Activated Brain Endothelium Research Articles

Nanomedicine for Ischemic Stroke.

Stroke is a severe brain disease leading to disability and death. Ischemic stroke dominates in stroke cases, and there are no effective therapies in clinic, partly due to the challenges in delivering therapeutics to ischemic sites in the brain. This review is focused on the current knowledge of pathogenesis in ischemic stroke, and its potential therapies and diagnosis. Furthermore, we present recent advances in developments of nanoparticle-based therapeutics for improved treatment of ischemic stroke using polymeric NPs, liposomes and cell-derived nanovesicles. We also address several critical questions in ischemic stroke, such as understanding how nanoparticles cross the blood brain barrier and developing in vivo imaging technologies to address this critical question. Finally, we discuss new opportunities in developing novel therapeutics by targeting activated brain endothelium and inflammatory neutrophils to improve the current therapies for ischemic stroke.

Pentraxin 3 regulates neutrophil infiltration to the brain during neuroinflammation

Introduction: The acute phase protein pentraxin 3 (PTX3) is known for its anti-inflammatory effects through downregulating neutrophil transmigration during peripheral inflammation. Furthermore, we have previously demonstrated a neuroprotective and neuroreparative effect of PTX3 after cerebral ischaemia. Here we investigated, to our knowledge for the first time, the role of PTX3 in neutrophil transmigration and neurotoxicity following lipopolysaccharide (LPS)-induced cerebral inflammation and cerebral ischaemia. Methods: Neutrophil transmigration through interleukin-1β (IL-1β) activated brain endothelium and neurotoxicity of neutrophils isolated from wild-type (WT) or PTX3 knock-out (KO) mice was assessed in vitro. Primary cortical neuronal death after treatment with transmigrated neutrophils was quantified by lactate dehydrogenase (LDH) assay. Cerebral inflammation or ischemia was induced in WT and PTX3 KO mice via intrastriatal LPS injection or by transient middle cerebral artery occlusion (MCAo) respectively. Subsequent neutrophil infiltration in the brain was assessed by immunohistochemistry and the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β by enzyme-linked immunosorbent assay (ELISA). Results: Neutrophils isolated from WT mice after intrastriatal LPS injection transmigrated significantly more through IL-1β activated brain endothelium compared to neutrophils from PTX3 KO mice. Transmigrated WT and PTX3 KO neutrophils were significantly more neurotoxic than corresponding non-transmigrated neutrophils; however, no significant differences in neurotoxicity between genotypes were observed. PTX3 reduced the number of transmigrated neutrophils to the brain after intrastriatal LPS injection. Furthermore, PTX3 KO mice showed significantly increased levels of neutrophils in the brain after LPS administration or in the ischaemic hemisphere after MCAo, compared to WT mice. Conclusion: Our study shows that PTX3 regulates neutrophil transmigration in the CNS during neuroinflammation, demonstrating the potential of PTX3 as an effective therapeutic target in neuroinflammatory conditions.

Pentraxin 3 regulates neutrophil infiltration to the brain during neuroinflammation.

Introduction: The acute phase protein pentraxin 3 (PTX3) is known for its anti-inflammatory effects through downregulating neutrophil transmigration during peripheral inflammation. Furthermore, we have previously demonstrated a neuroprotective and neuroreparative effect of PTX3 after cerebral ischaemia. Here we investigated, to our knowledge for the first time, the role of PTX3 in neutrophil transmigration and neurotoxicity following lipopolysaccharide (LPS)-induced cerebral inflammation and cerebral ischaemia. Methods: Neutrophil transmigration through interleukin-1β (IL-1β) activated brain endothelium and neurotoxicity of neutrophils isolated from wild-type (WT) or PTX3 knock-out (KO) mice was assessed in vitro. Primary cortical neuronal death after treatment with transmigrated neutrophils was quantified by lactate dehydrogenase (LDH) assay. Cerebral inflammation or ischemia was induced in WT and PTX3 KO mice via intrastriatal LPS injection or by transient middle cerebral artery occlusion (MCAo) respectively. Subsequent neutrophil infiltration in the brain was assessed by immunohistochemistry and the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β by enzyme-linked immunosorbent assay (ELISA). Results: Neutrophils isolated from WT mice after intrastriatal LPS injection transmigrated significantly more through IL-1β activated brain endothelium compared to neutrophils from PTX3 KO mice. Transmigrated WT and PTX3 KO neutrophils were significantly more neurotoxic than corresponding non-transmigrated neutrophils; however, no significant differences in neurotoxicity between genotypes were observed. PTX3 reduced the number of transmigrated neutrophils to the brain after intrastriatal LPS injection. Furthermore, PTX3 KO mice showed significantly increased levels of neutrophils in the brain after LPS administration or in the ischaemic hemisphere after MCAo, compared to WT mice. Conclusion: Our study shows that PTX3 regulates neutrophil transmigration in the CNS during neuroinflammation, demonstrating the potential of PTX3 as an effective therapeutic target in neuroinflammatory conditions.

SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells.

BackgroundHemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1—transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca2+-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process.MethodsCerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied in vitro, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways.ResultsCerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused de novo expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca2+-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion.ConclusionstPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.

Non-pathogenic tissue-resident CD8+ T cells uniquely accumulate in the brains of lupus-prone mice

Severe lupus often includes psychiatric and neurological sequelae, although the cellular contributors to CNS disease remain poorly defined. Using intravascular staining to discriminate tissue-localized from blood-borne cells, we find substantial accumulation of CD8+ T cells relative to other lymphocytes in brain tissue, which correlates with lupus disease and limited neuropathology. This is in contrast to all other affected organs, where infiltrating CD4+ cells are predominant. Brain-infiltrating CD8+ T cells represent an activated subset of those found in the periphery, having a resident-memory phenotype (CD69+CD122−PD1+CD44+CD62L−) and expressing adhesion molecules (VLA-4+LFA-1+) complementary to activated brain endothelium. Remarkably, infiltrating CD8+ T cells do not cause tissue damage in lupus-prone mice, as genetic ablation of these cells via β2 m deficiency does not reverse neuropathology, but exacerbates disease both in the brain and globally despite decreased serum IgG levels. Thus, lupus-associated inflammation disrupts the blood-brain barrier in a discriminating way biased in favor of non-pathogenic CD8+ T cells relative to other infiltrating leukocytes, perhaps preventing further tissue damage in such a sensitive organ.

Use of MR Cell Tracking to Evaluate Targeting of Glial Precursor Cells to Inflammatory Tissue by Exploiting the Very Late Antigen-4 Docking Receptor

To determine if glial precursor cells can be targeted to inflamed brain through overexpression of very late antigen-4 (VLA-4) and whether this docking process can be monitored with magnetic resonance (MR) cell tracking after intraarterial injection. All experimental procedures were performed between August 2010 and February 2012 and were approved by the institutional animal care and use committee. Human glial precursor cells (hGPs) were transfected with VLA-4 and labeled with superparamagnetic iron oxide that contained rhodamine. A microfluidic adhesion assay was used for assessing VLA-4 receptor-mediated cell docking in vitro. A rat model of global lipopolysaccharide (LPS)-mediated brain inflammation was used to induce global vascular cell adhesion molecule-1 (VCAM-1) expression. hGPs were infused into the carotid artery in four animal cohorts (consisting of three rats each): rats that received VLA-4-naive hGPs but did not receive LPS, rats that received VLA-4-expressing hGPs but not LPS, rats that received VLA-4-naive hGPs and LPS, and rats that received VLA-4-expressing hGPs and LPS. MR imaging was performed at 9.4 T before and 1, 10, 20, and 30 minutes after injection. Brain tissue was processed for histologic examination. Quantification of low-signal-intensity pixels was performed with pixel-by-pixel analysis for MR images obtained before and after cell injection. With use of the microfluidic adhesion assay, cell binding to activated brain endothelium significantly increased compared with VLA-4-naive control cells (71.5 cells per field of view±11.7 vs 36.4 cells per field of view±3.3, respectively; P<.05). Real-time quantitative in vivo MR cell tracking revealed that VLA-4-expressing cells docked exclusively within the vascular bed of the ipsilateral carotid artery and that VLA-4-expressing cells exhibited significantly enhanced homing as compared with VLA-4-naive cells (1448 significant pixels±366.5 vs 113.3 significant pixels±19.88, respectively; P<.05). Furthermore, MR cell tracking was crucial for correct cell delivery and proper ligation of specific arteries. Targeted intraarterial delivery and homing of VLA-4-expressing hGPs to inflamed endothelium is feasible and can be monitored in real time by using MR imaging in a quantitative, dynamic manner.

Intercellular Interactomics of Human Brain Endothelial Cells and Th17 Lymphocytes: A Novel Strategy for Identifying Therapeutic Targets of CNS Inflammation

Leukocyte infiltration across an activated brain endothelium contributes to the neuroinflammation seen in many neurological disorders. Recent evidence shows that IL-17-producing T-lymphocytes (e.g., Th17 cells) possess brain-homing capability and contribute to the pathogenesis of multiple sclerosis and cerebral ischemia. The leukocyte transmigration across the endothelium is a highly regulated, multistep process involving intercellular communications and interactions between the leukocytes and endothelial cells. The molecules involved in the process are attractive therapeutic targets for inhibiting leukocyte brain migration. We hypothesized and have been successful in demonstrating that molecules of potential therapeutic significance involved in Th17-brain endothelial cell (BEC) communications and interactions can be discovered through the combination of advanced membrane/submembrane proteomic and interactomic methods. We describe elements of this strategy and preliminary results obtained in method and approach development. The Th17-BEC interaction network provides new insights into the complexity of the transmigration process mediated by well-organized, subcellularly localized molecular interactions. These molecules and interactions are potential diagnostic, therapeutic, or theranostic targets for treatment of neurological conditions accompanied or caused by leukocyte infiltration.

Tickets to the brain: Role of CCR2 and CX 3CR1 in myeloid cell entry in the CNS

Myeloid cells are mediators of central nervous system (CNS) damage and recovery in neuroinflammatory and neurodegenerative disorders. Besides endogenous myelomonocytic cell populations that reside in the brain already during development, newly migrated leukocytes are considered as important disease modulators in the adult brain. Thus, understanding of myeloid cell recruitment is pivotal for manipulating immune cell entry into the CNS and potentially reducing disease burden. Before myeloid cells engraft in the brain, they first tether to and roll on the activated brain endothelium, then they firmly adhere and eventually transmigrate into the damaged brain where they execute effector functions and differentiate into cells with microglia-like features. These steps are mainly regulated by adhesion molecules and by chemokines and their cognate receptors. Due to recent advances in our understanding of monocyte heterogeneity, the interest in chemokine receptors has significantly increased. Among others, the presence of the chemokine receptors CCR2 and CX 3CR 1 is considered to be critical for both myeloid cell trafficking along inflamed vessels and subsequent accumulation in the brain. Therefore, these molecules present viable targets for therapeutic manipulations of myeloid cells destined for the CNS.

Platelet interleukin-1α drives cerebrovascular inflammation

AbstractWhite blood cell infiltration across an activated brain endothelium contributes to neurologic disease, including cerebral ischemia and multiple sclerosis. Identifying mechanisms of cerebrovascular activation is therefore critical to our understanding of brain disease. Platelet accumulation in microvessels of ischemic mouse brain was associated with endothelial activation in vivo. Mouse platelets expressed interleukin-1α (IL-1α), but not IL-1β, induced endothelial cell adhesion molecule expression (ICAM-1 and VCAM-1), and enhanced the release of CXC chemokine CXCL1 when incubated with primary cultures of brain endothelial cells from wild-type or IL-1α/β–deficient mice. A neutralizing antibody to IL-1α (but not IL-1β) or application of IL-1 receptor antagonist inhibited platelet-induced endothelial activation by more than 90%. Platelets from IL-1α/β–deficient mice did not induce expression of adhesion molecules in cerebrovascular endothelial cells and did not promote CXCL1 release in vitro. Conditioned medium from activated platelets induced an IL-1α–dependent activation of mouse brain endothelial cells and supported the transendothelial migration of neutrophils in vitro. Thus, we have identified platelets as a key source of IL-1α and propose that platelet activation of brain endothelium via IL-1α is a critical step for the entry of white blood cells, major contributors to inflammation-mediated injury in the brain.

Glyconanoparticles allow pre-symptomatic in vivo imaging of brain disease

Initial recruitment of leukocytes in inflammation associated with diseases such as multiple sclerosis (MS), ischemic stroke, and HIV-related dementia, takes place across intact, but activated brain endothelium. It is therefore undetectable to symptom-based diagnoses and cannot be observed by conventional imaging techniques, which rely on increased permeability of the blood-brain barrier (BBB) in later stages of disease. Specific visualization of the early-activated cerebral endothelium would provide a powerful tool for the presymptomatic diagnosis of brain disease and evaluation of new therapies. Here, we present the design, construction and in vivo application of carbohydrate-functionalized nanoparticles that allow direct detection of endothelial markers E-/P-selectin (CD62E/CD62P) in acute inflammation. These first examples of MRI-visible glyconanoparticles display multiple copies of the natural complex glycan ligand of selectins. Their resulting sensitivity and binding selectivity has allowed acute detection of disease in mammals with beneficial implications for treatment of an expanding patient population suffering from neurological disease.

Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes

Under inflammatory conditions (including HIV-1 encephalitis and multiple sclerosis), activated brain endothelium enhances the adhesion and transmigration of monocytes across the blood-brain barrier (BBB). Synthetic ligands that activate the peroxisome proliferator-activated receptors (PPARs) have anti-inflammatory properties, and PPAR stimulation prevents the interaction of leukocytes with cytokine stimulated-endothelium. However, the mechanism underlying these effects of PPAR ligands and their ability to intervene with leukocyte adhesion and migration across brain endothelial cells has yet to be explored. For the first time, using primary human brain endothelial cells (BMVEC), we demonstrated that monocyte adhesion and transendothelial migration across inflamed endothelium were markedly reduced by PPARgamma activation. In contrast to non-brain-derived endothelial cells, PPARalpha activation in the BMVEC had no significant effect on monocyte-endothelial interaction. Previously, our work indicated a critical role of Rho GTPases (like RhoA) in BMVEC to control migration of HIV-1 infected monocytes across BBB. In this study, we show that in the BMVEC PPARgamma stimulation prevented activation of two GTPases, Rac1 and RhoA, which correlated with decreased monocyte adhesion to and migration across brain endothelium. Relevant to HIV-1 neuropathogenesis, enhanced adhesion and migration of HIV-1 infected monocytes across the BBB were significantly reduced when BMVEC were treated with PPARgamma agonist. These findings indicate that Rac1 and RhoA inhibition by PPARgamma agonists could be a new approach for treatment of neuroinflammation by preventing monocyte migration across the BBB.