Characterization of Blood-Brain Barrier Opening Induced by Transcranial Histotripsy in Murine Brains.

OBJECTIVE: Transcranial histotripsy has shown promise as a non-invasive neurosurgical tool, as it has the ability to treat a wide range of locations in the brain without overheating the skull. One important effect of histotripsy in the brain is the blood-brain barrier (BBB) opening (BBBO) at the ablation site, but there is a knowledge gap concerning the extent of histotripsy-induced BBBO. Here we describe induction of BBBO by transcranial histotripsy and use of magnetic resonance imaging (MRI) and histology to quantify changes in BBBO at the periphery of the histotripsy ablation zone over time in the healthy mouse brain. METHODS: An eight-element, 1 MHz histotripsy transducer with a focal distance of 32.5 mm was used to treat the brains of 23 healthy female BL6 mice. T1-gadolinium (T1-Gd) MR images were acquired immediately following histotripsy treatment and during each of the subsequent 4 wk to quantify the size and intensity of BBB leakage. RESULTS: The T1-Gd MRI results revealed that the hyperintense BBBO volume increased over the first week and subsided gradually over the following 3 wk. Histology revealed complete loss of tight junction proteins and blood vessels in the center of the ablation region immediately after histotripsy, partial recovery in the periphery of the ablation zone 1 wk following histotripsy and near-complete recovery of tight junction complex after 4 wk. CONCLUSION: These results provide the first evidence of transcranial histotripsy-induced BBBO and repair at the periphery of the ablation zone.

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.

Epigenetics and stroke: role of DNA methylation and effect of aging on blood-brain barrier recovery.

Incomplete recovery of blood-brain barrier (BBB) function contributes to stroke outcomes. How the BBB recovers after stroke remains largely unknown. Emerging evidence suggests that epigenetic factors play a significant role in regulating post-stroke BBB recovery. This study aimed to evaluate the epigenetic and transcriptional profile of cerebral microvessels after thromboembolic (TE) stroke to define potential causes of limited BBB recovery. RNA-sequencing and reduced representation bisulfite sequencing (RRBS) analyses were performed using microvessels isolated from young (6 months) and old (18 months) mice seven days poststroke compared to age-matched sham controls. DNA methylation profiling of poststroke brain microvessels revealed 11,287 differentially methylated regions (DMR) in old and 9818 DMR in young mice, corresponding to annotated genes. These DMR were enriched in genes encoding cell structural proteins (e.g., cell junction, and cell polarity, actin cytoskeleton, extracellular matrix), transporters and channels (e.g., potassium transmembrane transporter, organic anion and inorganic cation transporters, calcium ion transport), and proteins involved in endothelial cell processes (e.g., angiogenesis/vasculogenesis, cell signaling and transcription regulation). Integrated analysis of methylation and RNA sequencing identified changes in cell junctions (occludin), actin remodeling (ezrin) as well as signaling pathways like Rho GTPase (RhoA and Cdc42ep4). Aging as a hub of aberrant methylation affected BBB recovery processes by profound alterations (hypermethylation and repression) in structural protein expression (e.g., claudin-5) as well as activation of a set of genes involved in endothelial to mesenchymal transformation (e.g., Sox9, Snai1), repression of angiogenesis and epigenetic regulation. These findings revealed that DNA methylation plays an important role in regulating BBB repair after stroke, through regulating processes associated with BBB restoration and prevalently with processes enhancing BBB injury.

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.

A tissue chip with integrated digital immunosensors: In situ brain endothelial barrier cytokine secretion monitoring.

Organ-on-a-chip platforms have potential to offer more cost-effective, ethical, and human-resembling models than animal models for disease study and drug discovery. Particularly, the Blood-Brain-Barrier-on-a-chip (BBB-oC) has emerged as a promising tool to investigate several neurological disorders since it promises to provide a model of the multifunctional tissue working as an important node to control pathogen entry, drug delivery and neuroinflammation. A comprehensive understanding of the multiple physiological functions of the tissue model requires biosensors detecting several tissue-secreted substances in a BBB-oC system. However, current sensor-integrated BBB-oC platforms are only available for tissue membrane integrity characterization based on permeability measurement. Protein secretory pathways are closely associated with the tissue's various diseased conditions. At present, no biosensor-integrated BBB-oC platform exists that permits in situ tissue protein secretion analysis over time, which prohibits researchers from fully understanding the time-evolving pathology of a tissue barrier. Herein, the authors present a platform named "Digital Tissue-BArrier-CytoKine-counting-on-a-chip (DigiTACK)," which integrates digital immunosensors into a tissue chip system and demonstrates on-chip multiplexed, ultrasensitive, longitudinal cytokine secretion profiling of cultured brain endothelial barrier tissues. The integrated digital sensors utilize a novel beadless microwell format to perform an ultrafast "digital fingerprinting" of the analytes while achieving a low limit of detection (LoD) around 100-500 fg/mL for mouse MCP1 (CCL2), IL-6 and KC (CXCL1). The DigiTACK platform is extensively applicable to profile temporal cytokine secretion of other barrier-related organ-on-a-chip systems and can provide new insight into the secretory dynamics of the BBB by sequentially controlled experiments.

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) [...].

Transcriptomic Profile of Blood-Brain Barrier Remodeling in Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a small vessel disease characterized by amyloid ß (Aß) peptide deposition within the walls of medium to small-caliber blood vessels, cerebral microhemorrhage, and blood-brain barrier (BBB) leakage. It is commonly associated with late-stage Alzheimer's disease. BBB dysfunction is indicated as a pathological substrate for CAA progression with hyperpermeability, enhancing the extravasation of plasma components and inducing neuroinflammation, further worsening BBB injury and contributing to cognitive decline. Although significant effort has been made in defining the gene mutations and risk factors involved in microvascular alterations with vascular dementia and Alzheimer's disease, the intra- and intercellular pathogenic mechanisms responsible for vascular hyperpermeability are still largely unknown. The present study aimed to elucidate the transcriptional profile of the cerebral microvessels (BBB) in a murine model with CAA vasculopathy to define potential causes and underlying mechanisms of BBB injury. A comprehensive RNA sequencing analysis was performed of CAA vasculopathy in Tg-SwDI mice at 6 and 18 months in comparison to age-matched wildtype controls to examine how age and amyloid accumulation impact the transcriptional signature of the BBB. Results indicate that Aß has a critical role in triggering brain endothelial cell and BBB dysfunction in CAA vasculopathy, causing an intense proinflammatory response, impairing oxidative metabolism, altering the coagulation status of brain endothelial cells, and remodeling barrier properties. The proinflammatory response includes both adaptive and innate immunity, with pronounced induction of genes that regulate macrophage/microglial activation and chemokines/adhesion molecules that support T and B cell transmigration. Age has an important impact on the effects of Aß, increasing the BBB injury in CAA vasculopathy. However, early inflammation, particularly microglia/macrophage activation and the mediators of B lymphocytes' activities are underlying processes of BBB hyperpermeability and cerebral microbleeds in the early stage of CAA vasculopathy. These findings reveal a specific profile of the CAA-associated BBB injury that leads to a full progression of CAA.

An In Vivo Mouse Model to Study Blood-Brain Barrier Destabilization in the Chronic Phase of Stroke.

Cerebral ischemic injury evokes a complex cascade of pathophysiological events at the blood-vascular-parenchymal interface. These evolve over time and space and result in progressive neurological damage. Emerging evidence suggests that blood-brain barrier (BBB) recovery and reestablishment of BBB impermeability are incomplete and that these could influence stroke injury recovery, increase the risk of new stroke occurrence, and be a solid substrate for developing vascular dementia. Recent work from the author's laboratory has established the existence of incomplete BBB recovery in chronic stroke conditions that was induced by structural alterations to brain endothelial junctional complexes and persistent BBB leakage. The experimental methodology presented here is focused on modelling chronic stroke injury using an in vivo thromboembolic mouse stroke model and how to evaluate the kinetics and magnitude of BBB hyperpermeability in chronic stroke conditions using a combination of magnetic resonance imaging, tracer studies, and immunohistochemistry.

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.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

Tight junctions in the blood-brain barrier promote edema formation and infarct size in stroke - Ambivalent effects of sealing proteins.

The outcome of stroke is greatly influenced by the state of the blood-brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.

Modeling blood-brain barrier pathology in cerebrovascular disease in vitro: current and future paradigms.

The complexity of the blood-brain barrier (BBB) and neurovascular unit (NVU) was and still is a challenge to bridge. A highly selective, restrictive and dynamic barrier, formed at the interface of blood and brain, the BBB is a "gatekeeper" and guardian of brain homeostasis and it also acts as a "sensor" of pathological events in blood and brain. The majority of brain and cerebrovascular pathologies are associated with BBB dysfunction, where changes at the BBB can lead to or support disease development. Thus, an ultimate goal of BBB research is to develop competent and highly translational models to understand mechanisms of BBB/NVU pathology and enable discovery and development of therapeutic strategies to improve vascular health and for the efficient delivery of drugs. This review article focuses on the progress being made to model BBB injury in cerebrovascular diseases in vitro.

A novel approach to treatment of thromboembolic stroke in mice: Redirecting neutrophils toward a peripherally implanted CXCL1-soaked sponge.

Neutrophils are considered key participants in post-ischemic stroke inflammation. They are the first white blood cells to arrive in ischemic brain and their presence in the brain tissue positively correlates with post-ischemic injury severity. CXCL1 is a neutrophil attractant chemokine and the present study evaluates whether redirecting neutrophil migration using a peripherally implanted CXCL1-soaked sponge can reduce brain inflammation and improve outcomes in a novel mouse model of thromboembolic (TE) stroke. TE stroke was induced by injection of a platelet-rich microemboli suspension into the internal carotid artery of adult C57BL/6 male mice. The model induced neuroinflammation that was associated with increases in multiple brain and serum cytokines/chemokines at the mRNA and protein levels, including very marked increases in CXCL1. In other groups of animals, an absorbable sterile hemostatic sponge, previously immersed in either saline (0.9%NaCl) or CXCL1, was implanted into subcutaneous pockets formed in the inguinal region on the left and right side following stroke surgery. Mice implanted with the sponge soaked with CXCL1 had significantly reduced neuroinflammation and infarct size after TE stroke compared to mice implanted with the sponge soaked with 0.9%NaCl. There was also reduced mortality and improved neurological deficits in the TE stroke + CXCL1 sponge group compared to the TE stroke +0.9%NaCl sponge group. In conclusion: redirecting bloodstream leukocytes toward a peripherally-implanted neutrophil chemokine CXCL1-soaked sponge improves outcomes in a novel mouse model of thromboembolic stroke. The present findings suggest a novel therapeutic strategy for patients with acute stroke.

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.

Involvement of Epigenetic Mechanisms and Non-coding RNAs in Blood-Brain Barrier and Neurovascular Unit Injury and Recovery After Stroke.

Cessation of blood flow leads to a complex cascade of pathophysiological events at the blood-vascular-parenchymal interface which evolves over time and space, and results in damage to neural cells and edema formation. Cerebral ischemic injury evokes a profound and deleterious upregulation in inflammation and triggers multiple cell death pathways, but it also induces a series of the events associated with regenerative responses, including vascular remodeling, angiogenesis, and neurogenesis. Emerging evidence suggests that epigenetic reprograming could play a pivotal role in ongoing post-stroke neurovascular unit (NVU) changes and recovery. This review summarizes current knowledge about post-stroke recovery processes at the NVU, as well as epigenetic mechanisms and modifiers (e.g., DNA methylation, histone modifying enzymes and microRNAs) associated with stroke injury, and NVU repair. It also discusses novel drug targets and therapeutic strategies for enhancing post-stroke recovery.

Decline in Sirtuin-1 expression and activity plays a critical role in blood-brain barrier permeability in aging.

Accumulating evidence suggest that cerebral microvascular disease increases with advancing age and is associated with lacunar stroke, leukoaraiosis, vascular dementia and Alzheimer disease. Increased blood brain barrier (BBB) permeability/leakage takes "center stage" in ongoing age-related vascular/brain parenchymal injury. Although significant effort has been made in defining the gene mutations and risk factors involved in microvascular alterations in vascular dementia and Alzheimer disease, the intra- and intercellular pathogenic mechanisms responsible for vascular hyperpermeability are still largely unknown. The present study aimed to reveal the ongoing senescence process in brain endothelial cells and its effect on BBB integrity in healthy/non-disease conditions. An analysis of BBB integrity during the life span of C56Bl6 mice (young, 2-6 months; middle-aged, 6-12, months; old, 16-22 months) showed increased BBB permeability for different molecular sized tracers (sodium fluorescein, inulin and 20 kDa dextran) in aged mice which was accompanied by modifications in tight junction (TJ) complex organization, manifested as altered TJ protein expression (particularly claudin-5). A gene screening analysis of aging associated markers in brain microvessels isolated from "aged" mice (C56Bl6, 18-20 months) and human brain samples showed a significant decline in sirtuin-1 expression (Sirt1; ~2.8-fold) confirmed at mRNA and protein levels and by activation assay. Experiments in Sirt1 transgenic mice and brain endothelial cell-specific Sirt1 knockout mice indicated that Sirt1 affects BBB integrity, with loss increasing permeability. Similarly, in vitro, overexpressing Sirt1 or increasing Sirt1 activity with an agonist (Sirt1720) protected against senescence-induced brain endothelial barrier hyperpermeability, stabilized claudin-5/ZO-1 interactions and rescued claudin-5 expression. These findings reveal a novel role of Sirt1 in modulating aging-associated BBB persistent leakage.

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.

Expression of periaxin (PRX) specifically in the human cerebrovascular system: PDZ domain-mediated strengthening of endothelial barrier function.

Regulation of cerebral endothelial cell function plays an essential role in changes in blood-brain barrier permeability. Proteins that are important for establishment of endothelial tight junctions have emerged as critical molecules, and PDZ domain containing-molecules are among the most important. We have discovered that the PDZ-domain containing protein periaxin (PRX) is expressed in human cerebral endothelial cells. Surprisingly, PRX protein is not detected in brain endothelium in other mammalian species, suggesting that it could confer human-specific vascular properties. In endothelial cells, PRX is predominantly localized to the nucleus and not tight junctions. Transcriptome analysis shows that PRX expression suppresses, by at least 50%, a panel of inflammatory markers, of which 70% are Type I interferon response genes; only four genes were significantly activated by PRX expression. When expressed in mouse endothelial cells, PRX strengthens barrier function, significantly increases transendothelial electrical resistance (~35%; p < 0.05), and reduces the permeability of a wide range of molecules. The PDZ domain of PRX is necessary and sufficient for its barrier enhancing properties, since a splice variant (S-PRX) that contains only the PDZ domain, also increases barrier function. PRX also attenuates the permeability enhancing effects of lipopolysaccharide. Collectively, these studies suggest that PRX could potentially regulate endothelial homeostasis in human cerebral endothelial cells by modulating inflammatory gene programs.

Brain endothelial cell junctions after cerebral hemorrhage: Changes, mechanisms and therapeutic targets.

Vascular disruption is the underlying cause of cerebral hemorrhage, including intracerebral, subarachnoid and intraventricular hemorrhage. The disease etiology also involves cerebral hemorrhage-induced blood-brain barrier (BBB) disruption, which contributes an important component to brain injury after the initial cerebral hemorrhage. BBB loss drives vasogenic edema, allows leukocyte extravasation and may lead to the entry of potentially neurotoxic and vasoactive compounds into brain. This review summarizes current information on changes in brain endothelial junction proteins in response to cerebral hemorrhage (and clot-related factors), the mechanisms underlying junction modification and potential therapeutic targets to limit BBB disruption and, potentially, hemorrhage occurrence. It also addresses advances in the tools that are now available for assessing changes in junctions after cerebral hemorrhage and the potential importance of such junction changes. Recent studies suggest post-translational modification, conformational change and intracellular trafficking of junctional proteins may alter barrier properties. Understanding how cerebral hemorrhage alters BBB properties beyond changes in tight junction protein loss may provide important therapeutic insights to prevent BBB dysfunction and restore normal function.

Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure.

Familial cerebral cavernous malformations type III (fCCM3) is a disease of the cerebrovascular system caused by loss-of-function mutations in ccm3 that result in dilated capillary beds that are susceptible to hemorrhage. Before hemorrhage, fCCM3 lesions are characterized by a hyperpermeable blood-brain barrier (BBB), the key pathologic feature of fCCM3. We demonstrate that connexin 43 (Cx43), a gap junction (GJ) protein that is incorporated into the BBB junction complex, is up-regulated in lesions of a murine model of fCCM3. Small interfering RNA-mediated ccm3 knockdown (CCM3KD) in brain endothelial cells in vitro increased Cx43 protein expression, GJ plaque size, GJ intracellular communication (GJIC), and barrier permeability. CCM3KD hyperpermeability was rescued by GAP27, a peptide gap junction and hemichannel inhibitor of Cx43 GJIC. Tight junction (TJ) protein, zonula occludens 1 (ZO-1), accumulated at Cx43 GJs in CCM3KD cells and displayed fragmented staining at TJs. The GAP27-mediated inhibition of Cx43 GJs in CCM3KD cells restored ZO-1 to TJ structures and reduced plaque accumulation at Cx43 GJs. The TJ protein, Claudin-5, was also fragmented at TJs in CCM3KD cells, and GAP27 treatment lengthened TJ-associated fragments and increased Claudin 5-Claudin 5 transinteraction. Overall, we demonstrate that Cx43 GJs are aberrantly increased in fCCM3 and regulate barrier permeability by a TJ-dependent mechanism.-Johnson, A. M., Roach, J. P., Hu, A., Stamatovic, S. M., Zochowski, M. R., Keep, R. F., Andjelkovic, A. V. Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure.