This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.
References
[1]
Nag S (2002) The blood-brain barrier and cerebral angiogenesis: lessons from the cold-injury. Trends Mol Med 8: 38–44.
[2]
Petty MA, Wettstein JG (2001) Elements of cerebral microvascular ischaemia. Brain Res Brain Res Rev 36: 23–34.
[3]
Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2: 285–293.
[4]
Alvarez JI, Dodelet-Devillers A, Kebir H, Ifergan I, Fabre PJ, et al. (2011) The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334: 1727–1731.
[5]
Dai R, Xia Y, Mao L, Mei Y, Xue Y, et al. (2011) Involvement of PI3K/Akt pathway in the neuroprotective effect of Sonic hedgehog on cortical neurons under oxidative stress. J Huazhong Univ Sci Technolog Med Sci 32: 856–860.
[6]
Xia YP, Dai RL, Li YN, Mao L, Xue YM, et al. (2012) The protective effect of sonic hedgehog is mediated by the phosphoinositide [corrected] 3-kinase/AKT/Bcl-2 pathway in cultured rat astrocytes under oxidative stress. Neuroscience 209: 1–11.
[7]
Sims JR, Lee SW, Topalkara K, Qiu J, Xu J, et al. (2009) Sonic hedgehog regulates ischemia/hypoxia-induced neural progenitor proliferation. Stroke 40: 3618–3626.
[8]
Ji H, Miao J, Zhang X, Du Y, Liu H, et al. (2012) Inhibition of sonic hedgehog signaling aggravates brain damage associated with the down-regulation of Gli1, Ptch1 and SOD1 expression in acute ischemic stroke. Neurosci Lett 506: 1–6.
[9]
？sterlund T, Kogerman P (2006) Hedgehog signalling: how to get from Smo to Ci and Gli. Trends Cell Biol 16: 176–180.
[10]
Krishnan V, Pereira FA, Qiu Y, Chen CH, Beachy PA, et al. (1997) Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase. Science 278: 1947–1950.
[11]
Gritli-Linde A, Bei M, Maas R, Zhang XM, Linde A, et al. (2002) Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 129: 5323–5337.
[12]
Fujii T, Kuwano H (2010) Regulation of the expression balance of angiopoietin-1 and angiopoietin-2 by Shh and FGF-2. In Vitro Cell Dev Biol Anim 46: 487–491.
[13]
Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, et al. (1996) Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87: 1161–1169.
[14]
Lee SW, Kim WJ, Choi YK, Song HS, Son MJ, et al. (2003) SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier. Nat Med 9: 900–906.
[15]
Thurston G, Suri C, Smith K, McClain J, Sato TN, et al. (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286: 2511–2514.
[16]
Gamble JR, Drew J, Trezise L, Underwood A, Parsons M, et al. (2000) Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions. Circ Res 87: 603–607.
[17]
Zhang ZG, Zhang L, Croll SD, Chopp M (2002) Angiopoietin-1 reduces cerebral blood vessel leakage and ischemic lesion volume after focal cerebral embolic ischemia in mice. Neuroscience 113: 683–687.
[18]
Lee SW, Moskowitz MA, Sims JR (2007) Sonic hedgehog inversely regulates the expression of angiopoietin-1 and angiopoietin-2 in fibroblasts. Int J Mol Med 19: 445–451.
[19]
Li Y, Xia Y, Wang Y, Mao L, Gao Y, et al. (2012) Sonic Hedgehog (Shh) Regulates the Expression of Angiogenic Growth Factors in Oxygen-Glucose-Deprived Astrocytes by Mediating the Nuclear Receptor NR2F2. Mol Neurobiol 47: 967–975.
[20]
Belayev L, Alonso OF, Busto R, Zhao W, Ginsberg MD (1996) Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. Stroke 27: 1616–1622.
[21]
Rosenstein JM, Mani N, Silverman WF, Krum JM (1998) Patterns of brain angiogenesis after vascular endothelial growth factor administration in vitro and in vivo. Proc Natl Acad Sci U S A 95: 7086–7091.
[22]
Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20: 84–91.
[23]
Shen F, Walker EJ, Jiang L, Degos V, Li J, et al. (2011) Coexpression of angiopoietin-1 with VEGF increases the structural integrity of the blood-brain barrier and reduces atrophy volume. J Cereb Blood Flow Metab 31: 2343–2351.
[24]
Diglio CA, Grammas P, Giacomelli F, Wiener J (1982) Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization. Lab Invest 46: 554–563.
[25]
Incardona JP, Gaffield W, Kapur RP, Roelink H (1998) The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 125: 3553–3562.
[26]
Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38: 323–337.
[27]
Lawson ND, Vogel AM, Weinstein BM (2002) sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 3: 127–136.
[28]
Chen W, Tang T, Eastham-Anderson J, Dunlap D, Alicke B, et al. (2011) Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells. Proc Natl Acad Sci U S A 108: 9589–9594.
[29]
Pola R, Ling LE, Silver M, Corbley MJ, Kearney M, et al. (2001) The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med 7: 706–711.
[30]
Hashizume A, Hieda Y (2006) Hedgehog peptide promotes cell polarization and lumen formation in developing mouse submandibular gland. Biochem Biophys Res Commun 339: 996–1000.
[31]
Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, et al. (2000) Tight junctions are membrane microdomains. J Cell Sci 113: 1771–1781.
[32]
Fischer S, Wobben M, Marti HH, Renz D, Schaper W (2002) Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1. Microvasc Res 63: 70–80.
[33]
Hirase T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, et al. (1997) Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 110: 1603–1613.
[34]
McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, et al. (1996) Occludin is a functional component of the tight junction. J Cell Sci 109: 2287–2298.
[35]
He QW, Xia YP, Chen SC, Wang Y, Huang M, et al. (2013) Astrocyte-Derived Sonic Hedgehog Contributes to Angiogenesis in Brain Microvascular Endothelial Cells via RhoA/ROCK Pathway After Oxygen-Glucose Deprivation. Mol Neurobiol 47: 976–987.
[36]
Sandoval KE, Witt KA (2008) Blood-brain barrier tight junction permeability and ischemic stroke. Neurobiol Dis 32: 200–219.
[37]
Teng H, Chopp M, Hozeska-Solgot A, Shen L, Lu M, et al. (2012) Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis. PLoS One 7: e33444.
[38]
Lee SW, Kim WJ, Jun HO, Choi YK, Kim KW (2009) Angiopoietin-1 reduces vascular endothelial growth factor-induced brain endothelial permeability via upregulation of ZO-2. Int J Mol Med 23: 279–284.
[39]
Hori S, Ohtsuki S, Hosoya K, Nakashima E, Terasaki T (2004) A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J Neurochem 89: 503–513.
[40]
Yu H, Wang P, An P, Xue Y (2012) Recombinant human angiopoietin-1 ameliorates the expressions of ZO-1, occludin, VE-cadherin, and PKCα signaling after focal cerebral ischemia/reperfusion in rats. J Mol Neurosci 46: 236–247.
[41]
Mammoto T, Parikh SM, Mammoto A, Gallagher D, Chan B, et al. (2007) Angiopoietin-1 requires p190 RhoGAP to protect against vascular leakage in vivo. J Biol Chem 282: 23910–23918.
