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

Abstract

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 pathophysiological 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.

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 ischemia 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 different mechanisms from the currently available drugs is essential.

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 pathophysiological 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 barrier 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⁾ disturbances of the functions of these cells cause BBB dysfunctions. When the BBB is disrupted by brain damage, its protective effects are disturbed, leading to severe pathophysiological conditions including brain edema, severe inflammatory damage, 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. Furthermore, 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 acceleration of microvascular permeability, which triggers severe conditions including brain edema, inflammatory damage, and hemorrhage (Fig. 1). In this section, these pathophysiological conditions are introduced.

Fig. 1. Pathophysiological Outcomes by BBB Disruption after Brain Damage

BBB disruption leads to severe conditions including brain edema, inflammatory damage, and hemorrhage.

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 characteristic pathophysiology of brain edema. Brain edema is a lethal pathological state characterized by an increase of brain volume 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 hernia. Although severe brain edema causes sudden death, coma and irreversible neuronal damage, there are few beneficial therapeutic drugs available. Brain edema is classified into vasogenic, cytotoxic and osmotic types.¹¹⁾ Vasogenic edema results 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 active substances including vascular endothelial growth factors (VEGFs) and matrix metalloproteinases (MMPs) are responsible for vasogenic edema formation through tight junction dysfunction.¹¹⁾

2.2. Inflammation

The BBB limits the passage of intravascular 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 activation via chemokine stimulation and leukocyte attachment to endothelial cells.¹⁴⁾ Leukocyte attachment occurs by interactions 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 fracture 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) related 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 junctions 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-derived transmitters.^6,26) After various types of brain damage, astrocytes convert from a resting form to a reactive form. Reactive 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 suggested that blockade of these factors attenuates BBB disruption and brain tissue damage. In this section, these findings are summarized.

Fig. 2. Constituents of the BBB Structure Including Endothelial Cells, Astrocytes, and Pericytes

Endothelial cells possess TJ proteins including CLN-5, OCLN and ZO-1, which form the TJ structure. Astrocytes and pericytes exist around endothelial cells and regulate endothelial barrier functions.

Fig. 3. Summary of BBB Disruption by Brain Damage

Production 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.

3.1. VEGFs

VEGFs including VEGF-A, B, C, and D are 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 models^33–35) 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 expressions.³⁸⁾

The beneficial effects of VEGF inhibition for BBB disruption 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 permeability, brain edema, and infarct volume.³⁹⁾ Furthermore, a VEGF receptor 2 (VEGF-R2) inhibitor SU5416 and VEGF-R2 knockdown also attenuated ischemic damage-induced BBB disruption.⁴⁰⁾ The beneficial effects of anti-VEGF neutralizing antibody 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 angiogenesis 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 beneficial effects of MMP inhibitors for BBB disruption were observed in several experimental animal models.^47–50) In cortical contusion rats, the MMP-inhibitor GM6001 reduced BBB disruption 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 ischemia 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 inflammatory 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 infiltration 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 proteins. 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 ischemia 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 vasoconstrictor 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 breakdown, brain edema, neurological deficits, and cognitive deficits.^62,63) ET-1 over-expression also increased MMP2 expression and decreased OCLN after ischemia.⁶²⁾ In a hemorrhage animal model, ET-1 over-expression resulted in BBB breakdown, 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 animal models. S-0139, an ET_A antagonist, reduced BBB permeability, brain edema formation, and infarct size after ischemic damage.⁶⁵⁾ Furthermore, Kim et al.^66,67) suggested that BQ788, an ET_B receptor antagonist, attenuated BBB disruption and vasogenic edema by inhibiting MMP-9 activation and ZO-1 protein degradation in experimental status epilepticus animals. 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, ET_B 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 damage 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 various 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 disruption are summarized in Table 1. Because VEGFs, MMPs, 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 tumors. Bosentan, a non-selective ET receptor antagonist, and ambrisentan and macitentan, selective ET_A antagonists, have also been used as therapeutic drugs for pulmonary hypertension. 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 ET_B receptor antagonists have not yet been used in the clinic, the development of an ET_B antagonist as a novel therapeutic drug is also expected. Interestingly, 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 hemorrhage, 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.

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 1
ET	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.

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.

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