Fc-saxatilin suppresses hypoxia-induced vascular leakage by regulating endothelial occludin expression

Hyun-Jung Choi; Il Kwon; Na-Eun Kim; Jayoung Kim; Sunho An; Sungsoo Kang; Sung-Yu Hong; Hyo Suk Nam; Ji Hoe Heo

doi:10.1160/TH16-06-0469

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2017; 117(03): 595-605
DOI: 10.1160/TH16-06-0469

Stroke, Systemic or Venous Thromboembolism

Schattauer GmbH

Fc-saxatilin suppresses hypoxia-induced vascular leakage by regulating endothelial occludin expression

Hyun-Jung Choi

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

,

Il Kwon

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

³Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea

,

Na-Eun Kim

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

,

Jayoung Kim

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

⁵Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, Korea

,

Sunho An

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

⁵Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, Korea

,

Sungsoo Kang

⁴Cardiovascular Product Evaluation Center, Yonsei University College of Medicine, Seoul, Korea

,

Sung-Yu Hong

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

⁴Cardiovascular Product Evaluation Center, Yonsei University College of Medicine, Seoul, Korea

,

Hyo Suk Nam

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

²Department of Neurology, Yonsei University College of Medicine, Seoul, Korea

,

Ji Hoe Heo

¹Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases, Yonsei University, College of Medicine, Seoul, Korea

²Department of Neurology, Yonsei University College of Medicine, Seoul, Korea

³Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea

⁵Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, Korea

› Author Affiliations

Abstract

Summary

Vascular leakage due to compromised integrity of the endothelial barrier is closely associated with brain damage in several neurological disorders, including ischaemic stroke. Saxatilin, a snake venom disintegrin containing the Arg-Gly-Asp (RGD) motif, exerts thrombolytic and antiplatelet effects by interacting with multiple integrins on platelets. Integrin signalling is indispensable for regulation of endothelial permeability. Saxatilin may play a role in vascular leakage after ischaemia because it has high affinity for endothelial integrins. Here, we determined whether Fc-saxatilin, an Fc-fusion protein of saxatilin, could prevent vascular leakage under hypoxic or ischaemic conditions. In mouse brain microvascular endothelial cells, hypoxia increased the permeability to FITC-dextran, and this effect was attenuated by Fc-saxatilin treatment. Fc-saxatilin also blocked vascular leakage of Evans Blue in the ischaemic brain induced by middle cerebral artery occlusion in mice. Furthermore, the expression of occludin, a tight junction protein, was reduced by hypoxia in endothelial cells. This downregulation of occludin was attenuated by Fc-saxatilin treatment. We also determined the activity of matrix metalloproteinases (MMPs) 2 and 9 because they are implicated in the degradation of occludin and of the microvascular basal lamina. Hypoxia increased MMP-9 activity, and this increase was attenuated by Fc-saxatilin treatment. Fc-saxatilin specifically bound to integrin αvβ3 of the endothelial cells and inhibited hypoxia-induced activation of FAK, a downstream signalling molecule in integrin-dependent signal transduction. Taken together, these results provide new insights into the mechanism via which Fc-saxatilin, as an integrin antagonist, prevents vascular leakage under ischemic conditions by regulating occludin expression in endothelial tight junctions.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Fc-saxatilin - disintegrin - occluding - endothelial permeability

Full Text

References

References
1 Hong SY, Koh YS, Chung KH. et al. Snake venom disintegrin, saxatilin, inhibits platelet aggregation, human umbilical vein endothelial cell proliferation, and smooth muscle cell migration. Thromb Res 2002; 105: 79-86.
2 Hong SY, Sohn YD, Chung KH. et al. Structural and functional significance of disulfide bonds in saxatilin, a 7.7 kDa disintegrin. Biochem Biophys Res Commun 2002; 293: 530-536.
3 Kwon I, Hong SY, Kim YD. et al. Thrombolytic effects of the snake venom disintegrin saxatilin determined by novel assessment methods: a FeCl3-induced thrombosis model in mice. PloS One 2013; 08: e81165.
4 Phillips DR, Charo IF, Parise LV. et al. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: 831-843.
5 Abou-Chebl A, Bajzer CT, Krieger DW. et al. Multimodal therapy for the treatment of severe ischaemic stroke combining GPIIb/IIIa antagonists and angioplasty after failure of thrombolysis. Stroke 2005; 36: 2286-2288.
6 Eckert B, Koch C, Thomalla G. et al. Aggressive therapy with intravenous abciximab and intra-arterial rtPA and additional PTA/stenting improves clinical outcome in acute vertebrobasilar occlusion: combined local fibrinolysis and intravenous abciximab in acute vertebrobasilar stroke treatment (FAST): results of a multicenter study. Stroke 2005; 36: 1160-1165.
7 Seitz RJ, Meisel S, Moll M. et al. The effect of combined thrombolysis with rtPA and tirofiban on ischaemic brain lesions. Neurology 2004; 62: 2110-2112.
8 Jang YJ, Jeon OH, Kim DS. Saxatilin, a snake venom disintegrin, regulates platelet activation associated with human vascular endothelial cell migration and invasion. J Vasc Res 2007; 44: 129-137.
9 Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 1994; 264: 569-571.
10 Nisato RE, Tille JC, Jonczyk A. et al. alphav beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro. Angiogenesis 2003; 06: 105-119.
11 Byzova TV, Goldman CK, Pampori N. et al. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell 2000; 06: 851-860.
12 Suzuma K, Takagi H, Otani A. et al. Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells. Invest Ophthalmol Visual Sci 1998; 39: 1028-1035.
13 Sloan EK, Pouliot N, Stanley KL. et al. Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone. Breast Cancer Res 2006; 08: R20.
14 Varner JA, Cheresh DA. Integrins and cancer. Curr Opin Cell Biol 1996; 08: 724-730.
15 Abumiya T, Lucero J, Heo JH. et al. Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischaemia. J Cerebral Blood Flow Metabol 1999; 19: 1038-1050.
16 Okada Y, Copeland BR, Hamann GF. et al. Integrin alphavbeta3 is expressed in selected microvessels after focal cerebral ischaemia. Am J Pathol 1996; 149: 37-44.
17 Tagaya M, Haring HP, Stuiver I. et al. Rapid loss of microvascular integrin expression during focal brain ischaemia reflects neuron injury. J Cerebral Blood Flow Metabol 2001; 21: 835-846.
18 Shimamura N, Matchett G, Solaroglu I. et al. Inhibition of integrin alphavbeta3 reduces blood-brain barrier breakdown in focal ischaemia in rats. J Neurosci Res 2006; 84: 1837-1847.
19 Shimamura N, Matchett G, Yatsushige H. et al. Inhibition of integrin alphavbeta3 ameliorates focal cerebral ischaemic damage in the rat middle cerebral artery occlusion model. Stroke 2006; 37: 1902-1909.
20 Abumiya T, Fitridge R, Mazur C. et al. Integrin alpha(IIb)beta(3) inhibitor preserves microvascular patency in experimental acute focal cerebral ischaemia. Stroke 2000; 31: 1402-1410.
21 Robinson SD, Reynolds LE, Wyder L. et al. Beta3-integrin regulates vascular endothelial growth factor-A-dependent permeability. Arterioscl Thromb Vasc Biol 2004; 24: 2108-2114.
22 Bauer AT, Burgers HF, Rabie T. et al. Matrix metalloproteinase-9 mediates hypoxia-induced vascular leakage in the brain via tight junction rearrangement. J Cerebral Blood Flow Metabol 2010; 30: 837-848.
23 Yang Y, Estrada EY, Thompson JF. et al. Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischaemia in rat. J Cerebral Blood Flow Metabol 2007; 27: 697-709.
24 Kago T, Takagi N, Date I. et al. Cerebral ischaemia enhances tyrosine phosphorylation of occludin in brain capillaries. Biochem Biophys Res Commun 2006; 339: 1197-1203.
25 Lohmann C, Krischke M, Wegener J. et al. Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways. Brain Res 2004; 995: 184-196.
26 Takenaga Y, Takagi N, Murotomi K. et al. Inhibition of Src activity decreases tyrosine phosphorylation of occludin in brain capillaries and attenuates increase in permeability of the blood-brain barrier after transient focal cerebral ischaemia. J Cerebral Blood Flow Metabol 2009; 29: 1099-1108.
27 Chen XL, Nam JO, Jean C. et al. VEGF-induced vascular permeability is mediated by FAK. Develop Cell 2012; 22: 146-157.
28 Jean C, Chen XL, Nam JO. et al. Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function. J Cell Biol 2014; 204: 247-263.
29 Wu MH. Endothelial focal adhesions and barrier function. J Physiol 2005; 569: 359-366.
30 Jin YJ, Park I, Hong IK. et al. Fibronectin and vitronectin induce AP-1-mediated matrix metalloproteinase-9 expression through integrin alpha(5)beta(1)/alpha(v)beta(3)-dependent Akt, ERK and JNK signalling pathways in human umbilical vein endothelial cells. Cell Signal 2011; 23: 125-134.
31 Rosenberg GA. Matrix metalloproteinases and their multiple roles in neurodegenerative diseases. Lancet Neurol 2009; 08: 205-216.
32 Cunningham LA, Wetzel M, Rosenberg GA. Multiple roles for MMPs and TIMPs in cerebral ischaemia. Glia 2005; 50: 329-339.
33 Kilkenny C, Browne WJ, Cuthill IC. et al. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J Pharmacol Pharmacotherap 2010; 01: 94-99.
34 Fisher M, Feuerstein G, Howells DW. et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009; 40: 2244-2250.
35 Heo JH, Lucero J, Abumiya T. et al. Matrix metalloproteinases increase very early during experimental focal cerebral ischaemia. J Cerebral Blood Flow Metabol 1999; 19: 624-633.
36 Nam HS, Kwon I, Lee BH. et al. Effects of Mesenchymal Stem Cell Treatment on the Expression of Matrix Metalloproteinases and Angiogenesis during Ischaemic Stroke Recovery. PloS One 2015; 10: e0144218.
37 Liu J, Jin X, Liu KJ. et al. Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischaemic stroke stage. J Neurosci 2012; 32: 3044-3057.
38 Skuli N, Monferran S, Delmas C. et al. Alphavbeta3/alphavbeta5 integrins-FAK-RhoB: a novel pathway for hypoxia regulation in glioblastoma. Cancer Res 2009; 69: 3308-3316.
39 Sandoval KE, Witt KA. Blood-brain barrier tight junction permeability and ischaemic stroke. Neurobiol Dis 2008; 32: 200-219.
40 Hirase T, Staddon JM, Saitou M. et al. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 1997; 110: 1603-1613.
41 Yamashita T, Abe K. Therapeutic approaches to vascular protection in ischaemic stroke. Acta Medica Okayama 2011; 65: 219-223.
42 Eliceiri BP, Puente XS, Hood JD. et al. Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signalling. J Cell Biol 2002; 157: 149-160.
43 Schwartz MA. Integrin signalling revisited. Trends Cell Biol 2001; 11: 466-470.
44 Kaur J, Zhao Z, Klein GM. et al. The neurotoxicity of tissue plasminogen activator? J Cerebral Blood Flow Metabol. 2004; 24: 945-963.
45 Jin R, Yang G, Li G. Molecular insights and therapeutic targets for blood-brain barrier disruption in ischaemic stroke: critical role of matrix metalloproteinases and tissue-type plasminogen activator. Neurobiol Dis 2010; 38: 376-385.

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