Summary

The roles of actin microfilaments, microtubules, and intermediate filaments in EC function are becoming increasingly defined and appreciated as increasingly complex. The actin cytoskeleton is intimately involved in numerous endothelial cell biologic processes of critical importance to the function of the organ and the entire organism as well. Historically viewed as separate and distinct cytoskeletal systems, microtubules and actin filaments are now known to interact functionally during dynamic cellular processes. Microtubule disruption induces rapid assembly of actin filaments and focal adhesions, isometric cellular contraction that correlates with the level of MLC phosphorylation, increased permeability across endothelial cell monolayers, and increased transendothelial leukocyte migration, while microtubule stabilization attenuates these effects. The mechanisms involved in these effects are poorly understood but are likely to be mediated through interaction with actin filaments, suggesting significant microfilament-microtubule crosstalk and an intriguing role for the microtubule cytoskeleton in EC barrier regulation. The exact roles of intermediate filaments such as vimentin await further exploration.

Glossary

Cadherin: A cell type specific calcium-dependent transmembrane protein present in adherens junctions.

Cortactin: An actin-bundling protein that is phosphorylated by tyrosine kinase pp60src.

Endothelial permeability: Dynamic process that involves alteration and contraction of cytoskeletal structure as well as loosened adhesive junctions to allow intercellular passage of fluid.

MLCK: ATP- and Ca2+/calmodulin-dependent kinase that phosphory-lates myosin light chain.

Rac GTPase: Members of large family of signaling proteins, which hydrolyze GTP to GDP and have roles in regulation of motility.

Rho GTPase: Members of large family of signaling proteins which hydrolyze GTP to GDP and are involved in signaling cascades linking extracellular stimuli to dynamic actin cytoskeletal rearrangement including stress fiber formation.

Tubulin: Globular protein that exists as a heterodimer in polar linear polymers that compose microtubules.

Vimentin: Type III intermediate filament protein expressed in cells of mesenchymal origin.

References

1. Dudek, S. M., and Garcia, J. G. (2001). Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. 91(4), 1487-1500.

Maintenance of the pulmonary vascular barrier is critically important to lung function. Regulation of vascular permeability is dependent on the integrity of the endothelial cell layer, which is a complex balance between intracellular contractility and cell—matrix tethering forces mediated by the dynamics of the actomyosin-based endothelial cell cytoskeletal network. Actin network contractility is mediated in part by Rho kinase/myosin light chain kinase (MLCK) dependent and independent pathways. The actin network is inserted into cell—cell and cell—matrix adhesive protein linkages that are highly regulated to produce a dynamic endothelial cell network.

2. Manjno, G., and Palade, G. (1961). Studies on inflammation. 1. Effect of histamine and serotonin on vascular permeability: An electron microscopic study. J. Biophys. Biochem. Cytol. 11, 571-605.

3. Garcia, J. G. N., Liu, F., Wang, P., Verin, A. D., Dechert, M. A., Gerthoffer, W. T., and English, D. K. (2001). Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J. Clin. Invest. 108(5), 689-701.

4. Rodriguez, O. C., Schaefer, A. W., Mandato, C. A., Forscher, P., Bement, W. M., Waterman-Storer, C. M. (2003). Conserved micro-tubule-actin interactions in cell movement and morphogenesis. Nat. Cell Biol. 5(7), 599-609.

5. Birukova, A. A., Liu, F., Garcia, J. G. N., Verin, A. D. (2004). Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am. J. Physiol. Lung Molec. Physiol. 287, L86-L93.

6. Galbraith, C. G., Skalak, R., and Chien, S. (1998). Shear stress induces spatial reorganization of the endothelial cell cytoskeleton. Cell. Motil. Cytoskeleton 40(4), 317-330.

7. Bayless, K. J., and Davis, G. E. Microtubule depolymerization rapidly collapses capillary tube networks in vitro and angiogenic vessels in vivo through the small GTPase Rho. J. Biol. Chem. Apr 20;276(16), 13483-13489. Epub 2001 Jan 25.

8. Helfand, B. T., Chang, L., and Goldman, R. D. (2004). Intermediate filaments are dynamic and motile elements of cellular architecture. J. Cell Sci. 2004. 117(Pt 2), 133-141. Intermediate filaments (IFs) are dynamic motile cytoskeletal elements that assemble in three distinct structural forms, but are most frequently observed as an extensive network of IFs extending from the perinuclear region to the endothelial cell surface. Vimentin IFs in each of the structural forms are motile and travel via microtubule-based motors along microtubule tracks (MT) tracks. IF motility speed appears to be based on both the type of IF and the state of assembly. Cross-linking between the MT and IF network appears to be critical to cell function, since disruption of IF motility results in significant pathological conditions in multiple cell types including neurons.

9. Dejana, E. (2004). Endothelial cell-cell junctions: Happy together. Nat. Rev. Mol. Cell. Biol. 5(4), 261-270. Endothelial cells contain cell—cell attachment sites known as adherens and tight junctions that not only have structural roles, but also participate in endothelial cell specific functions such as control of vascular permeability, angiogene-sis, and leukocyte diapedisis. Multiple transmembrane proteins such as VE-cadherins, claudins, and their associated intracellular proteins compose junctional complexes that trigger intracellular signaling cascades, which mediate a balance between endothelial cells composing a stable vascular barrier and activated endothelial cells undergoing angiogenesis or leukocyte diapedisis.

10. Tsuruta, D., and Jones, J. C. (2003). The vimentin cytoskeleton regulates focal contact size and adhesion of endothelial cells subjected to shear stress. J. Cell Sci 116(Pt 24), 4977-4984.

Capsule Biography

Dr. Joe G. N. Garcia is the Dr. David Marine Professor of Medicine, Director of the Division of Pulmonary and Critical Care Medicine, and Director of the Center for Translational Respiratory Medicine at The Johns Hopkins University School of Medicine in Baltimore, Maryland. One of his primary research interests include the regulation of the pulmonary microvascular endothelial barrier by the endothelial cytoskeleton.

Dr. Laura Linz McGillem is a postdoctoral fellow in the department of Pulmonary and Critical Care Medicine. Her thesis work at Louisiana State University Health Sciences Center included isolation and characterization of a novel nonmuscle myosin from bovine retina. Her interests include endothelial cell physiology, cell-cell adhesions, and the cytoskeleton.

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