Vol. 40, No. 5 569Biol. Pharm. Bull. 40, 569–575 (2017)

© 2017 The Pharmaceutical Society of Japan

Protection of the Blood–Brain Barrier as a Therapeutic Strategy for Brain Damage

Shotaro Michinaga and Yutaka Koyama*Laboratory of Pharmacology, Faculty of Pharmacy, Osaka Ohtani University;

3–11–1 Nishikiori-Kita, Tonda-bayashi, Osaka 584–8540, Japan.Received December 15, 2016

Severe brain damage by trauma, ischemia, and hemorrhage lead to fatal conditions including sudden death, subsequent complications of the extremities and cognitive dysfunctions. Despite the urgent need for treatments for these complications, currently available therapeutic drugs are limited. Blood–brain barrier (BBB) disruption is a common pathogenic feature in many types of brain damage. The characteristic patho-physiological conditions caused by BBB disruption are brain edema resulting from an excessive increase of brain water content, inflammatory damage caused by infiltrating immune cells, and hemorrhage caused by the breakdown of microvessel structures. Because these pathogenic features induced by BBB disruption cause fatal conditions, their improvement is a desirable strategy. Many studies using experimental animal models have focused on molecules involved in BBB disruption, including vascular endothelial growth factors (VEGFs), matrix metalloproteinases (MMPs) and endothelins (ETs). The inhibition of these factors in several experimental animals was protective against BBB disruption caused by several types of brain damage, and ameliorated brain edema, inflammatory damage, and hemorrhagic transformation. In patients with brain damage, the up-regulation of these factors was observed and was related to brain damage severity. Thus, BBB protection by targeting VEGFs, MMPs, and ETs might be a novel strategy for the treatment of brain damage.

Key words blood–brain barrier; brain damage; tight junction protein

1. INTRODUCTION

The brain is the most important tissue for life maintenance,and disturbances of brain functions result in many deleterious symptoms in both physical and psychological systems. Brain damage caused by traumatic brain injury (TBI), cerebral isch-emia and hemorrhage cause various fatal conditions including sudden death, coma, and motor and cognitive dysfunctions.1–3) To prevent these conditions, early treatments and appropriate strategies to prevent further damage should be performed. However, the beneficial effects of current therapeutic drugs used in the clinic are limited. Thus, the development of novel therapeutic drugs that act on different targets and exert dif-ferent mechanisms from the currently available drugs is es-sential.

The pathogenesis of brain damage is complicated because several pathophysiological conditions including brain edema, inflammation, oxidative stress, hypoxia, hemorrhage, and excitotoxicity are observed in parallel.1–3) Some pathophysi-ological conditions are different dependent on the type of brain damage, while blood–brain barrier (BBB) disruption is a common pathogenic feature in many types of brain damage.4,5) The BBB is a structural and cellular barrier that forms a bar-rier between the peripheral blood system and the brain tissue. The BBB protects against the extravasation of intravascular contents and the infiltration of intravascular immune cells into the cerebral parenchyma. Because the barrier functions of the BBB rely on endothelial cells, astrocytes and pericytes,6) dis-

turbances of the functions of these cells cause BBB dysfunc-tions. When the BBB is disrupted by brain damage, its protec-tive effects are disturbed, leading to severe pathophysiological conditions including brain edema, severe inflammatory dam-age, and hemorrhagic transformation. Thus, therapeutic drugs for BBB disruption must be beneficial for many types of brain damage.

Many studies using experimental animal models have identified several molecules involved in BBB disruption. Fur-thermore, inhibition of these molecules attenuated the BBB disruption and protection of the BBB ameliorated the severe conditions caused by TBI, cerebral ischemia, or hemorrhage. In patients with brain damage, increased BBB disruption-related factors were observed and correlated with the severity of brain damage.7–9) In this review, current investigations for novel therapeutic drugs of BBB disruption are summarized.

2. BBB DISRUPTION-INDUCED PATHOGENESIS

BBB disruption is characterized by an excessive accel-eration of microvascular permeability, which triggers severe conditions including brain edema, inflammatory damage, and hemorrhage (Fig. 1). In this section, these pathophysiological conditions are introduced.

2.1. Brain Edema Disturbances of endothelial barrier function lead to the extravasation of intravascular fluid and serum proteins resulting in an abnormal accumulation of fluid into the cerebral parenchyma. This condition is a characteris-

* To whom correspondence should be addressed. e-mail: koyamay@osaka-ohtani.ac.jp

Current Topics

Brain Damage and Potential Therapeutic Interventions

Review

570 Vol. 40, No. 5 (2017)Biol. Pharm. Bull.

tic pathophysiology of brain edema. Brain edema is a lethal pathological state characterized by an increase of brain vol-ume resulting from excessive brain water content.10,11) Because the brain tissue is covered by a robust rigid skull, an increase in brain volume causes an elevation of intracranial pressure (ICP) resulting in a compression of brain tissue and brain her-nia. Although severe brain edema causes sudden death, coma and irreversible neuronal damage, there are few beneficial therapeutic drugs available. Brain edema is classified into va-sogenic, cytotoxic and osmotic types.11) Vasogenic edema re-sults from BBB disruption.11,12) During TBI, cerebral ischemia, or hemorrhage, all of which cause BBB disruption, vasogenic edema is commonly observed and persists for several days after brain damage.12,13) Severe damage to endothelial cells and a decrease of endothelial tight junction proteins results in BBB disruption leading to vasogenic edema.11) In common with many types of brain damage, several physiological ac-tive substances including vascular endothelial growth factors (VEGFs) and matrix metalloproteinases (MMPs) are respon-sible for vasogenic edema formation through tight junction dysfunction.11)

2.2. Inflammation The BBB limits the passage of intra-vascular contents and the infiltration of intravascular immune cells such as leukocytes. Thus, dysfunction of BBB leads to the abnormal transmigration and infiltration of leukocytes into the cerebral parenchyma.14) Leukocyte transmigration through the BBB is a multi-step process including leukocyte activa-tion via chemokine stimulation and leukocyte attachment to endothelial cells.14) Leukocyte attachment occurs by interac-tions with cell adhesion molecules (CAMs) on leukocytes and endothelial cells including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).14) BBB disruption alters the functions of CAMs. The expressions of CAMs and chemokines evaluated after brain damage were reported to accelerate leukocyte infiltration.15–17) Infiltrating leukocytes migrate into areas of damage and release various cytokines, chemokines, reactive oxygen species (ROS) and

proteases that cause brain tissue damage.18)

2.3. Hemorrhage The breakdown of microvascular structures by severe BBB disruption leads to hemorrhagic transformation.3,13) Cerebral hemorrhage is a lethal condition, which results in sudden death, paralysis of the extremities, and cognitive dysfunctions. Hemorrhage results from a frac-ture of microvessels by cerebral contusions.19) In cerebral ischemia, the enhanced expression of MMP-9 is associated with hemorrhagic transformation caused by BBB disruption.20)

As a deleterious effect of tissue-type plasminogen activator (t-PA) which is a therapeutic drug for thrombus, hemorrhagic transformation by BBB disruption has been shown.3,21) Abu Fanne et al.22) suggested that inhibiting t-PA improved brain tissue damage, edema, and mortality caused by increased BBB permeability in thromboembolic stroke rats. MMPs and VEGFs are involved in BBB disruption by t-PA.21)

3. TARGETS OF THERAPEUTIC DRUGS FOR BBB DISRUPTION

As shown in Fig. 2, the BBB is composed of endothelial cells, astrocytes and pericytes known as a neurovascular Unit (NVU).6,23) Endothelial cells possess tight junction (TJ) re-lated proteins including claudin-5 (CLN-5), occludin (OCLN) and Zonula occludens-1 (ZO-1) in the cellular membrane.24) To maintain barrier functions, the adjacent endothelial cells strictly bind via these membrane proteins to form tight junc-tions that preserve the extravasation of intravascular contents and the invasion of intravascular cells.24) Thus, decreases or dysfunctions of these TJ proteins lead to a disturbance of endothelial barrier functions resulting in BBB disruption. Pericytes exist around endothelial cells and strengthen the protective functions of the BBB by regulating the proliferation and differentiation of endothelial cells.25) Astrocytes also exist around endothelial cells and support endothelial functions through their end-feet.6) A key function of astrocytes is the regulation of cerebral microvessel permeability by astrocyte-

Fig. 1. Pathophysiological Outcomes by BBB Disruption after Brain DamageBBB disruption leads to severe conditions including brain edema, inflammatory damage, and hemorrhage.

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derived transmitters.6,26) After various types of brain damage, astrocytes convert from a resting form to a reactive form. Re-active astrocyte- and microglia-derived factors are responsible for the BBB disruption.27,28) The relationship between BBB disruption and several factors including VEGFs, MMPs, and

endothelins (ETs) are shown in Fig. 3. Many studies have sug-gested that blockade of these factors attenuates BBB disrup-tion and brain tissue damage. In this section, these findings are summarized.

3.1. VEGFs VEGFs including VEGF-A, B, C, and D are

Fig. 2. Constituents of the BBB Structure Including Endothelial Cells, Astrocytes, and PericytesEndothelial cells possess TJ proteins including CLN-5, OCLN and ZO-1, which form the TJ structure. Astrocytes and pericytes exist around endothelial cells and regu-

late endothelial barrier functions.

Fig. 3. Summary of BBB Disruption by Brain DamageProduction of VEGFs, MMPs and ETs were accelerated in several types of brain cell after brain damage and these factors disrupt the BBB through the dysfunction of

TJ proteins.

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common angiogenic factors that accelerate the proliferation and migration of endothelial cells.29) VEGFs are also known as vascular permeability factors. Several studies suggested that VEGF enhances BBB permeability.30–32) Increased VEGF expressions were observed after brain damage in experimental animal models33–35) and in patients with brain damage.8,36,37) Argaw et al.27) focused on astrocytic VEGF-A as a key driver of BBB permeability and confirmed that the inactivation of astrocytic VEGF-A reduced BBB breakdown, lymphocyte infiltration and inflammatory damage in a mouse model of multiple sclerosis (MS). BBB disruption was induced by the VEGF-induced down-regulation of CLN-5 and OCLN expres-sions.38)

The beneficial effects of VEGF inhibition for BBB disrup-tion have been reported in experimental brain injury animals. In a cerebral ischemic animal model, VEGF inhibition by an anti-VEGF neutralizing antibody reduced the BBB permeabil-ity, brain edema, and infarct volume.39) Furthermore, a VEGF receptor 2 (VEGF-R2) inhibitor SU5416 and VEGF-R2 knock-down also attenuated ischemic damage-induced BBB disrup-tion.40) The beneficial effects of anti-VEGF neutralizing anti-body on t-PA-induced BBB disruption and hemorrhage were also reported.41) As summarized by Lange et al.,42) VEGF was reported to be a therapeutic target for BBB disruption and a therapeutic target for several other neurological disorders.

3.2. MMPs MMPs are a family of zinc-endopeptidases that degrade extracellular matrix (ECM) molecules such as collagen, laminin, and fibronectin.43) MMPs evoke angiogen-esis in physiological states44) while several types of MMPs including MMP-2, -3, -9, and -10 disturb BBB integrity by degrading the basal lamina in cerebral microvessels resulting in BBB breakdown.45,46) Increased MMP-2 and -9 and the ben-eficial effects of MMP inhibitors for BBB disruption were ob-served in several experimental animal models.47–50) In cortical contusion rats, the MMP-inhibitor GM6001 reduced BBB dis-ruption and brain edema.51) Another MMP inhibitor BB-1101 also reduced excessive BBB permeability in cerebral ischemia model rats.52,53) Reduced BBB disruption after cerebral isch-emia was also reported in MMP-9 knockout mice.54,55)

The involvement of MMPs in leukocyte migration and the regulation of CAM expression in the BBB were reported. MMP inhibitors reduced the increased migration of inflamma-tory cells and increased ICAM-1 and VCAM-1 expressions in inflammatory model mice.56) Agrawal et al.57) indicated that the ablation of both MMP-2 and -9 prevented leukocyte infil-tration in a mouse model of MS. Furthermore, MMP was also responsible for hemorrhagic transformation after treatment with t-PA. MMP inhibition reduced t-PA-induced hemorrhage in cerebral ischemic animals.58)

The activation of MMPs degrades the ECM and TJ pro-

teins. Chen et al.59) indicated that the overexpression of MMP-9 in brain endothelial cells caused the degradation of CLN-5 and CCLN leading to endothelial barrier disruption. In cerebral ischemia animals, the MMP-induced degradation of CLN-5 and OCLN caused BBB disruption in cerebral isch-emia animals.60)

In patients with TBI or cerebral ischemia, the up-regulation of MMPs was related to the severity of brain damage.7–9) Thus, the beneficial effects of MMP inhibition for BBB disruption-induced brain edema, inflammatory damage, and hemorrhage should be investigated in clinical trials.

3.3. ETs ETs including ET-1, -2, and -3 are vasoconstric-tor peptides that exert multiple physiological and pathological actions other than vascular constriction in nonvascular tissues including central nervous tissues.61) In experimental ischemia animals, the over-expression of ET-1 aggravated BBB break-down, brain edema, neurological deficits, and cognitive defi-cits.62,63) ET-1 over-expression also increased MMP2 expres-sion and decreased OCLN after ischemia.62) In a hemorrhage animal model, ET-1 over-expression resulted in BBB break-down, brain edema, and severe neurological dysfunction.64)

ET receptors can be classified into two types including ETA and ETB receptors. The involvement of these receptors for ET-induced BBB disruption was shown in experimental ani-mal models. S-0139, an ETA antagonist, reduced BBB perme-ability, brain edema formation, and infarct size after ischemic damage.65) Furthermore, Kim et al.66,67) suggested that BQ788, an ETB receptor antagonist, attenuated BBB disruption and vasogenic edema by inhibiting MMP-9 activation and ZO-1 protein degradation in experimental status epilepticus ani-mals. We previously reported that BQ788 ameliorated cortical cold injury-induced BBB disruption and vasogenic edema.68) Moreover, BQ788 also reversed the increase in MMP-9 and VEGF-A expressions after cold injury.69) Although further investigations are needed using similar experimental models with clinical brain damage, ETB receptor antagonists might be a beneficial therapeutic drug target to prevent BBB disruption.

4. CONCLUSION AND FUTURE DIRECTIONS

Although several drugs have been used to treat brain dam-age in the clinic, these beneficial effects are extremely limited. Thus, the development of novel drugs that affect new targets involved in the pathogenesis of brain damage is essential. In this review, we focused on BBB disruption because it is observed in many types of brain damage, and causes vari-ous lethal conditions. As a strategy for BBB protection, the inhibition of causal factors of BBB disruption is desirable. Candidate molecules for therapeutic drugs targeting BBB dis-ruption are summarized in Table 1. Because VEGFs, MMPs,

Table 1. Summary of Candidate Targets and Drugs to Prevent BBB Disruption

Target molecules Experimental drugs Clinical drugs

VEGF SU5416, SU4312 (VEGF-R2 antagonist) Bevacizumab (VEGF neutralizing antibody)MMP GM6001, BB-1101 (MMP inhibitor)

MMP9 inhibitor 1ET S-0139, BQ-123, FR139317 (ETA antagonist)

BQ788, IRL-1038, IRL-2500 (ETB antagonist)Ambrisentan, Macitentan (ETA antagonist)

Bosentan (ETA and ETB antagonist)

Candidate drugs with inhibitory effects for VEGFs, MMPs, and ET receptors are summarized.

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and ETs cause the dysfunction of TJ proteins, the inhibition of these factors might protect endothelial barrier functions. Bevacizumab, a VEGF neutralizing antibody, has been used in the clinic to inhibit the proliferation and metastasis of tu-mors. Bosentan, a non-selective ET receptor antagonist, and ambrisentan and macitentan, selective ETA antagonists, have also been used as therapeutic drugs for pulmonary hyperten-sion. The inhibitory effects of cilostazol and fasudil on MMPs have also been reported.70) However, these clinical drugs have been not used for brain damage. Thus, the beneficial effects of these drugs for BBB disruption should be examined in clinical trials. Because selective ETB receptor antagonists have not yet been used in the clinic, the development of an ETB antagonist as a novel therapeutic drug is also expected. Inter-estingly, dexamethasone used to attenuate brain edema in the clinic downregulated VEGF and MMP-9 levels in vitro and in vivo experimental models.17,71) Thus, the anti-edema effects of dexamethasone may involve ameliorating BBB disruption that is commonly observed in TBI, cerebral ischemia, and hemor-rhage, and which leads to lethal conditions including brain edema, severe inflammatory damage, and hemorrhage. Thus, protection of the BBB must be a beneficial strategy for these pathophysiological conditions and a broad spectrum of brain damage.

Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research (C) from the JPSP (15K07981).

Conflict of Interest The authors declare no conflict of interest.

REFERENCES

1) Mracsko E, Veltkamp R. Neuroinflammation after intracerebral hemorrhage. Front. Cell. Neurosci., 8, 388 (2014).

2) Alluri H, Wiggins-Dohlvik K, Davis ML, Huang JH, Tharakan B. Blood–brain barrier dysfunction following traumatic brain injury. Metab. Brain Dis., 30, 1093–1104 (2015).

3) Turner RJ, Sharp FR. Implications of MMP9 for blood–brain bar-rier disruption and hemorrhagic transformation following ischemic stroke. Front. Cell. Neurosci., 10, 56 (2016).

4) Bramlett HM, Dietrich WD. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. J. Cereb. Blood Flow Metab., 24, 133–150 (2004).

5) Schoknecht K, Shalev H. Blood–brain barrier dysfunction in brain diseases: clinical experience. Epilepsia, 53 (Suppl. 6), 7–13 (2012).

6) Abbott NJ, Rönnbäck L, Hansson E. Astrocyte-endothelial inter-actions at the blood–brain barrier. Nat. Rev. Neurosci., 7, 41–53 (2006).

7) Grossetete M, Phelps J, Arko L, Yonas H, Rosenberg GA. Eleva-tion of matrix metalloproteinases 3 and 9 in cerebrospinal fluid and blood in patients with severe traumatic brain injury. Neurosurgery, 65, 702–708 (2009).

8) Hirose T, Matsumoto N, Tasaki O, Nakamura H, Akagaki F, Shima-zu T. Delayed progression of edema formation around a hematoma expressing high levels of VEGF and MMP-9 in a patient with trau-matic brain injury. Case report. Neurol. Med. Chir. (Tokyo), 53, 609–612 (2013).

9) Zheng K, Li C, Shan X, Liu H, Fan W, Wang Z, Zheng P. Matrix metalloproteinases and their tissue inhibitors in serum and cerebro-spinal fluid of patients with moderate and severe traumatic brain injury. Neurol. India, 61, 606–609 (2013).

10) Unterberg AW, Stover J, Kress B, Kiening KL. Edema and brain

trauma. Neuroscience, 129, 1019–1029 (2004).11) Nag S, Manias JL, Stewart DJ. Pathology and new players in the

pathogenesis of brain edema. Acta Neuropathol., 118, 197–217 (2009).

12) Stokum JA, Gerzanich V, Simard JM. Molecular pathophysiology of cerebral edema. J. Cereb. Blood Flow Metab., 36, 513–538 (2016).

13) Thrane AS, Rangroo Thrane V, Nedergaard M. Drowning stars: reassessing the role of astrocytes in brain edema. Trends Neurosci., 37, 620–628 (2014).

14) Lopes Pinheiro MA, Kooij G, Mizee MR, Kamermans A, Enzmann G, Lyck R, Schwaninger M, Engelhardt B, de Vries HE. Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke. Biochim. Biophys. Acta, 1862, 461–471 (2016).

15) Wang X, Feuerstein GZ. Induced expression of adhesion molecules following focal brain ischemia. J. Neurotrauma, 12, 825–832 (1995).

16) Pleines UE, Stover JF, Kossmann T, Trentz O, Morganti-Kossmann MC. Soluble ICAM-1 in CSF coincides with the extent of cerebral damage in patients with severe traumatic brain injury. J. Neurotrau-ma, 15, 399–409 (1998).

17) Yang JT, Lee TH, Lee IN, Chung CY, Kuo CH, Weng HH. Dexa-methasone inhibits ICAM-1 and MMP-9 expression and reduces brain edema in intracerebral hemorrhagic rats. Acta Neurochir. (Wien), 153, 2197–2203 (2011).

18) Das M, Mohapatra S, Mohapatra SS. New perspectives on central and peripheral immune responses to acute traumatic brain injury. J. Neuroinflammation, 9, 236 (2012).

19) Kurland D, Hong C, Aarabi B, Gerzanich V, Simard JM. Hemor-rhagic progression of a contusion after traumatic brain injury: a review. J. Neurotrauma, 29, 19–31 (2012).

20) Chaturvedi M, Kaczmarek L. Mmp-9 inhibition: a therapeutic strat-egy in ischemic stroke. Mol. Neurobiol., 49, 563–573 (2014).

21) Suzuki Y, Nagai N, Umemura K. A review of the mechanisms of blood–brain barrier permeability by tissue-type plasminogen acti-vator treatment for cerebral ischemia. Front. Cell. Neurosci., 10, 2 (2016).

22) Abu Fanne R, Nassar T, Yarovoi S, Rayan A, Lamensdorf I, Kara-koveski M, Vadim P, Jammal M, Cines DB, Higazi AA. Blood–brain barrier permeability and tPA-mediated neurotoxicity. Neuro-pharmacology, 58, 972–980 (2010).

23) Wolburg H, Noell S, Mack A, Wolburg-Buchholz K, Fallier-Becker P. Brain endothelial cells and the glio-vascular complex. Cell Tissue Res., 335, 75–96 (2009).

24) Stamatovic SM, Keep RF, Andjelkovic AV. Brain endothelial cell–cell junctions: how to open the blood–brain barrier. Curr. Neuro-pharmacol., 6, 179–192 (2008).

25) ElAli A, Thériault P, Rivest S. The role of pericytes in neuro-vascular unit remodeling in brain disorders. Int. J. Mol. Sci., 15, 6453–6474 (2014).

26) Alvarez JI, Katayama T, Prat A. Glial influence on the blood–brain barrier. Glia, 61, 1939–1958 (2013).

27) Argaw AT, Asp L, Zhang J, Navrazhina K, Pham T, Mariani JN, Mahase S, Dutta DJ, Seto J, Kramer EG, Ferrara N, Sofroniew MV, John GR. Astrocyte-derived VEGF-A drives blood–brain bar-rier disruption in CNS inflammatory disease. J. Clin. Invest., 122, 2454–2468 (2012).

28) da Fonseca AC, Matias D, Garcia C, Amaral R, Geraldo LH, Freitas C, Lima FR. The impact of microglial activation on blood–brain barrier in brain diseases. Front. Cell. Neurosci., 8, 362 (2014).

29) Roy H, Bhardwaj S, Ylä-Herttuala S. Biology of vascular endothe-lial growth factors. FEBS Lett., 580, 2879–2887 (2006).

30) Zhang ZG, Zhang L, Jiang Q, Zhang R, Davies K, Powers C, Bruggen N, Chopp M. VEGF enhances angiogenesis and promotes blood–brain barrier leakage in the ischemic brain. J. Clin. Invest., 106, 829–838 (2000).

574 Vol. 40, No. 5 (2017)Biol. Pharm. Bull.

31) Ay I, Francis JW, Brown RH Jr. VEGF increases blood–brain barri-er permeability to Evans blue dye and tetanus toxin fragment C but not adeno-associated virus in ALS mice. Brain Res., 1234, 198–205 (2008).

32) Jiang S, Xia R, Jiang Y, Wang L, Gao F. Vascular endothelial growth factors enhance the permeability of the mouse blood–brain barrier. PLoS ONE, 9, e86407 (2014).

33) Jośko J. Cerebral angiogenesis and expression of VEGF after sub-arachnoid hemorrhage (SAH) in rats. Brain Res., 981, 58–69 (2003).

34) Sköld MK, von Gertten C, Sandberg-Nordqvist AC, Mathiesen T, Holmin S. VEGF and VEGF receptor expression after experimental brain contusion in rat. J. Neurotrauma, 22, 353–367 (2005).

35) Lee C, Agoston DV. Vascular endothelial growth factor is involved in mediating increased de novo hippocampal neurogenesis in response to traumatic brain injury. J. Neurotrauma, 27, 541–553 (2010).

36) Shore PM, Jackson EK, Wisniewski SR, Clark RS, Adelson PD, Kochanek PM. Vascular endothelial growth factor is increased in cerebrospinal fluid after traumatic brain injury in infants and chil-dren. Neurosurgery, 54, 605–611, discussion, 611–612 (2004).

37) Matsuo R, Ago T, Kamouchi M, Kuroda J, Kuwashiro T, Hata J, Sugimori H, Fukuda K, Gotoh S, Makihara N, Fukuhara M, Awano H, Isomura T, Suzuki K, Yasaka M, Okada Y, Kiyohara Y, Kitazo-no T. Clinical significance of plasma VEGF value in ischemic stroke-research for biomarkers in ischemic stroke (REBIOS) study. BMC Neurol., 13, 32 (2013).

38) Argaw AT, Gurfein BT, Zhang Y, Zameer A, John GR. VEGF-mediated disruption of endothelial CLN-5 promotes blood–brain barrier breakdown. Proc. Natl. Acad. Sci. U.S.A., 106, 1977–1982 (2009).

39) Kimura R, Nakase H, Tamaki R, Sakaki T. Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke, 36, 1259–1263 (2005).

40) Reeson P, Tennant KA, Gerrow K, Wang J, Weiser Novak S, Thompson K, Lockhart KL, Holmes A, Nahirney PC, Brown CE. Delayed inhibition of VEGF signaling after stroke attenuates blood–brain barrier breakdown and improves functional recovery in a comorbidity-dependent manner. J. Neurosci., 35, 5128–5143 (2015).

41) Kanazawa M, Igarashi H, Kawamura K, Takahashi T, Kakita A, Takahashi H, Nakada T, Nishizawa M, Shimohata T. Inhibition of VEGF signaling pathway attenuates hemorrhage after tPA treat-ment. J. Cereb. Blood Flow Metab., 31, 1461–1474 (2011).

42) Lange C, Storkebaum E, de Almodóvar CR, Dewerchin M, Carme-liet P. Vascular endothelial growth factor: a neurovascular target in neurological diseases. Nat. Rev. Neurol., 12, 439–454 (2016).

43) Mott JD, Werb Z. Regulation of matrix biology by matrix metal-loproteinases. Curr. Opin. Cell Biol., 16, 558–564 (2004).

44) Pepper MS. Role of the matrix metalloproteinase and plasminogen activator–plasmin systems in angiogenesis. Arterioscler. Thromb. Vasc. Biol., 21, 1104–1117 (2001).

45) Seo JH, Guo S, Lok J, Navaratna D, Whalen MJ, Kim KW, Lo EH. Neurovascular matrix metalloproteinases and the blood–brain bar-rier. Curr. Pharm. Des., 18, 3645–3648 (2012).

46) Lakhan SE, Kirchgessner A, Tepper D, Leonard A. Matrix metal-loproteinases and blood–brain barrier disruption in acute ischemic stroke. Front Neurol., 4, 32 (2013).

47) Wang J, Tsirka SE. Neuroprotection by inhibition of matrix metallo-proteinases in a mouse model of intracerebral haemorrhage. Brain, 128, 1622–1633 (2005).

48) Zhao BQ, Wang S, Kim HY, Storrie H, Rosen BR, Mooney DJ, Wang X, Lo EH. Role of matrix metalloproteinases in delayed cor-tical responses after stroke. Nat. Med., 12, 441–445 (2006).

49) Sifringer M, Stefovska V, Zentner I, Hansen B, Stepulak A, Knaute C, Marzahn J, Ikonomidou C. The role of matrix metalloprotein-ases in infant traumatic brain injury. Neurobiol. Dis., 25, 526–535

(2007).50) Tejima E, Zhao BQ, Tsuji K, Rosell A, van Leyen K, Gonzalez RG,

Montaner J, Wang X, Lo EH. Astrocytic induction of matrix metal-loproteinase-9 and edema in brain hemorrhage. J. Cereb. Blood Flow Metab., 27, 460–468 (2007).

51) Shigemori Y, Katayama Y, Mori T, Maeda T, Kawamata T. Matrix metalloproteinase-9 is associated with blood–brain barrier opening and brain edema formation after cortical contusion in rats. Acta Neurochir. Suppl., 96, 130–133 (2006).

52) Rosenberg GA, Estrada EY, Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood–brain barrier opening after reperfusion in rat brain. Stroke, 29, 2189–2195 (1998).

53) Sood RR, Taheri S, Candelario-Jalil E, Estrada EY, Rosenberg GA. Early beneficial effect of matrix metalloproteinase inhibition on blood–brain barrier permeability as measured by magnetic resonance imaging countered by impaired long-term recovery after stroke in rat brain. J. Cereb. Blood Flow Metab., 28, 431–438 (2008).

54) Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood–brain barrier and white matter compo-nents after cerebral ischemia. J. Neurosci., 21, 7724–7732 (2001).

55) Svedin P, Hagberg H, Sävman K, Zhu C, Mallard C. Matrix metal-loproteinase-9 gene knock-out protects the immature brain after cerebral hypoxia-ischemia. J. Neurosci., 27, 1511–1518 (2007).

56) Lee KS, Jin SM, Kim HJ, Lee YC. Matrix metalloproteinase inhibi-tor regulates inflammatory cell migration by reducing ICAM-1 and VCAM-1 expression in a murine model of toluene diisocyanate-induced asthma. J. Allergy Clin. Immunol., 111, 1278–1284 (2003).

57) Agrawal S, Anderson P, Durbeej M, van Rooijen N, Ivars F, Opde-nakker G, Sorokin LM. Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasa-tion in experimental autoimmune encephalomyelitis. J. Exp. Med., 203, 1007–1019 (2006).

58) Lapchak PA, Chapman DF, Zivin JA. Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)-induced hemor-rhage after thromboembolic stroke. Stroke, 31, 3034–3040 (2000).

59) Chen F, Ohashi N, Li W, Eckman C, Nguyen JH. Disruptions of oc-cludin and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure. Hepatology, 50, 1914–1923 (2009).

60) Yang Y, Estrada EY, Thompson JF, Liu W, Rosenberg GA. Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J. Cereb. Blood Flow Metab., 27, 697–709 (2007).

61) Schinelli S. Pharmacology and physiopathology of the brain endo-thelin system: An overview. Curr. Med. Chem., 13, 627–638 (2006).

62) Leung JW, Chung SS, Chung SK. Endothelial endothelin-1 over-ex-pression using receptor tyrosine kinase tie-1 promoter leads to more severe vascular permeability and blood–brain barrier breakdown after transient middle cerebral artery occlusion. Brain Res., 1266, 121–129 (2009).

63) Zhang X, Yeung PK, McAlonan GM, Chung SS, Chung SK. Trans-genic mice overexpressing endothelial endothelin-1 show cognitive deficit with blood–brain barrier breakdown after transient ischemia with long-term reperfusion. Neurobiol. Learn. Mem., 101, 46–54 (2013).

64) Yeung PK, Shen J, Chung SS, Chung SK. Targeted over-expression of endothelin-1 in astrocytes leads to more severe brain damage and vasospasm after subarachnoid hemorrhage. BMC Neurosci., 14, 131 (2013).

65) Matsuo Y, Mihara S, Ninomiya M, Fujimoto M. Protective effect of endothelin type A receptor antagonist on brain edema and injury after transient middle cerebral artery occlusion in rats. Stroke, 32, 2143–2148 (2001).

66) Kim JE, Ryu HJ, Kang TC. Status epilepticus induces vasogenic

Vol. 40, No. 5 (2017) 575Biol. Pharm. Bull.

edema via tumor necrosis factor-α/endothelin-1-mediated two dif-ferent pathways. PLoS ONE, 8, e74458 (2013).

67) Kim JY, Ko AR, Hyun HW, Kang TC. ETB receptor-mediated MMP-9 activation induces vasogenic edema via ZO-1 protein deg-radation following status epilepticus. Neuroscience, 304, 355–367 (2015).

68) Michinaga S, Nagase M, Matsuyama E, Yamanaka D, Seno N, Fuka M, Yamamoto Y, Koyama Y. Amelioration of cold injury-induced cortical brain edema formation by selective endothelin ETB recep-tor antagonists in mice. PLoS ONE, 9, e102009 (2014).

69) Michinaga S, Seno N, Fuka M, Yamamoto Y, Minami S, Kimura A, Hatanaka S, Nagase M, Matsuyama E, Yamanaka D, Koyama Y.

Improvement of cold injury-induced mouse brain edema by endo-thelin ETB antagonists is accompanied by decreases in matrixme-talloproteinase 9 and vascular endothelial growth factor-A. Eur. J. Neurosci., 42, 2356–2370 (2015).

70) Lakhan SE, Kirchgessner A, Tepper D, Leonard A. Matrix metal-loproteinases and blood–brain barrier disruption in acute ischemic stroke. Front Neurol., 4, 32 (2013).

71) Kim H, Lee JM, Park JS, Jo SA, Kim YO, Kim CW, Jo I. Dexa-methasone coordinately regulates angiopoietin-1 and VEGF: a mechanism of glucocorticoid-induced stabilization of blood–brain barrier. Biochem. Biophys. Res. Commun., 372, 243–248 (2008).