Human apolipoprotein E ɛ4 expression impairs cerebral vascularization and blood-brain barrier function in mice.
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ABSTRACT:
SUBMITTER: Alata W
PROVIDER: S-EPMC4296574 | biostudies-other | 2015 Jan
REPOSITORIES: biostudies-other
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Project description:BackgroundTraumatic Brain Injury (TBI) is a major cause of disability and mortality, to which there is currently no comprehensive treatment. Blood Brain Barrier (BBB) dysfunction is well documented in human TBI patients, yet the molecular mechanisms that underlie this neurovascular unit (NVU) pathology remains unclear. The apolipoprotein-E (apoE) protein has been implicated in controlling BBB integrity in an isoform dependent manner, via suppression of Cyclophilin A (CypA)-Matrix metallopeptidase-9 (MMP-9) signaling cascades, however the contribution of this pathway in TBI-induced BBB permeability is not fully investigated.MethodsWe exposed C57Bl/6 mice to controlled cortical impact and assessed NVU and BBB permeability responses up to 21 days post-injury. We pharmacologically probed the role of the CypA-MMP-9 pathway in BBB permeability after TBI using Cyclosporin A (CsA, 20 mg/kg). Finally, as the apoE4 protein is known to be functionally deficient compared to the apoE3 protein, we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI.ResultsIn C57Bl/6 mice there was an inverse relationship between soluble apoE and BBB permeability, such that damaged BBB stabilizes as apoE levels increase in the days following TBI. TBI mice displayed acute pericyte loss, increased MMP-9 production and activity, and reduced tight-junction expression. Treatment with the CypA antagonist CsA in C57Bl/6 mice attenuates MMP-9 responses and enhances BBB repair after injury, demonstrating that MMP-9 plays an important role in the timing of spontaneous BBB repair after TBI. We also show that apoe mRNA is present in both astrocytes and pericytes after TBI. We report that APOE3 and APOE4 mice have similar acute BBB responses to TBI, but APOE3 mice display faster spontaneous BBB repair than APOE4 mice. Isolated microvessel analysis reveals delayed pericyte repopulation, augmented and sustained MMP-9 expression at the NVU, and impaired stabilization of Zonula Occludens-1, Occludin and Claudin-5 expression at tight junctions in APOE4 mice after TBI compared to APOE3 mice.ConclusionsThese data confirm apoE as an important modulator of spontaneous BBB stabilization following TBI, and highlights the APOE4 allele as a risk factor for poor outcome after TBI.
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Project description:Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.
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Project description:Insults to the developing brain often result in irreparable damage resulting in long-term deficits in motor and cognitive functions. The only treatment today for hypoxic-ischemic encephalopathy (HIE) in newborns is hypothermia, which has limited clinical benefit. We have studied changes to the blood-brain barriers (BBB) as well as regional cerebral blood flow (rCBF) in a neonatal model of HIE to further understand the underlying pathologic mechanisms. Nine-day old mice pups, brain roughly equivalent to the near-term human fetus, were subjected to hypoxia-ischemia. Hypoxia-ischemia increased BBB permeability to small and large molecules within hours after the insult, which normalized in the following days. The opening of the BBB was associated with changes to BBB protein expression whereas gene transcript levels were increased showing direct molecular damage to the BBB but also suggesting compensatory mechanisms. Brain pathology was closely related to reductions in rCBF during the hypoxia as well as the areas with compromised BBB showing that these are intimately linked. The transient opening of the BBB after the insult is likely to contribute to the pathology but at the same time provides an opportunity for therapeutics to better reach the infarcted areas in the brain.
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Project description:Blood-brain barrier (BBB) dysfunction, e.g., increase in BBB permeability, has been reported to contribute to cognitive impairment. However, the effects of anesthesia and surgery on BBB permeability, the underlying mechanisms, and associated cognitive function remain largely to be determined. Here, we assessed the effects of surgery (laparotomy) under 1.4% isoflurane anesthesia (anesthesia/surgery) for 2?h on BBB permeability, levels of junction proteins and cognitive function in both 9- and 18-month-old wild-type mice and 9-month-old interleukin (IL)-6 knockout mice. BBB permeability was determined by dextran tracer (immunohistochemistry imaging and spectrophotometric quantification), and protein levels were measured by Western blot and cognitive function was assessed by using both Morris water maze and Barnes maze. We found that the anesthesia/surgery increased mouse BBB permeability to 10-kDa dextran, but not to 70-kDa dextran, in an IL-6-dependent and age-associated manner. In addition, the anesthesia/surgery induced an age-associated increase in blood IL-6 level. Cognitive impairment was detected in 18-month-old, but not 9-month-old, mice after the anesthesia/surgery. Finally, the anesthesia/surgery decreased the levels of ?-catenin and tight junction protein claudin, occludin and ZO-1, but not adherent junction protein VE-cadherin, E-cadherin, and p120-catenin. These data demonstrate that we have established a system to study the effects of perioperative factors, including anesthesia and surgery, on BBB and cognitive function. The results suggest that the anesthesia/surgery might induce an age-associated BBB dysfunction and cognitive impairment in mice. These findings would promote mechanistic studies of postoperative cognitive impairment, including postoperative delirium.
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Project description:Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler syndrome by inactivating Flvcr2 in mice. We showed that Flvcr2 was necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood-brain barrier. Endothelial cells lacking Flvcr2 had altered expression of angiogenic factors, failed to adopt tip cell properties, and displayed reduced sprouting, leading to vascular malformations similar to those seen in humans with Fowler syndrome. Brain hypovascularization was associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier was maintained in Flvcr2 mutant mice. Our Fowler syndrome model therefore defined the pathobiology of this disease and provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
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Project description:Plasmodium falciparum malaria is responsible for nearly one million annual deaths worldwide. Because of the difficulty in monitoring the pathogenesis of cerebral malaria in humans, we conducted a study in various mouse models to better understand disease progression in experimental cerebral malaria (ECM). We compared the effect on the integrity of the blood brain barrier (BBB) and the histopathology of the brain of P. berghei ANKA, a known ECM model, P. berghei NK65, generally thought not to induce ECM, P. yoelii 17XL, originally reported to induce human cerebral malaria-like histopathology, and P. yoelii YM. As expected, P. berghei ANKA infection caused neurological signs, cerebral hemorrhages, and BBB dysfunction in CBA/CaJ and Swiss Webster mice, while Balb/c and A/J mice were resistant. Surprisingly, PbNK induced ECM in CBA/CaJ mice, while all other mice were resistant. P. yoelii 17XL and P. yoelii YM caused lethal hyperparasitemia in all mouse strains; histopathological alterations, BBB dysfunction, or neurological signs were not observed. Intravital imaging revealed that infected erythrocytes containing mature parasites passed slowly through capillaries making intimate contact with the endothelium, but did not arrest. Except for relatively rare microhemorrhages, mice with ECM presented no obvious histopathological alterations that would explain the widespread disruption of the BBB. Intravital imaging did reveal, however, that postcapillary venules, but not capillaries or arterioles, from mice with ECM, but not hyperparasitemia, exhibit platelet marginalization, extravascular fibrin deposition, CD14 expression, and extensive vascular leakage. Blockage of LFA-1 mediated cellular interactions prevented leukocyte adhesion, vascular leakage, neurological signs, and death from ECM. The endothelial barrier-stabilizing mediators imatinib and FTY720 inhibited vascular leakage and neurological signs and prolonged survival to ECM. Thus, it appears that neurological signs and coma in ECM are due to regulated opening of paracellular-junctional and transcellular-vesicular fluid transport pathways at the neuroimmunological BBB.
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Project description:Cerebral complications in preeclampsia contribute substantially to maternal mortality and morbidity. There is a lack of reliable and accessible predictors for preeclampsia-related cerebral complications. In this study, plasma from women with preeclampsia (n = 28), women with normal pregnancies (n = 28) and non-pregnant women (n = 16) was analyzed for concentrations of the cerebral biomarkers neurofilament light (NfL), tau, neuron-specific enolase (NSE) and S100B. Then, an in vitro blood-brain barrier (BBB) model, based on the human cerebral microvascular endothelial cell line (hCMEC/D3), was employed to assess the effect of plasma from the three study groups. Transendothelial electrical resistance (TEER) was used as an estimation of BBB integrity. NfL and tau are proteins expressed in axons, NSE in neurons and S100B in glial cells and are used as biomarkers for neurological injury in other diseases such as dementia, traumatic brain injury and hypoxic brain injury. Plasma concentrations of NfL, tau, NSE and S100B were all higher in women with preeclampsia compared with women with normal pregnancies (8.85 vs. 5.25 ng/L, p < 0.001; 2.90 vs. 2.40 ng/L, p < 0.05; 3.50 vs. 2.37 µg/L, p < 0.001 and 0.08 vs. 0.05 µg/L, p < 0.01, respectively). Plasma concentrations of NfL were also higher in women with preeclampsia compared with non-pregnant women (p < 0.001). Higher plasma concentrations of the cerebral biomarker NfL were associated with decreased TEER (p = 0.002) in an in vitro model of the BBB, a finding which indicates that NfL could be a promising biomarker for BBB alterations in preeclampsia.
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Project description:Treatment of many pathologies of the brain could be improved markedly by the development of noninvasive therapeutic approaches that elicit robust, endothelial cell-selective gene expression in specific brain regions that are targeted under MR image guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a noninvasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce an MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e., "sonoselective" transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing MBs with pulsed low-pressure (0.1 MPa) 1.1-MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e., 0.3-MPa and 0.4-MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low-pressure regimen does not up-regulate inflammatory or immune responses. Single-cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.
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Project description:Cerebral malaria claims the lives of over 600,000 African children every year. To better understand the pathogenesis of this devastating disease, we compared the cellular dynamics in the cortical microvasculature between two infection models, Plasmodium berghei ANKA (PbA) infected CBA/CaJ mice, which develop experimental cerebral malaria (ECM), and P. yoelii 17XL (PyXL) infected mice, which succumb to malarial hyperparasitemia without neurological impairment. Using a combination of intravital imaging and flow cytometry, we show that significantly more CD8(+) T cells, neutrophils, and macrophages are recruited to postcapillary venules during ECM compared to hyperparasitemia. ECM correlated with ICAM-1 upregulation on macrophages, while vascular endothelia upregulated ICAM-1 during ECM and hyperparasitemia. The arrest of large numbers of leukocytes in postcapillary and larger venules caused microrheological alterations that significantly restricted the venous blood flow. Treatment with FTY720, which inhibits vascular leakage, neurological signs, and death from ECM, prevented the recruitment of a subpopulation of CD45(hi) CD8(+) T cells, ICAM-1(+) macrophages, and neutrophils to postcapillary venules. FTY720 had no effect on the ECM-associated expression of the pattern recognition receptor CD14 in postcapillary venules suggesting that endothelial activation is insufficient to cause vascular pathology. Expression of the endothelial tight junction proteins claudin-5, occludin, and ZO-1 in the cerebral cortex and cerebellum of PbA-infected mice with ECM was unaltered compared to FTY720-treated PbA-infected mice or PyXL-infected mice with hyperparasitemia. Thus, blood brain barrier opening does not involve endothelial injury and is likely reversible, consistent with the rapid recovery of many patients with CM. We conclude that the ECM-associated recruitment of large numbers of activated leukocytes, in particular CD8(+) T cells and ICAM(+) macrophages, causes a severe restriction in the venous blood efflux from the brain, which exacerbates the vasogenic edema and increases the intracranial pressure. Thus, death from ECM could potentially occur as a consequence of intracranial hypertension.
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Project description:Rationale: The blood-brain barrier (BBB) prevents the effective delivery of therapeutic molecules to the central nervous system (CNS). A recently generated adeno-associated virus (AAV)-based vector, AAV-PHP.eB, has been found to penetrate the BBB more efficiently than other vectors including AAV-PHP.B. However, little is known about the mechanisms. In this study, we investigated how AAV-PHP.eB penetrates the BBB in mice. Methods: We injected AAV-PHP.eB into the bloodstream of wild-type C57BL/6 and BALB/c mice as well as mouse strains carrying genetic mutation in apolipoprotein E gene (Apoe) or low-density lipoprotein receptor gene (Ldlr), or lacking various components of the immune system. Then, we evaluated AAV-PHP.eB transduction to the brain and spinal cord in these mice. Results: We found that the transduction to the CNS of intravenous AAV-PHP.eB was more efficient in C57BL/6 than BALB/c mice, and significantly reduced in Apoe or Ldlr knockout C57BL/6 mice compared to wild-type C57BL/6 mice. Moreover, poor CNS transduction in BALB/c mice was dramatically increased by B-cell or natural killer-cell depletion. Conclusions: Our findings demonstrate that the ApoE-LDLR pathway underlies the CNS tropism of AAV-PHP.eB and that the immune system contributes to the strain specificity of AAV-PHP.eB.
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