Endothelial peroxiredoxin-4 is indispensable for blood-brain barrier integrity and long-term functional recovery after ischemic stroke.

The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury.

Circadian Rhythms of the Blood-Brain Barrier and Drug Delivery.

The blood-brain barrier (BBB) is a critical interface separating the central nervous system from the peripheral circulation, ensuring brain homeostasis and function. Recent research has unveiled a profound connection between the BBB and circadian rhythms, the endogenous oscillations synchronizing biological processes with the 24-hour light-dark cycle. This review explores the significance of circadian rhythms in the context of BBB functions, with an emphasis on substrate passage through the BBB. Our discussion includes efflux transporters and the molecular timing mechanisms that regulate their activities. A significant focus of this review is the potential implications of chronotherapy, leveraging our knowledge of circadian rhythms for improving drug delivery to the brain. Understanding the temporal changes in BBB can lead to optimized timing of drug administration, to enhance therapeutic efficacy for neurological disorders while reducing side effects. By elucidating the interplay between circadian rhythms and drug transport across the BBB, this review offers insights into innovative therapeutic interventions.

Blood-Brain Barrier Dysfunction in Normal Aging and Neurodegeneration: Mechanisms, Impact, and Treatments.

Cerebral endothelial cells and their linking tight junctions form a unique, dynamic and multi-functional interface, the blood-brain barrier (BBB). The endothelium is regulated by perivascular cells and components forming the neurovascular unit. This review examines BBB and neurovascular unit changes in normal aging and in neurodegenerative disorders, particularly focusing on Alzheimer disease, cerebral amyloid angiopathy and vascular dementia. Increasing evidence indicates BBB dysfunction contributes to neurodegeneration. Mechanisms underlying BBB dysfunction are outlined (endothelium and neurovascular unit mediated) as is the BBB as a therapeutic target including increasing the uptake of systemically delivered therapeutics across the BBB, enhancing clearance of potential neurotoxic compounds via the BBB, and preventing BBB dysfunction. Finally, a need for novel biomarkers of BBB dysfunction is addressed.

Brain edema formation and therapy after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) accounts for about 10% of all strokes in the United States of America causing a high degree of disability and mortality. There is initial (primary) brain injury due to the mechanical disruption caused by the hematoma. There is then secondary injury, triggered by the initial injury but also the release of various clot-derived factors (e.g., thrombin and hemoglobin). ICH alters brain fluid homeostasis. Apart from the initial hematoma mass, ICH causes blood-brain barrier disruption and parenchymal cell swelling, which result in brain edema and intracranial hypertension affecting patient prognosis. Reducing brain edema is a critical part of post-ICH care. However, there are limited effective treatment methods for reducing perihematomal cerebral edema and intracranial pressure in ICH. This review discusses the mechanisms underlying perihematomal brain edema formation, the effects of sex and age, as well as how edema is resolved. It examines progress in pharmacotherapy, particularly focusing on drugs which have been or are currently being investigated in clinical trials.

Mechanisms of cerebrospinal fluid and brain interstitial fluid production.

Fluid homeostasis is fundamental for brain function with cerebral edema and hydrocephalus both being major neurological conditions. Fluid movement from blood into brain is one crucial element in cerebral fluid homeostasis. Traditionally it has been thought to occur primarily at the choroid plexus (CP) as cerebrospinal fluid (CSF) secretion due to polarized distribution of ion transporters at the CP epithelium. However, there are currently controversies as to the importance of the CP in fluid secretion, just how fluid transport occurs at that epithelium versus other sites, as well as the direction of fluid flow in the cerebral ventricles. The purpose of this review is to evaluate evidence on the movement of fluid from blood to CSF at the CP and the cerebral vasculature and how this differs from other tissues, e.g., how ion transport at the blood-brain barrier as well as the CP may drive fluid flow. It also addresses recent promising data on two potential targets for modulating CP fluid secretion, the Na+/K+/Cl- cotransporter, NKCC1, and the non-selective cation channel, transient receptor potential vanilloid 4 (TRPV4). Finally, it raises the issue that fluid secretion from blood is not constant, changing with disease and during the day. The apparent importance of NKCC1 phosphorylation and TRPV4 activity at the CP in determining fluid movement suggests that such secretion may also vary over short time frames. Such dynamic changes in CP (and potentially blood-brain barrier) function may contribute to some of the controversies over its role in brain fluid secretion.

20 kDa isoform of connexin-43 augments spatial reorganization of the brain endothelial junctional complex and lesion leakage in cerebral cavernous malformation type-3.

Cerebral cavernous malformation type-3 (CCM3) is a type of brain vascular malformation caused by mutations in programmed cell death protein-10 (PDCD10). It is characterized by early life occurrence of hemorrhagic stroke and profound blood-brain barrier defects. The pathogenic mechanisms responsible for microvascular hyperpermeability and lesion progression in CCM3 are still largely unknown. The current study examined brain endothelial barrier structural defects formed in the absence of CCM3 in vivo and in vitro that may lead to CCM3 lesion leakage. We found significant upregulation of a 20 kDa isoform of connexin 43 (GJA1-20 k) in brain endothelial cells (BEC) in both non-leaky and leaky lesions, as well as in an in vitro CCM3 knockdown model (CCM3KD-BEC). Morphological, biochemical, FRET, and FRAP analyses of CCM3KD-BEC found GJA1-20 k regulates full-length GJA1 biogenesis, prompting uncontrolled gap junction growth. Furthermore, by binding to a tight junction scaffolding protein, ZO-1, GJA1-20 k interferes with Cx43/ZO-1 interactions and gap junction/tight junction crosstalk, promoting ZO-1 dissociation from tight junction complexes and diminishing claudin-5/ZO-1 interaction. As a consequence, the tight junction complex is destabilized, allowing "replacement" of tight junctions with gap junctions leading to increased brain endothelial barrier permeability. Modifying cellular levels of GJA1-20 k rescued brain endothelial barrier integrity re-establishing the spatial organization of gap and tight junctional complexes. This study highlights generation of potential defects at the CCM3-affected brain endothelial barrier which may underlie prolonged vascular leakiness.

Proton-Coupled Oligopeptide Transport (Slc15) in the Brain: Past and Future Research.

This mini-review describes the role of the solute carrier (SLC)15 family of proton-coupled oligopeptide transporters (POTs) and particularly Pept2 (Slc15A2) and PhT1 (Slc15A4) in the brain. That family transports endogenous di- and tripeptides and peptidomimetics but also a number of drugs. The review focuses on the pioneering work of David E. Smith in the field in identifying the impact of PepT2 at the choroid plexus (the blood-CSF barrier) as well as PepT2 and PhT1 in brain parenchymal cells. It also discusses recent findings and future directions in relation to brain POTs including cellular and subcellular localization, regulatory pathways, transporter structure, species differences and disease states.

Novel Approach to Visualize Microglia Death and Proliferation After Intracerebral Hemorrhage in Mice.

BACKGROUND: Microglia are important brain immune cells. However, it is difficult to differentiate microglia from monocyte-derived macrophages. To visualize microglia changes following intracerebral hemorrhage (ICH), we utilized a genetic knock-in mouse line, Tmem119 (transmembrane protein 119)-EGFP (enhanced green fluorescent protein), which expresses EGFP specifically in microglia. METHODS: There were 2 parts in this study. First, autologous blood was injected into the right basal ganglia to model ICH in Tmem119-EGFP mice. Mice were euthanized at 4 hours, days 1, 3, and 7 after ICH. Sham animals were used as controls. Second, Tmem119-EGFP mice were injected with iron or thrombin, factors involved in ICH-induced injury, and were euthanized at 4 hours. Naïve mice were controls. Brains were harvested for histology. RESULTS: The number of perihematomal microglia significantly decreased 1 day after ICH, but markedly increased by days 3 and 7. Microglia death was also induced by intracerebral iron injection while microglia proliferation was found with intracerebral thrombin injection. CONCLUSIONS: Perihematomal microglia death and proliferation after ICH are visualized in vivo with a Tmem119-EGFP transgenic mouse line. Iron and thrombin may contribute to ICH-induced microglia death and proliferation, respectively.

Cerebral Cavernous Malformation Pathogenesis: Investigating Lesion Formation and Progression with Animal Models.

Cerebral cavernous malformation (CCM) is a cerebromicrovascular disease that affects up to 0.5% of the population. Vessel dilation, decreased endothelial cell-cell contact, and loss of junctional complexes lead to loss of brain endothelial barrier integrity and hemorrhagic lesion formation. Leakage of hemorrhagic lesions results in patient symptoms and complications, including seizures, epilepsy, focal headaches, and hemorrhagic stroke. CCMs are classified as sporadic (sCCM) or familial (fCCM), associated with loss-of-function mutations in KRIT1/CCM1, CCM2, and PDCD10/CCM3. Identifying the CCM proteins has thrust the field forward by (1) revealing cellular processes and signaling pathways underlying fCCM pathogenesis, and (2) facilitating the development of animal models to study CCM protein function. CCM animal models range from various murine models to zebrafish models, with each model providing unique insights into CCM lesion development and progression. Additionally, these animal models serve as preclinical models to study therapeutic options for CCM treatment. This review briefly summarizes CCM disease pathology and the molecular functions of the CCM proteins, followed by an in-depth discussion of animal models used to study CCM pathogenesis and developing therapeutics.

Molecular Mechanisms of Cerebrovascular Diseases.

Cerebrovascular disease involves a range of conditions including ischemic and hemorrhagic stroke, vascular malformations, and vascular cognitive impairment and dementia (VCID) [...].

Delayed Minocycline Treatment Ameliorates Hydrocephalus Development and Choroid Plexus Inflammation in Spontaneously Hypertensive Rats.

Hydrocephalus is a complicated disorder that affects both adult and pediatric populations. The mechanism of hydrocephalus development, especially when there is no mass lesion present causing an obstructive, is poorly understood. Prior studies have demonstrated that spontaneously hypertensive rats (SHRs) develop hydrocephalus by week 7, which was attenuated with minocycline. The aim of this study was to determine sex differences in hydrocephalus development and to examine the effect of minocycline administration after hydrocephalus onset. Male and female Wistar-Kyoto rats (WKYs) and SHRs underwent magnetic resonance imaging at weeks 7 and 9 to determine ventricular volume. Choroid plexus epiplexus cell activation, cognitive deficits, white matter atrophy, and hippocampal neuronal loss were examined at week 9. In the second phase of the experiment, male SHRs (7 weeks old) were treated with either saline or minocycline (20 mg/kg) for 14 days, and similar radiologic, histologic, and behavior tests were performed. Hydrocephalus was present at week 7 and increased at week 9 in both male and female SHRs, which was associated with greater epiplexus cell activation than WKYs. Male SHRs had greater ventricular volume and epiplexus cell activation compared to female SHRs. Minocycline administration improved cognitive function, white matter atrophy, and hippocampal neuronal cell loss. In conclusion, while both male and female SHRs developed hydrocephalus and epiplexus cell activation by week 9, it was more severe in males. Delayed minocycline treatment alleviated hydrocephalus, epiplexus macrophage activation, brain pathology, and cognitive impairment in male SHRs.

Role of Complement Component 3 in Early Erythrolysis in the Hematoma After Experimental Intracerebral Hemorrhage.

Background and Purpose: Early erythrolysis occurs within the hematoma following intracerebral hemorrhage (ICH), and the release of erythrocyte cytoplasmic proteins such as hemoglobin and Prx2 (peroxiredoxin 2) can cause brain injury. Complement activation can induce erythrolysis. This study determined the function of complement component 3 (C3) in erythrolysis in hematoma and brain injury after ICH in mice. Methods: This study has 3 parts. First, ICH was induced in adult male C3-sufficient and deficient mice and animals were euthanized on days 1, 3, 7, and 28 for immunohistochemistry after magnetic resonance imaging and behavioral testing. Second, C3-sufficient and deficient mice with ICH were euthanized on day 1 for Western blot analysis. Third, C3-sufficient mice received injections of PBS and Prx2. Mice underwent both magnetic resonance imaging and behavioral tests on day 1 and were then euthanized. Brains were harvested for immunohistochemistry and Fluoro-Jade C staining. Results: Erythrolysis occurred in the hematoma in C3-sufficient and deficient mice on day 3 following ICH. C3-deficient mice had less erythrolysis, brain swelling, and neuronal degeneration in the acute phase and less brain atrophy in the chronic phase. There were fewer neurological deficits on days 3, 7, and 28 in C3-deficient mice. C3-deficient mice also had less extracellular Prx2 release. Moreover, Prx2 induced brain edema and brain injury and recruited macrophage scavenger receptor-1- and CD4-positive cells following ICH in mice. Conclusions: C3-deficient mice had less severe erythrolysis and brain injury following ICH compared with C3-sufficient mice. Prx2 released after erythrolysis can cause brain damage and neuroinflammation in mice.

Ultra-Early Cerebral Thrombosis Formation After Experimental Subarachnoid Hemorrhage Detected on T2* Magnetic Resonance Imaging.

BACKGROUND AND PURPOSE: The mechanisms of brain damage during ultra-early subarachnoid hemorrhage (SAH) have not been well studied. The current study examined the SAH-induced hyperacute brain damage at 4 hours using magnetic resonance imaging and brain histology in a mouse model. METHODS: SAH was induced by endovascular perforation in adult mice. First, adult male wild-type mice underwent magnetic resonance imaging T2 and T2* 4 hours after an endovascular perforation or a sham operation and were euthanized to assess brain histology. Second, male and female adult lipocalin-2 knockout mice had SAH. All animals underwent magnetic resonance imaging at 4 hours, and the brains were harvested for brain histology. RESULTS: T2* hypointensity vessels were observed in the brain 4 hours after SAH in male wild-type mice. The numbers of T2*-positive vessels were significantly higher in SAH brains than in sham-operated mice. Brain histology showed thrombosis and erythrocyte plugs in the T2*-positive cerebral vessels which may be venules. The number of T2*-positive vessels correlated with SAH grade and the presence of T2 lesions. Brain thrombosis was also accompanied by albumin leakage and neuronal injury. LCN2 deficient male mice had lower numbers of T2*-positive vessels after SAH compared with wild-type male mice. CONCLUSIONS: SAH causes ultra-early brain vessel thrombosis that can be detected by T2* gradient-echo sequence at 4 hours after SAH. LCN2 deficiency decreased the number of T2*-positive vessels.

CD47 blocking antibody accelerates hematoma clearance and alleviates hydrocephalus after experimental intraventricular hemorrhage.

Background CD47, a glycoprotein on red blood cell membranes, inhibits phagocytosis via interaction with signal regulatory protein α on phagocytes. Our previous research has demonstrated that blocking CD47 accelerates hematoma clearance and reduces brain injury after intracerebral hemorrhage. The current study investigated whether phagocytosis or erythrocyte CD47 impacts hematoma resolution and hydrocephalus development after intraventricular hemorrhage (IVH). Methods Adult (3-month-old) male Fischer 344 rats were intraventricularly injected with 200 µl autologous blood, mixed with either CD47 blocking antibody or isotype IgG, or 200 µl saline as control. In subgroups of CD47 blocking antibody treated rats, clodronate liposomes (to deplete microglia/monocyte-derived macrophages) or control liposomes were co-injected. Magnetic resonance imaging (MRI) was used to evaluate ventricular volume and intraventricular T2* lesion volume (estimating hematoma volume). The brains were harvested after 4 or 72 h for histology to evaluate phagocytosis. Results In adult male rats, CD47 blocking antibody alleviated hydrocephalus development by day 3. In addition, the CD47 blocking antibody reduced intraventricular T2* lesion and T2* non-hypointense lesion size after IVH through day 1 to day 3. Erythrophagocytosis was observed as soon as 4 h after IVH and was enhanced on day 3. Furthermore, intra-hematoma infiltration of CD68, heme oxygenase-1 and ferritin positive phagocytes were upregulated by CD47 blockade by day 3. Clodronate liposomes co-injection caused more severe hydrocephalus and weight loss. Conclusion Blocking CD47 in the hematoma accelerated hematoma clearance and alleviated hemolysis and hydrocephalus development after IVH, suggesting CD47 might be valuable in the future treatment for IVH.

Cerebrospinal Fluid from Aneurysmal Subarachnoid Hemorrhage Patients Leads to Hydrocephalus in Nude Mice.

OBJECTIVE: Our prior studies have found that intracerebroventricular injection of blood components can cause hydrocephalus and choroid plexus epiplexus cell activation in rats. To minimize the cross-species reaction, the current study examines whether intraventricular injection of acellular components of cerebrospinal fluid (CSF) from subarachnoid hemorrhage patients can cause hydrocephalus and epiplexus macrophage activation in nude mice which lack a T cell inflammatory response. METHODS: Adult male nude mice received intraventricular injections of acellular CSF from subarachnoid hemorrhage patients or a control patient. All mice had preoperative magnetic resonance imaging as baseline and postoperative scans at 24 h after CSF injection to determine ventricular volume. Brains were harvested at 24 h for brain histology, immunohistochemistry, and electron microscopy. RESULTS: Intraventricular injection of CSF from two of five subarachnoid hemorrhage patients obtained < 48 h from ictus resulted in ventricular enlargement at 24 h. CSF-related hydrocephalus was associated with activation of epiplexus macrophages and ependymal injury. CONCLUSIONS: Components of the acellular CSF of subarachnoid hemorrhage patients can cause epiplexus macrophage activation, ependymal cell damage, and ventricular enlargement in nude mice. This may serve as a unique model to study mechanisms of hydrocephalus development following subarachnoid hemorrhage.

Hydrocephalus Following Experimental Subarachnoid Hemorrhage in Rats with Different Aerobic Capacity.

Low aerobic capacity is considered to be a risk factor for stroke, while the mechanisms underlying the phenomenon are still unclear. The current study looked into the impacts of different aerobic capacities on early brain injury in a subarachnoid hemorrhage (SAH) model using rats bred for high and low aerobic capacity (high-capacity runners, HCR; low-capacity runners, LCR). SAH was modeled with endovascular perforation in HCR and LCR rats. Twenty-four hours after SAH, the rats underwent behavioral testing and MRI, and were then euthanized. The brains were used to investigate ventricular wall damage, blood-brain barrier breakdown, oxidative stress, and hemoglobin scavenging. The LCR rats had worse SAH grades (p < 0.01), ventricular dilatation (p < 0.01), ventricular wall damage (p < 0.01), and behavioral scores (p < 0.01). The periventricular expression of HO-1 and CD163 was significantly increased in LCR rats (p < 0.01 each). CD163-positive cells were co-localized with HO-1-positive cells. The LCR rats had greater early brain injuries than HCR rats. The LCR rats had more serious SAH and extensive ventricular wall damage that evolved more frequently into hydrocephalus. This may reflect changes in iron handling and neuroinflammation.

Claudin-1-Dependent Destabilization of the Blood-Brain Barrier in Chronic Stroke.

Recent evidence suggests that blood-brain barrier (BBB) recovery and reestablishment of BBB impermeability after stroke is incomplete. This could influence stroke recovery, increase the risk of repeat stroke, and be a solid substrate for developing vascular dementia. Although accumulating evidence has defined morphological alterations and underlying mechanisms of tight junction (TJ) changes during BBB breakdown in acute stroke, very little is known about the type of alterations and mechanisms in BBB "leakage" found subacutely or chronically. The current study examined BBB structural alterations during the "BBB leakage" associated with the chronic phase of stroke in male mice and both genders of humans. We found significant upregulation of claudin-1 mRNA and protein, a nonspecific claudin for blood vessels, and downregulation in claudin-5 expression. Morphological and biochemical as well as fluorescence resonance energy transfer and fluorescence recovery after photobleaching analysis of postischemic brain endothelial cells and cells overexpressing claudin-1 indicated that newly synthesized claudin-1 was present on the cell membrane (∼45%), was incorporated into the TJ complex with established interaction with zonula occludens-1 (ZO-1), and was building homophilic cis- and trans-interactions. The appearance of claudin-1 in the TJ complex reduced claudin-5 strands (homophilic claudin-5 cis- and trans-interactions) and claudin-5/ZO-1 interaction affecting claudin-5 incorporation into the TJ complex. Moreover, claudin-1 induction was associated with an endothelial proinflammatory phenotype. Targeting claudin-1 with a specific C1C2 peptide improved brain endothelial barrier permeability and functional recovery in chronic stroke condition. This study highlights a potential "defect" in postischemic barrier formation that may underlie prolonged vessel leakiness.SIGNIFICANCE STATEMENT Although rarely expressed at the normal blood-brain barrier (BBB), claudin-1 is expressed in pathological conditions. Analyzing poststroke human and mouse blood microvessels we have identified that claudin-1 is highly expressed in leaky brain microvessels. Our results reveal that claudin-1 is incorporated in BBB tight junction complex, impeding BBB recovery and causing BBB leakiness during poststroke recovery. Targeting claudin-1 with a claudin-1 peptide improves brain endothelial barrier permeability and consequently functional neurological recovery after stroke.

Prx2 (Peroxiredoxin 2) as a Cause of Hydrocephalus After Intraventricular Hemorrhage.

Background and Purpose- Our recent study demonstrated that release of Prx2 (peroxiredoxin 2) from red blood cells (RBCs) is involved in the inflammatory response and brain injury after intracerebral hemorrhage. The current study investigated the role of extracellular Prx2 in hydrocephalus development after experimental intraventricular hemorrhage. Methods- There were 4 parts in this study. First, Sprague-Dawley rats received an intraventricular injection of lysed RBC or saline and were euthanized at 1 hour for Prx2 measurements. Second, rats received an intraventricular injection of Prx2, deactivated Prx2, or saline. Third, lysed RBC was coinjected with conoidin A, a Prx2 inhibitor, or vehicle. Fourth, rats received Prx2 injection and were treated with minocycline or saline (i.p.). The effects of Prx2 and the inhibitors were examined using magnetic resonance imaging assessing ventriculomegaly, histology assessing ventricular wall damage, and immunohistochemistry to assess inflammation, particularly at the choroid plexus. Results- Intraventricular injection of lysed RBC resulted in increased brain Prx2 and hydrocephalus. Intraventricular injection of Prx2 alone caused hydrocephalus, ventricular wall damage, activation of choroid plexus epiplexus cells (macrophages), and an accumulation of neutrophils. Conoidin A attenuated lysed RBC-induced injury. Systemic minocycline treatment reduced the epiplexus cell activation and hydrocephalus induced by Prx2. Conclusions- Prx2 contributed to the intraventricular hemorrhage-induced hydrocephalus, probably by inducing inflammatory responses in choroid plexus and ventricular wall damage.

Endothelial Targets in Stroke: Translating Animal Models to Human.

Cerebral ischemia (stroke) induces injury to the cerebral endothelium that may contribute to parenchymal injury and worsen outcome. This review focuses on current preclinical studies examining how to prevent ischemia-induced endothelial dysfunction. It particularly focuses on targets at the endothelium itself. Those include endothelial tight junctions, transcytosis, endothelial cell death, and adhesion molecule expression. It also examines how such studies are being translated to the clinic, especially as adjunct therapies for preventing intracerebral hemorrhage during reperfusion of the ischemic brain. Identification of endothelial targets may prove valuable in a search for combination therapies that would specifically protect different cell types in ischemia.

Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood-brain barrier disruption after ischemic brain injury.

The damage borne by the endothelial cells (ECs) forming the blood-brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs-namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30-60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs-but not within neurons-ameliorated BBB impairment 1-24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown.