Conclusions and Future Studies

SlP induces cell migration, but as emphasized in this review, SlP also promotes the barrier function of the vascular endothelium. SlP activates both Racl and Rho, and both small GTPases have been implicated in the physiological processes of cell migration and endothelial barrier function (see Figure l). At least for Racl, this apparent discrepancy may depend on whether Racl is localized at cell-cell junctions and involved in barrier function or at the leading edge of a wounded cell and promoting cell migration. To complicate our understanding, these two GTPases have also been associated with processes that loosen the endothelial barrier. Therefore, the current take-home message is that the integrity of the adherens junction, at least in endothelial

Sphingosine 1-phosphate

Actin

Strong

Actin

Strong

Actin

Cortical Actin

Sphingosine 1-phosphate

Cortical Actin

Adherens Junction

Actin

Adherens Junction

Actin

Endothelial Barrier Function

Figure 1 Proposed cellular targets and signaling pathways by which sphingosine l-phosphate (SlP) tightens the endothelial barrier. SlP binds to endothelial differentiation gene receptors (Edg). Edg l couples primarily with Gj and Edg 3 and Edg 5 with Gq and Gl2/l3. SlP activates Racl, and many of the physiological outcomes of SlP and Racl are linked to Gj. Down-regulation of Edg l or Edg 3 or inhibition of Gj prevents the SlP-induced enhancement of the endothelial barrier. In epithelial cells, Racl sequesters IQGAPl and removes this proposed negative regulator from adherens junction proteins. The resultant linkage to the actin cytoskeleton facilitates the integrity of the adherens junction, although this sequence of events has not been described in endothelial cells. SlP and active Racl also remodel the actin cytoskeleton as evidenced by a thickened, cortical actin and stress fibers. Active Racl appears to affect cortical actin via activation of PAK and the subsequent inactivation of cofilin. The formation of actin stress fibers involves Rho and Rho kinase, and inhibition of Rho kinase also prevents the barrier-enhancing property of SlP The linkage of Rho with Gl2/l3 and Edg 3 and Edg 5 requires demonstration. To complicate matters, both Racl and Rho have also been associated with loosening of the endothelial barrier. Thus, subtle changes in the activation of Racl and Rho may profoundly influence endothelial barrier function. VE, vascular endothelial cadherin; pl20, pl20-catenin; a, a-catenin; b, b-catenin; g, g-catenin or plakoglobin; p2l-activated kinase. (see color insert)

cells, may be influenced by subtle changes in the Rho family of GTPases, Rho, Racl, and Cdc42. The cellular targets that regulate endothelial barrier function, whether adherens junction proteins or the actin cytoskeleton located at adhesions between cells and cell to substratum, need to be elucidated and require further study.

Glossary

Barrier function: The semipermeable nature of the endothelium to the passage of water and protein. Includes the movement of water and protein by diffusion and by convection (or bulk flow of water).

Permeability: The process whereby water or proteins diffuses from a higher concentration in the blood through the junctions between endothe-lial cells to a lower concentration in the tissue.

Rac and Rho: Members of the Rho family of small GTPases that function as intracellular signaling molecules.

Thrombocytopenia: Platelet count in the blood of 50,000/||L or less. Normal platelet count is 150,000 to 300,000 cells per microliter of blood.

Acknowledgments

This work was supported by a National Institutes of Health grant, HL-68079, and by an American Heart Association grant, AHA-97-127A.

Further Reading

1. Patil, S., Kaplan, J. E., and Minnear, F. L. (1997). Protein, not adenosine or adenine nucleotides, mediates platelet decrease in endothelial permeability. Am. J. Physiol. Heart Circ. Physiol. 273, H2304-H2311.

2. Haselton, F. R., and Alexander, J. S. (1992). Platelets and a platelet-released factor enhance endothelial barrier. Am. J. Physiol. Lung Cell Mol. Physiol. 263, L670-L678.

3. Alexander, J. S., Patton, W. F., Christman, B. W., Cuiper, L. L., and Haselton, F. R. (1998). Platelet-derived lysophosphatidic acid decreases endothelial permeability in vitro. Am. J. Physiol. Heart Circ. Physiol. 274, H115-H122. The first report that a phospholipid(s), possibly lysophosphatidic acid (LPA), is the active platelet factor.

4. Minnear, F. L., Patil, S., Bell, D., Gainor, J. P., Morton, C. A. (2001). Platelet lipid(s) bound to albumin increases endothelial electrical resistance: Mimicked by LPA. Am. J. Physiol. Lung Cell Mol. Physiol. 281, L1337-L1344.

5. Garcia, J. G. N., Liu, F., Verin, A. D., Birukova, A., Dechert, M. A., Gerthoffer, W. T., Bamburg, J. R., and English, D. (2001). Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J. Clin. Invest. 108, 689-701. S1P increases endothelial permeability across cell monolayers derived from bovine and human pulmonary arteries and human umbilical vein. Depletion of Edg-1 and Edg-3 receptors and pertussis toxin inhibit the activity of S1P. Both p21-activated kinase (PAK) and cofilin translocate to the cell periphery after treatment with S1P. Expression of a dominantnegative PAK-1 or wild-type cofilin reduces the increase in cortical actin, and the latter also blunts the increase in endothelial electrical resistance induced by S1P.

6. Lee, M.-J., Thangada, S., Claffey, K. P., Ancellin, N., Liu, C. H., Kluk, M., Volpi, M., Sha'afi, R. I., and Hla, T. (1999). Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingo-sine-1-phosphate. Cell 99, 301-312.

7. Gainor, J. P., Morton, C. A., Roberts, J. T., Vincent, P. A., and Minnear, F. L. (2001). Platelet-conditioned medium increases endothelial electrical resistance independently of cAMP/PKA and cGMP/PKG. Am. J. Heart Circ. Physiol. 281, H1992-H2001. Platelet-conditioned medium (PCM) and LPA rapidly increase endothelial electrical resistance via a novel, signaling pathway involving tyrosine kinases, the Gt protein, and PI-3 kinase. This pathway is independent of protein kinases A and G.

8. Fukata, M., Nakagawa, M., Itoh, N., Kawajiri, A., Yamaga, M., Kuroda, S., and Kaibuchi, K. (2001). Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell-cell dissociation during cell scattering. Mol. Cell Biol. 21, 2165-2183.

9. Wojciak-Stothard, B., Potempa, S., Eichholtz, T., and Ridley, A. J. (2001). Rho and Rac but not Cdc42 regulate endothelial permeability. J. Cell Sci. 114, 1343-1355.

Capsule Biography

Dr. Minnear is Professor of Physiology and Pharmacology, Assistant Dean of Graduate Studies, and Director of the M.D./Ph.D. Scholars Program at West Virginia University School of Medicine. His research focuses on regulation of the vascular endothelial barrier to the passage of water and protein with emphasis on the adherens junction and the actin cytoskeleton. His work is supported by grants from the National Heart, Lung and Blood Institute and the American Heart Association.

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