Blood-brain Barrier Preservation Research Articles

Autotaxin-Lysophosphatidic Acid Axis: Key Regulator of Blood-Brain Barrier Integrity and Stroke Outcomes during Cerebral Ischemia-Reperfusion

Background: Endothelial hyperpermeability subsequent to ischemic stroke poses severe ramifications for patient outcomes, with the preservation of blood-brain barrier (BBB) integrity being paramount. The pathological role of the autotaxin-lysophosphatidic acid (ATX-LPA) axis in various disorders is established. While LPA's detrimental effects in stroke pathology have been documented, the mechanistic role of its enzymatic precursor, autotaxin (ATX), especially in the context of BBB disruption and mitochondrial dysfunction, necessitates deeper exploration.Objective: Our research aimed to dissect the intricate interplay of the ATX-LPA axis in modulating BBB integrity and mitochondrial bioenergetics during cerebral ischemia-reperfusion injury.Methods: Employing well-characterized murine models, ischemic strokes were meticulously induced, succeeded by reperfusion. A gamut of advanced assays—including the electrical cell-substrate impedance sensor, Seahorse XF24 analyzer, and AR-2 probe fluorescence—were systematically deployed. Moreover, to attain a nuanced understanding, we genetically engineered a mouse model exhibiting endothelial-specific ATX deletion, facilitating a direct interrogation of ATX's endothelial role.Results: Post-ischemic reperfusion culminated in a statistically significant upregulation of ATX mRNA (1.7-fold, p<0.05) and a quadrupled surge in its enzymatic activity (p<0.01). Disturbingly, the I/R cohort manifested augmented LPA concentrations, concomitant with a pronounced mitochondrial bioenergetic collapse, evidenced by metrics such as the oxygen consumption rate and ATP synthesis (both p<0.01). BBB integrity was critically compromised, a derangement that ATX inhibitors ameliorated (p<0.05). Intriguingly, mice with endothelial-specific ATX ablation exhibited remarkably attenuated permeability (p<0.05) and infarct volumetric reductions (p<0.01), while preserving cerebral perfusion (p<0.05).Conclusion: Our findings underscore the ATX-LPA axis as a linchpin in the pathophysiological cascade following ischemic stroke, specifically impinging on BBB and mitochondrial health. Deciphering and subsequently targeting this nexus could herald a paradigm shift in therapeutic strategies for ischemic stroke. This research was supported by National Institutes of Health grants HL141998, HL141998-01S1, AA025744, AA026708, and AA025744-02S1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

WITHDRAWN: Recombinant osteopontin stabilizes the blood brain barrier following subarachnoid hemorrhage in rats

Background and PurposeRecombinant osteopontin (rOPN) decreases neuronal cell death and brain edema and improves the neurological scores following subarachnoid hemorrhage (SAH) in rats. The molecular mechanisms of the preservation of blood-brain barrier of rOPN following SAH in rats were examined in this study. MethodsAdult rats were divided into 3 groups: sham, vehicle, and treatment groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the brains was harvested at 24 hours for analysis. For the mechanism part of the study two additional groups were operated and treated with Fib-14 and Wortmannin additional to rOPN. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 hours after SAH induction. The blood brain barrier (BBB) permeability was evaluated using Evans Blue dye extravasation. The expressions of tight junction proteins claudin-3, claudin-5, occludin and the phosphorylation of β-catenin were examined using Western blot analysis. ResultsrOPN improved neurological scores and reduced brain edema at 24 hours after SAH. The BBB preservation action of rOPN is mediated in part through stabilizing β-catenin and increasing expression of tight junction proteins, claudin-3, claudin-5, and occludin. rOPN is thought to exert its effects through FAK activated PI3K-Akt pathway. ConclusionSince BBB disruption is one of the major pathophysiology of EBI after SAH, rOPN may be a promising drug to maintain BBB integrity and functions in SAH treatment.

Human-induced pluripotent stem cell-derived neural stem cell exosomes improve blood-brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis.

JOURNAL/nrgr/04.03/01300535-202502000-00029/figure1/v/2024-05-28T214302Z/r/image-tiff Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis. Human-induced pluripotent stem cell-derived neural stem cell exosomes (hiPSC-NSC-Exos) have shown potential for brain injury repair in central nervous system diseases. In this study, we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism. Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits, enhanced blood-brain barrier integrity, and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage. Additionally, hiPSC-NSC-Exos decreased immune cell infiltration, activated astrocytes, and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and tumor necrosis factor-α post-intracerebral hemorrhage, thereby improving the inflammatory microenvironment. RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion, thereby improving blood-brain barrier integrity. Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects. In summary, our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity, in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.

High-Mobility Group Box 1-Signaling Inhibition With Glycyrrhizin Prevents Cerebral T-Cell Infiltration After Cardiac Arrest.

Background High-mobility group box 1 (HMGB1) is a major promotor of ischemic injuries and aseptic inflammatory responses. We tested its inhibition on neurological outcome and systemic immune response after cardiac arrest (CA) in rabbits. Methods and Results After 10 minutes of ventricular fibrillation, rabbits were resuscitated and received saline (control) or the HMGB1 inhibitor glycyrrhizin. A sham group underwent a similar procedure without CA. After resuscitation, glycyrrhizin blunted the successive rises in HMGB1, interleukin-6, and interleukin-10 blood levels as compared with control. Blood counts of the different immune cell populations were not different in glycyrrhizin versus control. After animal awakening, neurological outcome was improved by glycyrrhizin versus control, regarding both clinical recovery and histopathological damages. This was associated with reduced cerebral CD4+ and CD8+ T-cell infiltration beginning 2 hours after CA. Conversely, granulocytes' attraction or loss of microglial cells or cerebral monocytes were not modified by glycyrrhizin after CA. These modifications were not related to the blood-brain barrier preservation with glycyrrhizin versus control. Interestingly, the specific blockade of the HMGB1 receptor for advanced glycation end products by FPS-ZM1 recapitulated the neuroprotective effects of glycyrrhizin. Conclusions Our findings support that the early inhibition of HMGB1-signaling pathway prevents cerebral chemoattraction of T cells and neurological sequelae after CA. Glycyrrhizin could become a clinically relevant therapeutic target in this situation.

Cytoskeletal protein breakdown and serum albumin extravasation in MRI DWI-T2WI mismatch area in acute murine cerebral ischemia

MRI diffusion-weighted imaging (DWI)-FLAIR mismatch is known as predictive of symptom onset within 4.5 h. This study assessed the breakdown of cytoskeletal protein and blood-brain barrier (BBB) in DWI-T2 mismatch. We employed occlusion of middle cerebral artery (MCAO) in C57BL/6 mice. We serially measured MRI including DWI and T2WI. After MRI, we prepared brain sections or samples and examined microtubule-associated protein 2 (MAP2) expression, alpha-fodrin degradation, extravasation of albumin and claudin-5 expression. In permanent or transient MCAO for 45 min, DWI hyperintensities was already found at 60 min without change of T2, showing DWI-T2 mismatch. In permanent MCAO, MAP2 expressions were preserved, and no extravasation of albumin was observed. In transient MCAO, MAP2 immunoreaction was already lost in the lateral part of the striatum. In both models, alpha-fodrin degradation was already detected. At 180 min, T2 hyperintensities appeared, where MAP2 signal was lost and albumin extravasation was found. At 24 h, hyperintensities of DWI and T2WI was found in the whole MCA territory, where MAP2 signal was completely lost with marked albumin extravasation and alpha-fodrin degradation. Immunoreaction for claudin-5 was preserved up to 180 min. DWI-T2 mismatch area may not always indicate intactness of cytoskeletal protein but shows preservation of BBB.

Role of pericytes in blood–brain barrier preservation during ischemia through tunneling nanotubes

Crosstalk mechanisms between pericytes, endothelial cells, and astrocytes preserve integrity and function of the blood-brain-barrier (BBB) under physiological conditions. Long intercellular channels allowing the transfer of small molecules and organelles between distant cells called tunneling nanotubes (TNT) represent a potential substrate for energy and matter exchanges between the tripartite cellular compartments of the BBB. However, the role of TNT across BBB cells under physiological conditions and in the course of BBB dysfunction is unknown. In this work, we analyzed the TNT’s role in the functional dialog between human brain endothelial cells, and brain pericytes co-cultured with human astrocytes under normal conditions or after exposure to ischemia/reperfusion, a condition in which BBB breakdown occurs, and pericytes participate in the BBB repair. Using live time-lapse fluorescence microscopy and laser-scanning confocal microscopy, we found that astrocytes form long TNT with pericytes and endothelial cells and receive functional mitochondria from both cell types through this mechanism. The mitochondrial transfer also occurred in multicellular assembloids of human BBB that reproduce the three-dimensional architecture of the BBB. Under conditions of ischemia/reperfusion, TNT formation is upregulated, and astrocytes exposed to oxygen-glucose deprivation were rescued from apoptosis by healthy pericytes through TNT-mediated transfer of functional mitochondria, an effect that was virtually abolished in the presence of TNT-destroying drugs. The results establish a functional role of TNT in the crosstalk between BBB cells and demonstrate that TNT-mediated mitochondrial transfer from pericytes rescues astrocytes from ischemia/reperfusion-induced apoptosis. Our data confirm that the pericytes might play a pivotal role in preserving the structural and functional integrity of BBB under physiological conditions and participate in BBB repair in brain diseases.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood–brain barrier in acute ischemic stroke

Blood–brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database (https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

Activation of Frizzled-7 attenuates blood\u2013brain barrier disruption through Dvl/\u03b2-catenin/WISP1 signaling pathway after intracerebral hemorrhage in mice

BackgroundDestruction of blood–brain barrier (BBB) ​​is one of the main mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Frizzled-7 is a key protein expressed on the surface of endothelial cells that controls vascular permeability through the Wnt-canonical pathway involving WNT1-inducible signaling pathway protein 1 (WISPI). This study aimed to investigate the role of Frizzled-7 signaling in BBB preservation after ICH in mice.MethodsAdult CD1 mice were subjected to sham surgery or collagenase-induced ICH. Frizzled-7 activation or knockdown was performed by administration of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) by intracerebroventricular injection at 48 h before ICH induction. WISP1 activation or WISP1 knockdown was performed to evaluate the underlying signaling pathway. Post-ICH assessments included neurobehavior, brain edema, BBB permeability, hemoglobin level, western blot and immunofluorescence.ResultsThe brain expressions of Frizzled-7 and WISP1 significantly increased post-ICH. Frizzled-7 was expressed in endothelial cells, astrocytes, and neurons after ICH. Activation of Frizzled-7 significantly improved neurological function, reduced brain water content and attenuated BBB permeability to large molecular weight substances after ICH. Whereas, knockdown of Frizzled-7 worsened neurological function and brain edema after ICH. Activation of Frizzled-7 significantly increased the expressions of Dvl, β-Catenin, WISP1, VE-Cadherin, Claudin-5, ZO-1 and reduced the expression of phospho-β-Catenin. WISP1 knockdown abolished the effects of Frizzled-7 activation on the expressions of VE-Cadherin, Claudin-5 and ZO-1 at 24 h after ICH.ConclusionsFrizzled-7 activation potentially attenuated BBB permeability and improved neurological deficits after ICH through Dvl​​/β-Catenin/WISP1 pathway. Frizzled-7 may be a potential target for the development of ICH therapeutic drugs.

Mesenchymal Stem/Stromal Cell Therapy in Blood-Brain Barrier Preservation Following Ischemia: Molecular Mechanisms and Prospects.

Ischemic stroke is the leading cause of mortality and long-term disability worldwide. Disruption of the blood–brain barrier (BBB) is a prominent pathophysiological mechanism, responsible for a series of subsequent inflammatory cascades that exacerbate the damage to brain tissue. However, the benefit of recanalization is limited in most patients because of the narrow therapeutic time window. Recently, mesenchymal stem cells (MSCs) have been assessed as excellent candidates for cell-based therapy in cerebral ischemia, including neuroinflammatory alleviation, angiogenesis and neurogenesis promotion through their paracrine actions. In addition, accumulating evidence on how MSC therapy preserves BBB integrity after stroke may open up novel therapeutic targets for treating cerebrovascular diseases. In this review, we focus on the molecular mechanisms of MSC-based therapy in the ischemia-induced prevention of BBB compromise. Currently, therapeutic effects of MSCs for stroke are primarily based on the fundamental pathogenesis of BBB breakdown, such as attenuating leukocyte infiltration, matrix metalloproteinase (MMP) regulation, antioxidant, anti-inflammation, stabilizing morphology and crosstalk between cellular components of the BBB. We also discuss prospective studies to improve the effectiveness of MSC therapy through enhanced migration into defined brain regions of stem cells. Targeted therapy is a promising new direction and is being prioritized for extensive research.

Interactions of Lipids, Lipoproteins, and Apolipoproteins with the Blood-Brain Barrier.

Lipids and lipoproteins are a diverse group of substances and their interactions with the blood-brain barrier (BBB) is similarly diverse. Some lipoproteins such as high density lipoprotein (HDL), apolipoprotein (apo) A-I, apoJ, some free fatty acids, and triglycerides cross the BBB whereas others such as apoE do not. Some forms of cholesterol can cross the BBB and others do not. Lipids can have effects on BBB preservation and function: HDL may protect the BBB during multiple sclerosis, cholesterol can disrupt the BBB, and triglycerides inhibit the transport of leptin across the BBB and the activation of the hypothalamic leptin receptor. ApoE is associated with many effects on the BBB, with the specific isoform apoE4 having detrimental effects. In summary, the diverse ways in which lipids, lipoproteins, and apolipoproteins interact with the BBB is important in both health and disease.

Targeting Caveolin-1 and Claudin-5 with AY9944, Improve Blood-Brain Barrier Permeability; Computational Simulation and Experimental Study.

The current study aimed to determine the protective effect of AY9944 related to Caveolin-1 and Claudin-5 role in lipid raft, which can rescue the blood-brain barrier from enhanced permeability. Therefore, in vivo analyses were performed following ischemia in normal, ischemic, and AY9944-treated animal groups. The results revealed that AY9944 reduced the infarct size, edema, and brain water content. The extravasation of Alb-Alexa 594 and biocytin-TMR was minimum in the AY9944-treated animals. The results showed a significant decrease in the expression level of Caveolin-1 over 8h and 48h and a remarkable increase in the level of Claudin-5 over 48h following ischemia in AY9944-treated animals. Molecular docking simulation demonstrated that AY9944 exerts a possible protective role via attenuating the interaction of the Caveolin-1 and cholesterol in lipid raft. These findings point out that AY9944 plays a protective role in stroke by means of blood-brain barrier preservation. Proper neural function essentially needs a constant homeostatic brain environment which is provided by the blood-brain barrier. Rescuing blood-brain barrier from enhanced permeability via inducing the protective effect of AY9944 related to caveolin-1 and claudin-5 role in lipid raft was the aim of the current study.

The single intravenous administration of methylene blue after traumatic brain injury diminishes neurological deficit, blood-brain barrier disruption and decrease in the expression of S100 protein in rats

The protective effect of methylene blue (MB) was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus, an increase in the expression of S100 protein and blood-brain barrier (BBB) permeability in the ipsilateral hemisphere. The single intravenous injection of MB (1 mg/kg body weight) 30 min after TBI significantly reduced the limb function impairment as well as a TBI-induced increase in the expression of inflammatory marker S100 protein, and BBB permeability. When modeling inflammation in vitro, MB was found to protect cultured neurons from the toxic effects of lipopolysaccharide.In conclusion, the preservation of blood-brain barrier and a decrease in the expression of S100 protein may be an important mechanism by means of which MB improves neurological outcome.Our data demonstrate that MB can be a very promising pharmacological compound with neuroprotective properties for TBI treatment.

Endothelium-targeted overexpression of Krüppel-like factor 11 protects the blood-brain barrier function after ischemic brain injury.

Microvascular endothelial cell (EC) injury and the subsequent blood-brain barrier (BBB) breakdown are frequently seen in many neurological disorders, including stroke. We have previously documented that peroxisome proliferator-activated receptor gamma (PPARγ)-mediated cerebral protection during ischemic insults needs Krüppel-like factor 11 (KLF11) as a critical coactivator. However, the role of endothelial KLF11 in cerebrovascular function and stroke outcome is unclear. This study is aimed at investigating the regulatory role of endothelial KLF11 in BBB preservation and neurovascular protection after ischemic stroke. EC-targeted overexpression of KLF11 significantly mitigated BBB leakage in ischemic brains, evidenced by significantly reduced extravasation of BBB tracers and infiltration of peripheral immune cells, and less brain water content. Endothelial cell-selective KLF11 transgenic (EC-KLF11 Tg) mice also exhibited smaller brain infarct and improved neurological function in response to ischemic insults. Furthermore, EC-targeted transgenic overexpression of KLF11 preserved cerebral tight junction (TJ) levels and attenuated the expression of pro-inflammatory factors in mice after ischemic stroke. Mechanistically, we demonstrated that KLF11 directly binds to the promoter of major endothelial TJ proteins including occludin and ZO-1 to promote their activities. Our data indicate that KLF11 functions at the EC level to preserve BBB structural and functional integrity, and therefore, confers brain protection in ischemic stroke. KLF11 may be a novel therapeutic target for the treatment of ischemic stroke and other neurological conditions involving BBB breakdown and neuroinflammation.

Microthrombus-Targeting Micelles for Neurovascular Remodeling and Enhanced Microcirculatory Perfusion in Acute Ischemic Stroke.

Reperfusion injury exists as the major obstacle to full recovery of neuron functions after ischemic stroke onset and clinical thrombolytic therapies. Complex cellular cascades including oxidative stress, neuroinflammation, and brain vascular impairment occur within neurovascular units, leading to microthrombus formation and ultimate neuron death. In this work, a multitarget micelle system is developed to simultaneously modulate various cell types involved in these events. Briefly, rapamycin is encapsulated in self-assembled micelles that are consisted of reactive oxygen species (ROS)-responsive and fibrin-binding polymers to achieve micelle retention and controlled drug release within the ischemic lesion. Neuron survival is reinforced by the combination of micelle facilitated ROS elimination and antistress signaling pathway interference under ischemia conditions. In vivo results demonstrate an overall remodeling of neurovascular unit through micelle polarized M2 microglia repair and blood-brain barrier preservation, leading to enhanced neuroprotection and blood perfusion. This strategy gives a proof of concept that neurovascular units can serve as an integrated target for ischemic stroke treatment with nanomedicines.

Canonical Wnt Pathway Maintains Blood-Brain Barrier Integrity upon Ischemic Stroke and Its Activation Ameliorates Tissue Plasminogen Activator Therapy.

Stroke induces blood-brain barrier (BBB) breakdown, which promotes complications like oedema and hemorrhagic transformation. Administration of recombinant tissue plasminogen activator (rtPA) within a therapeutic time window of 4.5h after stroke onset constitutes the only existing treatment. Beyond this time window, rtPA worsens BBB breakdown. Canonical Wnt pathway induces BBB formation and maturation during ontogeny. We hypothesized that the pathway is required to maintain BBB functions after stroke; thus, its activation might improve rtPA therapy. Therefore, we first assessed pathway activity in the brain of mice subjected to transient middle cerebral artery occlusion (MCAo). Next, we evaluated the effect of pathway deactivation early after stroke onset on BBB functions. Finally, we assessed the impact of pathway activation on BBB breakdown associated to delayed administration of rtPA. Our results show that pathway activity is induced predominately in endothelial cells early after ischemic stroke. Early deactivation of the pathway using a potent inhibitor, XAV939, aggravates BBB breakdown and increases hemorrhagic transformation incidence. On the other hand, pathway activation using a potent activator, 6-bromoindirubin-3'-oxime (6-BIO), reduces the incidence of hemorrhagic transformation associated to delayed rtPA administration by attenuating BBB breakdown via promotion of tight junction formation and repressing endothelial basal permeability independently of rtPA proteolytic activity. BBB preservation upon pathway activation limited the deleterious effects of delayed rtPA administration. Our study demonstrates that activation of the canonical Wnt pathway constitutes a clinically relevant strategy to extend the therapeutic time window of rtPA by attenuating BBB breakdown via regulation of BBB-specific mechanisms.

Magnesium: Pathophysiological mechanisms and potential therapeutic roles in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) remains the second-most common form of stroke with high morbidity and mortality. ICH can be divided into two pathophysiological stages: an acute primary phase, including hematoma volume expansion, and a subacute secondary phase consisting of blood-brain barrier disruption and perihematomal edema expansion. To date, all major trials for ICH have targeted the primary phase with therapies designed to reduce hematoma expansion through blood pressure control, surgical evacuation, and hemostasis. However, none of these trials has resulted in improved clinical outcomes. Magnesium is a ubiquitous element that also plays roles in vasodilation, hemostasis, and blood-brain barrier preservation. Animal models have highlighted potential therapeutic roles for magnesium in neurological diseases specifically targeting these pathophysiological mechanisms. Retrospective studies have also demonstrated inverse associations between admission magnesium levels and hematoma volume, hematoma expansion, and clinical outcome in patients with ICH. These associations, coupled with the multifactorial role of magnesium that targets both primary and secondary phases of ICH, suggest that magnesium may be a viable target of study in future ICH studies.

Loss of Regulator of G-Protein Signaling 5 Leads to Neurovascular Protection in Stroke.

Background and Purpose- In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Pericyte detachment contributes to BBB disruption and neurovascular dysfunction, but little is known about its regulation in stroke. Here, we investigated how loss of RGS5 (regulator of G protein signaling 5) in pericytes affects BBB breakdown in stroke and its consequences. Method- We used RGS5 knockout and control mice and applied a permanent middle cerebral occlusion model. We analyzed pericyte numbers, phenotype, and vessel morphology using immunohistochemistry and confocal microscopy. We investigated BBB breakdown by measuring endothelial coverage, tight junctions, and AQP4 (aquaporin 4) in addition to BBB permeability (fluorescent-conjugated dextran extravasation). Tissue hypoxia was assessed with pimonidazole hydrochloride and neuronal death quantified with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results- We demonstrate that loss of RGS5 increases pericyte numbers and their endothelial coverage, which is associated with higher capillary density and length, and significantly less BBB damage after stroke. Loss of RGS5 in pericytes results in reduced vascular leakage and preserved tight junctions and AQP4, decreased cerebral hypoxia, and partial neuronal protection in the infarct area. Conclusions- Our findings show that loss of RGS5 affects pericyte-related BBB preservation in stroke and identifies RGS5 as an important target for neurovascular protection.

Lithium Posttreatment Alleviates Blood–Brain Barrier Injury After Intracerebral Hemorrhage in Rats

Vasogenic cerebral edema formation after blood–brain barrier (BBB) damage aggravates the devastating consequences of intracerebral hemorrhage (ICH). The present study aims to probe into a therapeutic method on BBB preservation after ICH with a glycogen synthase kinase-3β (GSK-3β) inhibitor, lithium. Intrastriatal infusion of semicoagulated autologous whole blood or sham surgery was performed on male Sprague-Dawley (SD) rats (n = 208). Experimental animals received administration of 4,6-disubstitutedpyrrolo-pyrimidine (TWS119), lithium alone or in combination with a phosphatidylinositol 3-kinase inhibitor, wortmannin, after ICH. Behavioral tests, brain edema, and BBB permeability were determined at 24 and 72 h after surgery. Expressions of Akt, GSK-3β, β-catenin, claudin-1 and claudin-3 were evaluated via Western blots. Our results showed lithium alone posttreatment activated GSK-3β, therefore increasing active β-catenin and claudin-1 and claudin-3 expressions, which were accompanied with improved BBB integrity and ameliorated sensorimotor deficits and brain edema in ICH animals. We concluded that lithium alone reduced BBB damage after ICH, likely through regulating Akt/GSK-3β pathway and stabilizing β-catenin.

Somatostatin Maintains Permeability and Integrity of Blood-Brain Barrier in β-Amyloid Induced Toxicity.

In Alzheimer's disease (AD), the impaired clearance of β-amyloid peptide (Aβ) due to disrupted tight junction and transporter proteins is the prominent cause of disease progression. Somatostatin (SST) blocks the aggregation of Aβ and inflammation whereas reduction of SST levels in the CSF and brain tissue is associated with impaired cognitive function and memory loss. However, the role of SST in preservation of blood-brain barrier (BBB) integrity and functionality in Aβ-induced toxicity is not known. In the present study using human CMEC/D3 cells, we demonstrate that SST prevents Aβ-induced BBB permeability by regulating LRP1 and RAGE expression and improving the disrupted tight junction proteins. Furthermore, SST abrogates Aβ-induced JNK phosphorylation and expression of MMP2. Taken together, results presented here suggest that SST might serve as a therapeutic intervention in AD via targeting multiple pathways responsible for neurotoxicity, impaired BBB function, and disease progression.

Targeted homing of CCR2-overexpressing mesenchymal stromal cells to ischemic brain enhances post-stroke recovery partially through PRDX4-mediated blood-brain barrier preservation.

Rationale: Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic strategy for the acute ischemic stroke (AIS). However, the poor targeted migration and low engraftment in ischemic lesions restrict their treatment efficacy. The ischemic brain lesions express a specific chemokine profile, while cultured MSCs lack the set of corresponding receptors. Thus, we hypothesize that overexpression of certain chemokine receptor might help in MSCs homing and improve therapeutic efficacy. Methods: Using the middle cerebral artery occlusion (MCAO) model of ischemic stroke, we identified that CCL2 is one of the most highly expressed chemokines in the ipsilateral hemisphere. Then, we genetically transduced the corresponding receptor, CCR2 to the MSCs and quantified the cell retention of MSCCCR2 compared to the MSCdtomato control. Results: MSCCCR2 exhibited significantly enhanced migration to the ischemic lesions and improved the neurological outcomes. Brain edema and blood-brain barrier (BBB) leakage levels were also found to be much lower in the MSCCCR2-treated rats than the MSCdtomato group. Moreover, this BBB protection led to reduced inflammation infiltration and reactive oxygen species (ROS) generation. Similar results were also confirmed using the in vitro BBB model. Furthermore, genome-wide RNA sequencing (RNA-seq) analysis revealed that peroxiredoxin4 (PRDX4) was highly expressed in MSCs, which mainly contributed to their antioxidant impacts on MCAO rats and oxygen-glucose deprivation (OGD)-treated endothelium. Conclusion: Taken together, this study suggests that overexpression of CCR2 on MSCs enhances their targeted migration to the ischemic hemisphere and improves the therapeutic outcomes, which is attributed to the PRDX4-mediated BBB preservation.