CNS Endothelial Cells Research Articles

Elevated Levels of Serum Biomarkers Associated with Damage to the CNS Neurons and Endothelial Cells Are Linked with Changes in Brain Connectivity in Breast Cancer Patients with Vestibulo-Atactic Syndrome

Vestibulo-atactic syndrome (VAS), which represents a combination of motor and vestibular disorders, can be manifested as a clinical complication of breast cancer treatment and has a significant impact on patients’ quality of life. The identification of novel potential biomarkers that might help to predict the onset of VAS and its progression could improve the management of this group of patients. In the current study, the levels of intercellular cell adhesion molecule 1 (ICAM-1), platelet/endothelial cell adhesion molecule 1 (PECAM-1), NSE (neuron-specific enolase), and the antibodies recognizing NR-2 subunit of NMDA receptor (NR-2-ab) were measured in the blood serum of BC survivor patients with vestibulo-atactic syndrome (VAS) and associated with the brain connectome data obtained via functional magnetic resonance imaging (fMRI) studies. A total of 21 patients were registered in this open, single-center trial and compared to age-matched healthy female volunteers (control group) (n = 17). BC patients with VAS demonstrated higher serum levels of ICAM-1, PECAM-1, and NSE and a lower value of NR-2-ab, with values of 654.7 ± 184.8, 115.3 ± 37.03, 49.9 ± 103.9, and 0.5 ± 0.3 pg/mL, respectively, as compared to the healthy volunteers, with 230.2 ± 44.8, 62.8 ± 15.6, 15.5 ± 6.4, and 1.4 ± 0.7 pg/mL. According to the fMRI data (employing seed-to-voxel and ROI-to-ROI methods), in BC patients with VAS, significant changes were detected in the functional connectivity in the areas involved in the regulation of postural-tonic reflexes, the coordination of movements, and the regulation of balance. In conclusion, the detected elevated levels of serum biomarkers may reveal damage to the CNS neurons and endothelial cells that is, in turn, associated with the change in the brain connectivity in this group of patients.

Short regulatory DNA sequences to target brain endothelial cells for gene therapy

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

Brain endothelial LRP1 maintains blood\u2013brain barrier integrity

The entry of blood-borne molecules into the brain is restricted by the blood–brain barrier (BBB). Various physical, transport and immune properties tightly regulate molecule movement between the blood and the brain to maintain brain homeostasis. A recent study utilizing a pan-endothelial, constitutive Tie2-Cre showed that paracellular passage of blood proteins into the brain is governed by endocytic and cell signaling protein low-density lipoprotein receptor–related protein 1 (LRP1). Taking advantage of conditional Slco1c1-CreERT2 specific to CNS endothelial cells and choroid plexus epithelial cells we now supplement previous results and show that brain endothelial Lrp1 ablation results in protease-mediated tight junction degradation, P-glycoprotein (P-gp) reduction and a loss of BBB integrity.

Immune Activation of the Blood Brain Barrier and Implications for Neuroinflammation in Schizophrenia

Abstract Neuroimmune dysregulation has been implicated in various neuropsychiatric disorders, including schizophrenia (SZ). Multiple studies indicate peripheral inflammation, including increased serum IL-6 and circulating Th17 cells, and neuroinflammation in the context of psychosis. Reports that the blood brain barrier (BBB), the highly specialized CNS endothelial cells, is disrupted in patients suggest that vascular alterations contribute to neuroimmune dysfunction in SZ. Therefore, we investigated whether the BBB is intrinsically compromised in the most common genetic risk factor for SZ, the hemizygous deletion of chromosome 22q11.2 (22qDS). We employed an innovative approach based on the differentiation of human induced pluripotent stem cells (iPSCs) from 22qDS patients with SZ into BBB-like endothelium and further substantiated our findings in vivo in a mouse model of 22qDS. We demonstrate that barrier function and immune privilege properties of the BBB are compromised in 22qDS, as we found compromised expression of the tight junction protein claudin-5 and upregulation of the cell adhesion molecule ICAM-1 (CD54). To assess the immunological consequences of such imbalances, we determined the impact of the BBB on transmigrating immune cells and found that the 22qDS BBB supported leukocyte migration and activation. Furthermore, we observed increased astrocyte activation and IL-6 expression in 22qDS mice and post-mortem 22qDS patients, suggesting that compromised BBB may promote astrocyte-mediated neuroinflammation in 22qDS. These results indicate that 22qDS is associated with changes in the BBB, and suggest that the BBB may play a role in the increased risk for neuropsychiatric disease in 22qDS patients.

Profiling the mouse brain endothelial transcriptome in health and disease models reveals a core blood-brain barrier dysfunction module.

Blood vessels in the central nervous system (CNS) form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells to peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizures, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB-dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders.

Blood-Brain Barrier Permeability Is Regulated by Lipid Transport-Dependent Suppression of Caveolae-Mediated Transcytosis

The blood-brain barrier (BBB) provides a constant homeostatic brain environment that is essential for proper neural function. An unusually low rate of vesicular transport (transcytosis) has been identified as one of the two unique properties of CNS endothelial cells, relative to peripheral endothelial cells, that maintain the restrictive quality of the BBB. However, it is not known how this low rate of transcytosis is achieved. Here we provide a mechanism whereby the regulation of CNS endothelial cell lipid composition specifically inhibits the caveolae-mediated transcytotic route readily used in the periphery. An unbiased lipidomic analysis reveals significant differences in endothelial cell lipid signatures from the CNS and periphery, which underlie a suppression of caveolae vesicle formation and trafficking in brain endothelial cells. Furthermore, lipids transported by Mfsd2a establish a unique lipid environment that inhibits caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensure BBB integrity.

FZD10-Gα13 signalling axis points to a role of FZD10 in CNS angiogenesis

Among the 10 Frizzled (FZD) isoforms belonging to the Class F of G protein-coupled receptors (GPCRs), FZD10 remains the most enigmatic. FZD10 shows homology to FZD4 and FZD9 and was previously implicated in both β-catenin-dependent and –independent signalling. In normal tissue, FZD10 levels are generally very low; however, its upregulation in synovial carcinoma has attracted some attention for therapy. Our findings identify FZD10 as a receptor interacting with and signalling through the heterotrimeric G protein Gα13 but not Gα12, Gαi1, GαoA, Gαs, or Gαq. Stimulation with the FZD agonist WNT induced the dissociation of the Gα13 protein from FZD10, and led to global Gα12/13–dependent cell changes assessed by dynamic mass redistribution measurements. Furthermore, we show that FZD10 mediates Gα12/13 activation-dependent induction of YAP/TAZ transcriptional activity. In addition, we show a distinct expression of FZD10 in embryonic CNS endothelial cells at E11.5–E14.5. Given the well-known importance of Gα13 signalling for the development of the vascular system, the selective expression of FZD10 in brain vascular endothelial cells points at a potential role of FZD10-Gα13 signalling in CNS angiogenesis.

Isolation of vascular endothelial cells from intact and injured murine brain cortex-technical issues and pitfalls in FACS analysis of the nervous tissue.

CNS endothelial cells (CNS-ECs), one of the main non-neural CNS cell populations, play a vital role in physiology, pathology, and regeneration of the nervous system. Therefore, there is an urgent need to enhance our knowledge on their biology to elucidate mechanisms responsible for the blood brain barrier function in normal and pathological conditions, interaction between brain endothelium and neural stem cells in the neurogenic niche, the paracrine processes in the brain and spinal cord, etc. Here, we described a novel, simple, and efficient protocol for isolation of endothelial, vessel-forming cells from the murine CNS, which is based on Sca-1 expression. Using this newly described protocol we were able to detect and sort viable, highly pure CNS-ECs with minimal contamination by cells of non-endothelial origin. This method will increase the availability of CNS-ECs for in vitro research. Moreover, we compared phenotype of CNS-ECs isolated from neonatal mice and adult intact and injured brain looking for the cells of endothelial precursor characteristic, such as those found in the bone marrow and circulating in the bloodstream after organ injuries. We have found that neonatal brain capillaries contain proportion of endothelial precursor cells (Sca-1(+) , CD45(-) , c-Kit(+) ). Such precursors were also found in adult brain cortex, both in intact and injured brain. Finally, we discuss several crucial technical issues concerning CNS tissue preparation for flow cytometry analysis and cell sorting as well as nonspecific antibody binding caused by inflammatory microglia/macrophages which should be avoided in order to reliable isolation of pure CNS cells for downstream procedures including cell transplantation-based translational studies.

Activation of adult rat CNS endothelial cells by opioid-induced toll-like receptor 4 (TLR4) signaling induces proinflammatory, biochemical, morphological, and behavioral sequelae

CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at toll-like receptor 4 (TLR4), presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (−)-morphine or its mu opioid receptor (MOR)-inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated nuclear factor kappaB (NF-κB), increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 (PGE2) from these cells. (−)-Morphine-induced upregulation of TNFα mRNA and PGE2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (−)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (−)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (−)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequelae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by presumptive glial inhibitors, as well as TLR4-mediated phenomena more broadly.

E- and P-Selectin Are Not Required for the Development of Experimental Autoimmune Encephalomyelitis in C57BL/6 and SJL Mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.

Intercellular adhesion molecule-1 gene polymorphism in Iranian patients with multiple sclerosis

Intercellular adhesion molecule-1 (ICAM-1), an important molecule in leucocyte activation and migration, is expressed on the CNS endothelial cells of patients with multiple sclerosis. This study was conducted to determine whether ICAM-1 gene polymorphism influences the risk of developing multiple sclerosis in an Iranian population. We studied 157 patients with definite multiple sclerosis and 156 ethnically matched controls. The patients and the controls were genotyped for ICAM-1 gene polymorphism at codons 241 (exon 4) and 469 (exon 6). G/R241 and K/E 469 allele and genotype distribution did not show any significant difference between patient and control groups. We concluded that role of the ICAM-1 gene polymorphisms in the pathogenesis of multiple sclerosis is still controversial and should be studied further.

CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis

The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.

Deficient p75 low-affinity neurotrophin receptor expression exacerbates experimental allergic encephalomyelitis in C57/BL6 mice

We have investigated the role of p75 NTR in inflammation in experimental allergic encephalomyelitis (EAE), a model for the human disease multiple sclerosis (MS). Induction of EAE in C57/BL6 wild-type mice resulted in expression of p75 NTR in endothelial cells in the CNS. In contrast to the clinical manifestation of EAE observed in wild-type C57/BL6 mice, mice deficient for p75 NTR (p75 NTR knockout mice) developed severe or lethal disease and concomitant increased levels of inflammation in the CNS. Our findings suggest a physiological significant role for p75 NTR in CNS endothelial cells during inflammation and involvement in preservation of blood–brain barrier integrity during a severe infiltrative attack.

Cytokine regulation of MCP-1 expression in brain and retinal microvascular endothelial cells

Chemokines have a pivotal role in the selective mediation and amplification of inflammation. The CNS vascular endothelial cells, which form part of the blood–brain barrier (BBB) and blood–retinal barrier (BRB), are ideally situated to present chemokines to circulating lymphocytes leading to their recruitment. Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, a potent chemoattractant of T cells and monocytes, has been implicated in inflammatory and angio-proliferative brain and retinal disease. In this study, MCP-1 expression by CNS endothelial cells was investigated in vitro. Rat brain (GP8/3.9) and retinal (JG2/1) vascular endothelial cell lines expressed MCP-1 constitutively in vitro as assessed by immunocytochemistry and enzyme linked immunosorbant assay (ELISA). Upregulation of secreted MCP-1 was observed following activation with the pro-inflammatory cytokines TNF-α, IL-1β and IFN-γ, and was reduced following dexamethasone treatment. Functional chemotactic activity of brain and retinal endothelial cell supernatants was demonstrated in an in vitro chemotaxis assay, which was inhibited by anti-MCP-1 antibodies. These findings suggest that endothelial cell-derived MCP-1 plays a key role in leukocyte recruitment across the blood–brain and blood–retinal barriers in vivo.

Cytokine regulation of E-selectin in rat CNS microvascular endothelial cells: differential response of CNS and non-CNS vessels

We have compared the induced expression of E-selectin in primary cultures of rat brain microvascular endothelial cells (EC), pericytes and in non-CNS microvascular endothelium stimulated with the cytokines, IL-1β (20 ng/ml), and tumor necrosis factor (TNF)-α (75 ng/ml). Expression was studied at both the protein and mRNA levels. Fluorescence in-situ hybridization (FISH) was used to examine de novo synthesis of E-selectin mRNA. Laser cytometric analysis was used as a novel approach to the quantitaion of FISH. In-situ hybridization was performed using two PCR-generated probes. The first probe (517 bp) spanned the lectin and epidermal growth factor (EGF)-like domain. The second probe (562 bp) spanned the CR3, 4, and 6 domains. E-selectin-specific mRNA was localized to the perinuclear regions of the EC. Both cytokines, IL-1β and TNF-α significantly increased E-selectin gene expression in CNS EC but not pericytes. IL-1β induced higher E-selectin mRNA levels than TNF-α. The maximum number of mRNA-positive cells was observed after stimulation for 4–6 h. Surface protein expression was sustained for up to 48 h following addition of cytokine. This was in contrast to the transient expression in non-CNS EC indicating that pure primary CNS EC display slightly different kinetics of E-selectin expression than non-CNS EC.

NG2 immunoreactivity on human brain endothelial cells.

In this study, we evaluated the expression of NG2 on human brain endothelial cells derived from temporal lobe tissue resected as a treatment for intractable epilepsy. Using dissociated cell cultures, we found expression of NG2 on both proliferating and non-proliferating cells, at the mRNA level by reverse transcription-polymerase chain reaction analyses, and at the protein level by immunocytochemistry and immunoblotting. We further observed that human cerebral microvessels in nonmalignant CNS tissues immunoreacted with NG2. NG2 protein was detected using both a rabbit antibody raised against the rodent NG2 and a monoclonal antibody raised against the human NG2 (9.2.27). Our findings further define the range of resident cells of the CNS that can express NG2 and indicate that expression of NG2 by endothelial cells is not restricted to proliferating CNS endothelial cells or to endothelial cells found in brain tumors.

Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium.

Matrix metalloproteinases (MMPs) have been implicated in the early breakdown of the blood-brain barrier in neuroinflammatory disease. Although expression of these enzymes by resident glial cells and recruited immune cells has been described, altered expression of MMPs by the CNS vascular endothelial cells may also contribute to barrier disruption. In the present study, the in vitro expression of MMP-2 and -9 as well as tissue inhibitor of metalloproteinase (TIMP)-2 by rat CNS microvascular endothelial cells has been determined and compared with that by endothelial cell lines derived from rat aorta and high endothelial venules. Primary cultures of rat brain microvascular endothelial cells as well as the rat brain (GP8/3.9) and rat retinal endothelial (JG2/1) cell lines constitutively expressed MMP-2, -9 and TIMP-2. In vitro activation of CNS endothelium with the pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta, resulted in selective upregulation of MMP-9 activity, whereas no significant changes were seen in MMP-2 or TIMP-2 levels at 24 h. The addition of dexamethasone partially inhibited the cytokine-induced upregulation of MMP-9. Treatment of GP8/3.9 brain endothelial cells with active MMP-9 caused subtle but distinct alterations in the expression of the junctional protein, ZO-1. Quantitative differences found between CNS and non-CNS endothelial cells in the expression of both MMP-2 and -9, and in the expression of TIMP-2 demonstrate that CNS vascular endothelium is functionally distinct from non-CNS endothelium. These results suggest that cytokine-induced upregulation of MMP-9 expression by the CNS vascular endothelium may play a role in the pathogenesis of blood-brain and blood-retinal barrier breakdown in vivo.

Molecular control of class II by CIITA in CNS endothelial cells

Molecular control of class II by CIITA in CNS endothelial cells

T-lymphocyte migration through CNS endothelial cells involves signaling through endothelial ICAM-1 via a rho-dependent pathway

T-lymphocyte migration through CNS endothelial cells involves signaling through endothelial ICAM-1 via a rho-dependent pathway

PECAM-1 (CD31) expression in the central nervous system and its role in experimental allergic encephalomyelitis in the rat

The role of T cell activation associated adhesion molecules on lymphocyte traffic and the initiation of inflammation has received considerable attention. This study, using a new monoclonal antibody (mAb) TLD-3A12, describes the distribution of PECAM-1 (CD31), an Ig supergene family adhesion molecule thought to be important in leukocyte transmigration during inflammation, in rat lymphoid organs and spinal cord. PECAM expression within the CNS is confined to endothelial cells of the blood brain barrier (BBB). Induction of inflammation within the CNS using the adoptive transfer of myelin reactive CD4+ T cells results in the de novo expression of immune adhesion and accessory molecules in the spinal cord, while the level of PECAM appeared only mildly increased. The distribution of PECAM on CNS endothelial cells became more diffuse during EAE induction, possibly the result of endothelial cell activation. In vitro studies demonstrate a partial inhibition of antigen-specific CD4+ T cell proliferation following anti-PECAM mAb treatment. Treatment of Lewis rats with TLD-3A12 antibody prior to T cell injection and throughout EAE induction does not result in a delay in the onset of clinical signs or weight loss, nor does it decrease the incidence and severity of disease. These data suggest that the expression of PECAM by CNS endothelial cells is not a requirement for the initiation of inflammation and clinical signs of EAE following the adoptive transfer of encephalitogenic lymphocytes. Thus, cells requiring PECAM-1 to migrate and perform their pathogenic functions are not critical to the development of rat EAE.