal isoactin reorganization by multiple mechanisms (e.g., ROCK has been shown to phosphorylate and thereby inactivate MLC phosphatase, thus resulting in an increase in phosphorylated MLC). In addition, ROCK1 can also play a pivotal role in generating contractile force by stabilizing filamentous actin cross-links through a LIM-kinase-depend-ent phosphorylation of the actin depolymerizing protein, ADF-cofilin, which is inactivated by phosphorylation. In turn, this may also influence cellular ATP levels, which may also be important in regulating cellular contractility, as has recently been demonstrated for retinal pericytes [14]. For the microvasculature, we believe that these molecular signaling cascades are essential elements controlling developmental and pathologic angiogenesis and offer a newfound awareness for the molecular and cellular mechanisms regulating microvascular morphogenesis.

Glossary

Capillary: A microvascular blood vessel interposed between the arterial and venous microcirculation, which has a lumenal diameter between 5 to 8 micrometers (i.e., wide enough to permit the passage of one red blood cell to pass, one at a time).

Cytoskeleton: The structural organelle of living cells, which comprises structural and contractile protein components. The cytoskeleton is usually comprised of three distinct filament systems: the thin filaments (actin filaments), the intermediate filaments (desmin or vimentin filaments), and the microtubules (made up of the protein, tubulin).

Morphogenesis: A term referring to the structural development of an organism or a part thereof (e.g., in the formation of the blood vascular system).

Pericyte: The mural (wall) cell of the capillary and postcapillary venule, which has been shown to possess contractile and structural properties similar to the vascular smooth muscle cell and which has been demonstrated to influence vascular endothelial cell growth.

References

1. Nguyen, L. L., and D'Amore, P. A. (2001). Cellular interactions in vascular growth and differentiation. Int. Rev. Cytol. 204, 1-48.

2. Attisano, L., and Wrana, J. L. (2002). Signal transduction by the TGF-beta superfamily. Science 296, 1646-1647.

3. Papetti, M., and Herman, I. M. (2002). Mechanisms of normal and tumor-derived angiogenesis. Am. J. Physiol. Cell Physiol. 282, C947-C970.

4. Darland, D. C., Massingham, L. J. et al. (2003). Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev. Biol. 264, 275-288. Interesting report revealing the role that pericytes play in regulating capillary morphogenesis.

5. Papetti, M., Shujath, J., Riley, K., and Herman, I. M. (2003). FGF-2 antagonizes the TGF-beta1-mediated induction of pericyte alpha-smooth muscle actin expression: A role for myf-5 and Smad-mediated signaling pathways. Invest. Ophthalmol. Vis. Sci. 44, 4994-5005.

Thorough accounting of the up- and downstream signaling pathways responsible for regulating pericyte growth and contractile phenotype.

6. Abramsson, A., Lindblom, P. et al. (2003). Endothelial and non-endothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors. J. Clin. Invest. 112, 1142-1151.

7. Cho, H., Kozasa, T. et al. (2003). Pericyte-specific expression of Rgs5: Implications for PDGF and EDG receptor signaling during vascular maturation. FASEB J. 17, 440-442.

8. Sieczkiewicz, G. J., and Herman, I. M. (2003). TGF^-1 signaling controls retinal pericyte contractile protein expression. Microvasc. Res. 66, 190-196.

9. Newcomb, P. M., and Herman, I. M. (1993). Pericyte growth and contractile phenotype: Modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle. J. Cell. Physiol. 155, 385-393.

10. Carlier, M. F. (1998). Control of actin dynamics. Curr. Opin. Cell. Biol. 10, 45-51.

11. Herman, I. M. (1993). Actin isoforms. Curr. Opin. Cell. Biol. 5, 48-55.

12. Etienne-Manneville, S., and Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629-635.

13. Kolyada, A. Y., Riley, K. N., and Herman, I. M. (2003). Rho GTPase signaling modulates cell shape and contractile phenotype in an isoactin-specific manner. Am. J. Physiol. Cell Physiol. 285, C1116-C1121. Demonstration that the Rho GTPase signaling pathway is active in pericytes and that modulation of cell shape and contractile potential is regulating in an isoactin-specific manner.

14. Kawamura, H., Sugiyama, T. et al. (2003). ATP: A vasoactive signal in the pericyte-containing microvasculature of the rat retina. J. Physiol. 551, 787-799. Recent findings revealing the role that ATP plays in controlling pericyte contractility.

Capsule Biography

Dr. Herman is currently Professor of Cellular and Molecular Physiology at Tufts University School of Medicine, where he also holds appointments as Professor of Cell Biology and Anatomy as well as Professor of Ophthalmology. For more than two decades, work in the Herman laboratory has been instrumental in revealing the molecular and cellular mechanisms regulating vascular remodeling during development and disease. For this work, Dr. Herman has been the recipient of numerous awards, including grants from the NIH, American Heart Association, and pharma.

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