doi: 10.1161/01.ATV.0000219612.70149.46
© 2006 American Heart Association, Inc.
Editorials |
Flow Perturbation Is Linked to Endothelial PAR Signaling
From The Scripps Research Institute, La Jolla, Calif.
Correspondence to Wolfram Ruf, Scripps Research Institute, Department of Immunology, La Jolla, CA 92037. E-mail ruf{at}scripps.edu
The vascular endothelium integrates a variety of humoral stimuli and biophysical forces to maintain patent microvascular and macrovacular beds. At the interface between the blood and vessel wall, endothelial cells communicate in both directions by the release of growth factors, cytokines, and bioactive mediators to maintain vascular homeostasis. Endothelial dysfunction is central for acute systemic inflammatory syndromes including sepsis and chronic inflammatory disorders such as atherosclerosis. Early atherosclerotic lesions preferentially develop at sites where laminar flow patterns are disturbed, and endothelial cells sense changes in biomechanical forces that result from flow perturbation. Exposure to shear forces rapidly activates G proteins, ion fluxes, and mechanosensing receptors, including integrins and a complex involving PECAM-1, VEGFR-2, and vascular endothelial cell cadherin.1 Activation of downstream signaling pathways results in cytoskeletal reorganization and a flow-adapted endothelial phenotype.2 Endothelial cells under laminar flow have an atheroprotected phenotype, but perturbation of laminar flow patterns induces a complex transcriptional response that favors the induction of adhesion molecules, proinflammatory cytokines, and procoagulant genes.
See page 1185
Dekker et al profiled the repertoire of laminar flow-regulated genes in endothelial cells and identified lung Krueppel-like factor (LKLF or KLF-2) that was selectively induced on flow adaptation and suppressed by the inflammatory mediator tumor necrosis factor (TNF)
.3 KLF2 was indeed tonically expressed in vivo in noninflamed large vessel endothelial cells. KLFs are zinc finger transcription factors similar to Sp1 and Egr-1 families that act as transcriptional repressors or activators by high affinity binding to a CACCC promoter/enhancer element. KLF2 was originally identified as a regulator of T cell activation.4 Maturation of T cells and thymocytes induces upregulation of KLF2, and deletion of KLF2 results in a persistently activated phenotype with increased susceptibility to apoptosis. Activation of mature T cells rapidly downregulates KLF2, further confirming that KLF2 acts as a key regulator of the activation state of differentiated cells. Of the 16 KLF family members, some are ubiquitously expressed, whereas others have more restricted expression in hematopoietic or vascular cells.5 KLFs typically orchestrate gene programs that regulate activation, differentiation, proliferation, and apoptosis.
Although several KLFs are expressed in the vasculature, so far only KLF2 deletion results in embryonic lethality attributable to vascular failure.5 The prominent abnormality in KLF2-deficient vessels is diminished recruitment or retention of vascular smooth muscle cells starting after embryonic day 12.5 with subsequent rupture of the thinning endothelial cell lining of larger vessels.6 Similar phenotypes of vascular failure result from deletion of PDGF-B7 or the sphingosine 1 phosphate receptor 1 (S1PR1, EDG1)8 both of which play critical roles in the communication of endothelial cells with mural cells. Indeed KLF2 upregulates PDGF-B expression.9 KLF2 is thus a crucial endothelial cell expressed transcription factor that regulates adaptation of the vessel wall to the increasing hemodynamic forces during cardiovascular development. A series of recent publications9–13 have now elucidated the central role of KLF2 as a master regulator that links mechanosensing to the fine tuning of inflammatory and procoagulant responses of the endothelium.
Perturbed flow at atherosclerosis prone sites indeed leads to downregulation of KLF2 with concordant loss of vascular protective mechanisms.9,11 Laminar flow induces KLF2 through activation of the MEK5-ERK5-MEF2C pathway,9 and overexpression of KLF2 to a large extent mimics the atheroprotective antiinflammatory, antithrombotic, and antiproliferative phenotype of differentiated endothelial cells under flow.9,11 KLF2 induces endothelial cell nitric oxide synthase (eNOS) and reduces caveolin levels that suppresses eNOS activity. KLF2 thus regulates vascular tone.13 Moreover, KLF2 specifically prevents the upregulation of vascular cell adhesion molecule (VCAM) and E-selectin in response to interleukin (IL)-1ß, TNF, and endotoxin by sequestering the NF-
B coactivator CBP/p300, thereby reducing leukocyte and T-cell recruitment and local inflammation.13 KLF2 is barrier protective and counteracts VEGF-induced hyperpermeability.14 Furthermore, KLF2 increases thrombomodulin and suppresses antifibrinolytic plasminogen activator inhibitor (PAI)-1 expression while preventing cytokine-induced tissue factor (TF) expression (Figure). Together, these changes maintain the anticoagulant balance of the endothelium.12 In this issue, Lin et al add to this emerging theme by describing an unexpected regulatory role KLF2 in suppressing thrombin signaling through protease activated receptor (PAR) 1.15
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Lin et al show that overexpression of KLF2 abolishes thrombin-dependent induction of inflammatory cytokines IL-8, MCP-1, and, to a lesser degree, IL-6. Importantly, thrombin-mediated TF upregulation is also suppressed by KLF2. Because thrombin synergizes with mediators released from activated platelets such as sphingosine 1 phosphate to induce TF,16 KLF2 may suppress a vicious cycle of sustained endothelium procoagulant alteration at sites susceptible to thrombosis. Unlike KLF2’s suppression of cytokine-induced VCAM and E-selectin expression through sequestration of NF-B coactivators, KLF2 attenuated thrombin signaling by downregulating mRNA levels and cell surface expression of the thrombin receptor PAR1. Conversely, knock-down of KLF2 upregulated PAR1 and increased thrombin-mediated TF induction. These experiments thus demonstrate control of PAR expression by the central mechanosensing pathway of endothelial cells.
However, suppression of thrombin signaling by KLF2 is not uniform, and thrombin still induces CD40L release, essentially reversing suppressed baseline secretion in KLF2 overexpressing cells. Consistent with a more complex regulation of PAR1 by shear forces, Dekker et al described that PAR1 mRNA levels are upregulated when endothelial cells are switched from static to flow conditions.3 Similarly, Weibel-Palade body release by thrombin is not inhibited by KLF2 overexpression, although a shift in the dose response for thrombin was observed.11 Taken together, these results indicate that KLF2 modulates, rather than shuts off, PAR1 signaling completely. It is of interest that KLF2 suppresses PAR2 mRNA expression even more pronounced than PAR1 levels.9 PAR2 is more susceptible to inflammatory cytokine upregulation, and PAR2 activation potently induces TF.17,18 In addition, PAR2 is involved in recruiting leukocytes to activated endothelium, thus closely linking PAR2 to inflammatory pathways.19 Although PAR2 is not cleaved by thrombin, PAR2 can be crossactivated by thrombin-cleaved PAR1.20 In addition, certain thrombin responses are dependent on synergistic effects of PAR1 and PAR2 activation.21,22 The significant downregulation of thrombin-induced proinflammatory and procoagulant responses by KLF2 in endothelial cells may thus result from the concordant modulation of PAR1 and PAR2 expression. TF-dependent PAR2 signaling regulates vascular regeneration,23 and downregulation of PAR2 as well as TF may be part of the coordinated program by which KLF2 suppresses angiogenesis.14
It will be of interest for further studies to evaluate whether the significant upregulation of thrombomodulin by KLF2 maintains the vascular protective signaling properties of the downstream activated protein C (APC) pathway. Endothelial protein C receptor (EPCR)-bound APC activates PAR1.24 APC signaling is barrier protective through sphingosine 1 phosphate receptor cross-activation,25 whereas thrombin signaling through PAR1 is barrier disruptive.26 The effect of KLF2 on thrombin-induced permeability is of particular interest when considering the strong barrier protective effects of KLF2 in VEGF-induced hyperpermeability.14 APC-PAR1 signaling induces an antiapoptotic and endothelial protective gene profile that is distinct from thrombin-mediated PAR1 signaling in cytokine stimulated endothelial cells.27 Whether suppression of the proinflammatory signaling of thrombin in endothelial cells by KLF2 shifts the balance favorably to the APC signaling pathway should be of interest. These future directions will add to our understanding on the role of mechanosensing pathways to regulate the proinflammatory signaling balance of the endothelium.
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Acknowledgments |
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The work in the author’s laboratory is supported by the National Heart, Lung, and Blood Institute.
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References |
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 Top References |
|---|
1. Orr AW, Helmke BP, Blackman BR, Schwartz MA. Mechanisms of mechanotransduction. Dev Cell. 2006; 10: 11–20.[CrossRef][Medline] [Order article via Infotrieve]
2. Li YS, Haga JH, Chien S. Molecular basis of the effects of shear stress on vascular endothelial cells. J Biomech. 2005; 38: 1949–1971.[CrossRef][Medline] [Order article via Infotrieve]
3. Dekker RJ, van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E, Pannekoek H, Horrevoets AJG. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2). Blood. 2002; 100: 1689–1698.
4. Kuo CT, Leiden JM. Transcriptional regulation of T lymphocyte development and function. Annu Rev Immunol. 1999; 17: 149–187.[CrossRef][Medline] [Order article via Infotrieve]
5. Suzuki T, Aizawa K, Matsumura T, Nagai R. Vascular implications of the Kruppel-like family of transcription factors. Arterioscler Thromb Vasc Biol. 2005; 25: 1135–1141.
6. Kuo CT, Veselits ML, Barton KP, Lu MM, Clendenin C, Leiden JM. The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes Dev. 1997; 11: 2996–3006.
7. Lindahl P, Johansson BR, Levéen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997; 277: 242–245.
8. Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, Hobson JP, Rosenfeldt HM, Nava VE, Chae SS, Lee MJ, Liu CH, Hla T, Spiegel S, Proia RL. Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest. 2000; 106: 951–961.[Medline] [Order article via Infotrieve]
9. Parmar KM, Larman HB, Dai G, Zhang Y, Wang ET, Moorthy SN, Kratz JR, Lin Z, Jain MK, Gimbrone MA Jr, Garcia-Cardena G. Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. J Clin Invest. 2006; 116: 49–58.[CrossRef][Medline] [Order article via Infotrieve]
10. Dekker RJ, van Thienen JV, Rohlena J, de Jager SC, Elderkamp YW, Seppen J, de Vries CJ, Biessen EA, van Berkel TJ, Pannekoek H, Horrevoets AJ. Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Am J Pathol. 2005; 167: 609–618.
11. Dekker RJ, Boon RA, Rondaij MG, Kragt A, Volger OL, Elderkamp YW, Meijers JC, Voorberg J, Pannekoek H, Horrevoets AJ. KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood. 2006.
12. Lin Z, Kumar A, SenBanerjee S, Staniszewski K, Parmar K, Vaughan DE, Gimbrone MA Jr, Balasubramanian V, Garcia-Cardena G, Jain MK. Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ Res. 2005; 96: e48–e57.
13. SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW, Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA Jr, Garcia-Cardena G, Jain MK. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med. 2004; 199: 1305–1315.
14. Bhattacharya R, SenBanerjee S, Lin Z, Mir S, Hamik A, Wang P, Mukherjee P, Mukhopadhyay D, Jain MK. Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2. J Biol Chem. 2005; 280: 28848–28851.
15. Lin Z, Hamik A, Jain R, Kumar A, Jain MK. Kruppel-like factor 2 inhibits protease activated receptor-1 expression and thrombin-mediated endothelial activation. Arterioscler Thromb Vasc Biol. 2006; 26; 1185–1189.
16. Takeya H, Gabazza EC, Aoki S, Ueno H, Suzuki K. Synergistic effect of sphingosine 1-phosphate on thrombin-induced tissue factor expression in endothelial cells. Blood. 2003; 102: 1693–1700.
17. Alm A-K, Norström E, Sundelin J, Nystedt S. Stimulation of proteinase activated receptor-2 causes endothelial cells to promote blood coagulation in vitro. Thromb Haemost. 1999; 81: 984–988.[Medline] [Order article via Infotrieve]
18. Nystedt S, Ramakrishnan V, Sundelin J. The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells—Comparison with the thrombin receptor. J Biol Chem. 1996; 271: 14910–14915.
19. Lindner JR, Kahn ML, Coughlin SR, Sambrano GR, Schauble E, Bernstein D, Foy D, Hafezi-Moghadam A, Ley K. Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol. 2000; 165: 6504–6510.
20. O’Brien PJ, Prevost N, Molino M, Hollinger MK, Woolkalis MJ, Woulfe DS, Brass LF. Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1. J Biol Chem. 2000; 275: 13502–13509.
21. Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM. Protease-activated receptor 1 (PAR1) and PAR2 contribute to tumor cell motility and metastasis. Mol Cancer Res. 2004; 2: 395–402.
22. Lidington EA, Steinberg R, Kinderlerer AR, Landis RC, Ohba M, Samarel A, Haskard DO, Mason JC. A role for proteinase-activated receptor 2 and PKC-epsilon in thrombin-mediated induction of decay-accelerating factor on human endothelial cells. Am J Physiol Cell Physiol. 2005; 289: C1437–C1447.
23. Belting M, Dorrell MI, Sandgren S, Aguilar E, Ahamed J, Dorfleutner A, Carmeliet P, Mueller BM, Friedlander M, Ruf W. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling. Nature Med. 2004; 10: 502–509.[CrossRef][Medline] [Order article via Infotrieve]
24. Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science. 2002; 296: 1880–1882.
25. Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005; 105: 3178–3184.
26. Vogel SM, Gao X, Mehta D, Ye RD, John TA, Andrade-Gordon P, Tiruppathi C, Malik AB. Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. Physiol Genomics. 2000; 4 (2): 137–45.
27. Riewald M, Ruf W. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem. 2005; 280: 19808–19814.
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