Endothelial-specific Crif1 deletion induces BBB maturation and disruption via the alteration of actin dynamics by impaired mitochondrial respiration.
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ABSTRACT:
SUBMITTER: Lee MJ
PROVIDER: S-EPMC7308523 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hypoxia-induced disruption of the blood-brain barrier (BBB) is the result of many different mechanisms, including alterations to the cytoskeleton. In this study, we identified actin-binding proteins involved in cytoskeletal dynamics with quantitative proteomics and assessed changes in subcellular localization of two proteins involved in actin polymerization [vasodilator-stimulated phosphoprotein (VASP)] and cytoskeleton-plasma membrane cross-linking (moesin). We found significant redistribution of both VASP and moesin to the cytoskeletal and membrane fractions of BBB endothelial cells after 1-h hypoxic stress. We also investigated activation of actin-myosin contraction through assessment of phosphorylated myosin light chain (pMLC) with confocal microscopy. Hypoxia caused a rapid and transient increase in pMLC. Blocking MLC phosphorylation through inhibition of myosin light chain kinase (MLCK) with ML-7 prevented hypoxia-induced BBB disruption and relocalization of the tight junction protein ZO-1. Finally, we implicate the transient receptor potential (TRP)C family of channels in mediating these events since blockade of TRPC channels and the associated calcium influx with SKF-96365 prevents hypoxia-induced permeability changes and the phosphorylation of MLC needed for actin-myosin contraction. These data suggest that hypoxic stress triggers alterations to cytoskeletal structure that contribute to BBB disruption and that calcium influx through TRPC channels contributes to these events.
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Project description:The CR6-interacting factor1 (CRIF1) mitochondrial protein is indispensable for peptide synthesis and oxidative phosphorylation. Cardiomyocyte-specific deletion of CRIF1 showed impaired mitochondrial function and cardiomyopathy. We developed an endothelial cell-specific CRIF1 deletion mouse to ascertain whether dysfunctional endothelial CRIF1 influences cardiac function and is mediated by the antioxidant protein sirtuin 1 (SIRT1). We also examined the effect of the potent SIRT1 activator SRT1720 on cardiac dysfunction. Mice with endothelial cell-specific CRIF1 deletion showed an increased heart-to-body weight ratio, increased lethality, and markedly reduced fractional shortening of the left ventricle, resulting in severe cardiac dysfunction. Moreover, endothelial cell-specific CRIF1 deletion resulted in mitochondrial dysfunction, reduced ATP levels, inflammation, and excessive oxidative stress in heart tissues, associated with decreased SIRT1 expression. Intraperitoneal injection of SRT1720 ameliorated cardiac dysfunction by activating endothelial nitric oxide synthase, reducing oxidative stress, and inhibiting inflammation. Furthermore, the decreased endothelial junction-associated protein zonula occludens-1 in CRIF1-deleted mice was significantly recovered after SRT1720 treatment. Our results suggest that endothelial CRIF1 plays an important role in maintaining cardiac function, and that SIRT1 induction could be a therapeutic strategy for endothelial dysfunction-induced cardiac dysfunction.
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Project description:The blood-brain barrier (BBB) is a highly selective barrier that ensures a homeostatic environment for the central nervous system (CNS). BBB dysfunction, inflammation, and immune cell infiltration are hallmarks of many CNS disorders, including multiple sclerosis and stroke. Physiologically relevant human in vitro models of the BBB are essential to improve our understanding of its function in health and disease, identify novel drug targets, and assess potential new therapies. We present a BBB-on-a-chip model comprising human brain microvascular endothelial cells (HBMECs) cultured in a microfluidic platform that allows parallel culture of 40 chips. In each chip, a perfused HBMEC vessel was grown against an extracellular matrix gel in a membrane-free manner. BBBs-on-chips were exposed to varying concentrations of pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) to mimic inflammation. The effect of the inflammatory conditions was studied by assessing the BBBs-on-chips' barrier function, cell morphology, and expression of cell adhesion molecules. Primary human T cells were perfused through the lumen of the BBBs-on-chips to study T cell adhesion, extravasation, and migration. Under inflammatory conditions, the BBBs-on-chips showed decreased trans-endothelial electrical resistance (TEER), increased permeability to sodium fluorescein, and aberrant cell morphology in a concentration-dependent manner. Moreover, we observed increased expression of cell adhesion molecules and concomitant monocyte adhesion. T cells extravasated from the inflamed blood vessels and migrated towards a C-X-C Motif Chemokine Ligand 12 (CXCL12) gradient. T cell adhesion was significantly reduced and a trend towards decreased migration was observed in presence of Natalizumab, an antibody drug that blocks very late antigen-4 (VLA-4) and is used in the treatment of multiple sclerosis. In conclusion, we demonstrate a high-throughput microfluidic model of the human BBB that can be used to model neuroinflammation and assess anti-inflammatory and barrier-restoring interventions to fight neurological disorders.
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Project description:BackgroundDisruption of the BBB is a harmful event after intracranial hemorrhage (ICH), and this disruption contributes to a series of secondary injuries. We hypothesized that FGF21 may have protective effects after intracranial hemorrhage (ICH) and investigated possible underlying molecular mechanisms.MethodsBlood samples of ICH patients were collected to determine the relationship between the serum level of FGF21 and the [Formula: see text]GCS%. Wild-type mice, SIRT6flox/flox mice, endothelial-specific SIRT6-homozygous-knockout mice (eSIRT6-/- mice) and cultured human brain microvascular endothelial cells (HCMECs) were used to determine the protective effects of FGF21 on the BBB.ResultsWe obtained original clinical evidence from patient data identifying a positive correlation between the serum level of FGF21 and [Formula: see text]GCS%. In mice, we found that FGF21 treatment is capable of alleviating BBB damage, mitigating brain edema, reducing lesion volume and improving neurofunction after ICH. In vitro, after oxyhemoglobin injury, we further explored the protective effects of FGF21 on endothelial cells (ECs), which are a significant component of the BBB. Mitochondria play crucial roles during various types of stress reactions. FGF21 significantly improved mitochondrial biology and function in ECs, as evidenced by alleviated mitochondrial morphology damage, reduced ROS accumulation, and restored ATP production. Moreover, we found that the crucial regulatory mitochondrial factor deacylase sirtuin 6 (SIRT6) played an irreplaceable role in the effects of FGF21. Using endothelial-specific SIRT6-knockout mice, we found that SIRT6 deficiency largely diminished these neuroprotective effects of FGF21. Then, we revealed that FGF21 might promote the expression of SIRT6 via the AMPK-Foxo3a pathway.ConclusionsWe provide the first evidence that FGF21 is capable of protecting the BBB after ICH by improving SIRT6-mediated mitochondrial homeostasis.
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Project description:BackgroundEndothelial cells (ECs) in cerebral vessels are considered the primary targets in acute hemorrhagic brain injuries. EC dysfunction can aggravate neuronal injuries by causing secondary inflammatory responses and blood-brain barrier (BBB) disruption. Previous studies have reported that enhancement of mitochondrial function within ECs may reduce BBB disruption and decrease the severity of acute brain injuries. However, the molecular signaling pathways through which enhanced EC mitochondrial function is enhanced to exert this BBB protective effect have not been fully elucidated.MethodsTo identify signaling pathways involved in linking EC-specific mitochondrial dysfunction and BBB disruption, we first performed RNA sequencing using isolated cerebral vessels from TEKCRIF1 KO mice, a mouse strain that displays EC-specific mitochondrial dysfunction. After identification, we assessed the significance of candidate signaling pathways using an intracerebral hemorrhage (ICH) mouse model. BBB integrity was assessed using an IgG leakage assay, and symptomatic changes were evaluated using behavioral assays.ResultsTranscriptome analyses of the TEKCRIF1 KO mouse revealed significant changes in Notch1 signaling, a pathway intimately involved in BBB maintenance. We also observed a decrease in Notch1 signaling and expression of the mitochondrial oxidative phosphorylation (OxPhos) complex in the ICH mouse model, which also exhibits BBB disruption. To further assess the function of Notch1 signaling in relation to BBB disruption, we injected ICH model mice with adropin, a protein that interacts with the Notch1 ligand NB-3 and activates Notch1 signaling. We found that adropin prevented BBB disruption and reduced the extent (area) of the injury compared with that in vehicle controls, in association with alteration of mitochondrial function.ConclusionThese results suggest that the Notch1 signaling pathway acts as an upstream regulator of DEGs and can be a target to regulate the changes involved with endothelial mitochondrial dysfunction-dependent BBB disruption. Thus, treatment methods that activate Notch1 may be beneficial in acute brain injuries by protecting BBB integrity.
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Project description:Mitochondrial dysfunction has been implicated in the pathophysiology of various cardiovascular diseases. CRIF1 is a protein present in the mitochondria associated with large mitoribosomal subunits, and CRIF1 knockdown induces mitochondrial dysfunction and promotes ROS production. p66shc is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals and, therefore, is a key factor for oxidative stress in endothelial cells. In this study, we investigated whether mitochondrial dysfunction induced by CRIF1 knockdown induces p66shc stimulation and plays any role in mitochondrial dysfunction-induced endothelial activation. Knockdown of CRIF1 decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, III and IV, leading to increased mitochondrial ROS (mtROS) and hyperpolarization of the mitochondrial membrane potential. Knockdown of CRIF1 also stimulated phosphorylation of p66shc and increased cytosolic ROS in endothelial cells. Furthermore, the expression of vascular cell adhesion molecule-1 and endoplasmic reticulum stress proteins were increased upon CRIF1 knockdown in endothelial cells. However, p66shc knockdown blunted the alteration in mitochondrial dynamics and ROS production in CRIF1 knockdown endothelial cells. In addition, p66shc knockdown reduced the CRIF1 knockdown-induced increases in adhesion between monocytes and endothelial cells. Taken together, these results suggest that CRIF1 knockdown partially induces endothelial activation via increased ROS production and phosphorylation of p66shc.
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Project description:Rho GDP-dissociation inhibitor (RhoGDI), a downregulator of Rho family GTPases, prevents nucleotide exchange and membrane association. It is responsible for the activation of Rho GTPases, which regulate a variety of cellular processes, such as migration. Although RhoGDI2 has been identified as a tumor suppressor gene involved in cellular migration and invasion, little is known about its role in vascular endothelial cell (EC) migration. CR6-interacting factor 1 (CRIF1) is a CR6/GADD45-interacting protein with important mitochondrial functions and regulation of cell growth. We examined the expression of RhoGDI2 in CRIF1-deficient human umbilical vein endothelial cells (HUVECs) and its role in cell migration. Expression of RhoGDI2 was found to be considerably higher in CRIF1-deficient HUVECs along with suppression of cell migration. Moreover, the phosphorylation levels of Akt and CREB were decreased in CRIF1-silenced cells. The Akt-CREB signaling pathway was implicated in the changes in endothelial cell migration caused by CRIF1 downregulation. In addition to RhoGDI2, we identified another factor that promotes migration and invasion of ECs. Adrenomedullin2 (ADM2) is an autocrine/paracrine factor that regulates vascular tone and other vascular functions. Endogenous ADM2 levels were elevated in CRIF1-silenced HUVECs with no effect on cell migration. However, siRNA-mediated depletion of RhoGDI2 or exogenous ADM2 administration significantly restored cell migration via the Akt-CREB signaling pathway. In conclusion, RhoGDI2 and ADM2 play important roles in the migration of CRIF1-deficient endothelial cells.
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Project description:The epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1 is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affects skin homeostasis and hair morphogenesis, we generated epidermis-specific Crif1 cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1 cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis of Crif1 cKO mice was disrupted by down-regulation of Wnt/β-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.
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Project description:Vascular smooth muscle cells (VSMCs) have been widely recognized as key players in regulating blood-brain barrier (BBB) function, and their roles are unclear in ischemic stroke. Myosin phosphatase target subunit 1 (MYPT1) is essential for VSMC contraction and maintaining healthy vasculature. We generated VSMC-specific MYPT1 knockout (MYPT1SMKO) mice and cultured VSMCs infected with Lv-shMYPT1 to explore phenotypic switching of VSMCs and the accompanied impacts on BBB integrity. We found that MYPT1 deficiency induced phenotypic switching of synthetic VSMCs, which aggravated BBB disruption. Proteomic analysis identified evolutionarily conserved signaling intermediates in Toll pathways (ECSIT) as a downstream molecule that promotes activation of synthetic VSMCs and contributed to IL-6 expression. Knocking down ECSIT rescued phenotypic switching of VSMCs and BBB disruption. Additionally, inhibition of IL-6 decreased BBB permeability. These findings reveal that MYPT1 deficiency activated phenotypic switching of synthetic VSMCs and induced BBB disruption through ECSIT-IL-6 signaling after ischemic stroke.
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Project description:The phosphorylation of the 20-kD myosin light chain (MLC) and actin filament formation play a key role in endothelial barrier disruption. MLC is either mono- or di-phosphorylated (pMLC and ppMLC) at T18 or S19. The present study investigated whether there are any distinct roles of pMLC and ppMLC in barrier disruption induced by thrombin. Thrombin induced a modest bi-phasic increase in pMLC and a robust mono-phasic increase in ppMLC. pMLC localized in the perinuclear cytoplasm during the initial phase, while ppMLC localized in the cell periphery, where actin bundles were formed. Later, the actin bundles were rearranged into stress fibers, where pMLC co-localized. Rho-kinase inhibitors inhibited thrombin-induced barrier disruption and peripheral localization of ppMLC and actin bundles. The double, but not single, mutation of phosphorylation sites abolished the formation of peripheral actin bundles and the barrier disruption, indicating that mono-phosphorylation of MLC at either T18 or S19 is functionally sufficient for barrier disruption. Namely, the peripheral localization, but not the degree of phosphorylation, is suggested to be essential for the functional effect of ppMLC. These results suggest that MLC phosphorylation and actin bundle formation in cell periphery are initial events during barrier disruption.
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