Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

Leukotriene B4 receptor 1 (BLT1) does not mediate disease progression in a mouse model of liver fibrosis.

MASH is a prevalent liver disease that can progress to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and ultimately death, but there are no approved therapies. Leukotriene B4 (LTB4) is a potent pro-inflammatory chemoattractant that drives macrophage and neutrophil chemotaxis, and genetic loss or inhibition of its high affinity receptor, leukotriene B4 receptor 1 (BLT1), results in improved insulin sensitivity and decreased hepatic steatosis. To validate the therapeutic efficacy of BLT1 inhibition in an inflammatory and pro-fibrotic mouse model of MASH and fibrosis, mice were challenged with a choline-deficient, L-amino acid defined high fat diet and treated with a BLT1 antagonist at 30 or 90 mg/kg for 8 weeks. Liver function, histology, and gene expression were evaluated at the end of the study. Treatment with the BLT1 antagonist significantly reduced plasma lipids and liver steatosis but had no impact on liver injury biomarkers or histological endpoints such as inflammation, ballooning, or fibrosis compared to control. Artificial intelligence-powered digital pathology analysis revealed a significant reduction in steatosis co-localized fibrosis in livers treated with the BLT1 antagonist. Liver RNA-seq and pathway analyses revealed significant changes in fatty acid, arachidonic acid, and eicosanoid metabolic pathways with BLT1 antagonist treatment, however, these changes were not sufficient to impact inflammation and fibrosis endpoints. Targeting this LTB4-BLT1 axis with a small molecule inhibitor in animal models of chronic liver disease should be considered with caution, and additional studies are warranted to understand the mechanistic nuances of BLT1 inhibition in the context of MASH and liver fibrosis.

Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease.

BACKGROUND: Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. METHODS: We use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue. RESULTS: Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects. CONCLUSIONS: Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects.

Astrocytes propel neurovascular dysfunction during cerebral cavernous malformation lesion formation.

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2- and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1α in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1α target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

Insulin and Metformin Control Cell Proliferation by Regulating TDG-Mediated DNA Demethylation in Liver and Breast Cancer Cells.

Type 2 diabetes mellitus (T2DM) is a frequent comorbidity of cancer. Hyperinsulinemia secondary to T2DM promotes cancer progression, whereas antidiabetic agents, such as metformin, have anticancer effects. However, the detailed mechanism for insulin and metformin-regulated cancer cell proliferation remains unclear. This study identified a mechanism by which insulin upregulated the expression of c-Myc, sterol regulatory element-binding protein 1 (SREBP1), and acetyl-coenzyme A (CoA) carboxylase 1 (ACC1), which are important regulators of lipogenesis and cell proliferation. Thymine DNA glycosylase (TDG), a DNA demethylase, was transactivated by c-Myc upon insulin treatment, thereby decreasing 5-carboxylcytosine (5caC) abundance in the SREBP1 promoter. On the other hand, metformin-activated AMP-activated protein kinase (AMPK) increased DNA methyltransferase 3A (DNMT3A) activity to increase 5-methylcytosine (5mC) abundance in the TDG promoter. This resulted in decreased TDG expression and enhanced 5caC abundance in the SREBP1 promoter. These findings demonstrate that c-Myc activates, whereas AMPK inhibits, TDG-mediated DNA demethylation of the SREBP1 promoter in insulin-promoted and metformin-suppressed cancer progression, respectively. This study indicates that TDG is an epigenetic-based therapeutic target for cancers associated with T2DM.

Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity.

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

AMPK promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors DNMT1, RBBP7, and HAT1.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) acts as a master regulator of cellular energy homeostasis by directly phosphorylating metabolic enzymes and nutrient transporters and by indirectly promoting the transactivation of nuclear genes involved in mitochondrial biogenesis and function. We explored the mechanism of AMPK-mediated induction of gene expression. We identified AMPK consensus phosphorylation sequences in three proteins involved in nucleosome remodeling: DNA methyltransferase 1 (DNMT1), retinoblastoma binding protein 7 (RBBP7), and histone acetyltransferase 1 (HAT1). DNMT1 mediates DNA methylation that limits transcription factor access to promoters and is inhibited by RBBP7. Acetylation of histones by HAT1 creates a more relaxed chromatin-DNA structure that favors transcription. AMPK-mediated phosphorylation resulted in the activation of HAT1 and inhibition of DNMT1. For DNMT1, this inhibition was both a direct effect of phosphorylation and the result of increased interaction with RBBP7. In human umbilical vein cells, pharmacological AMPK activation or pulsatile shear stress triggered nucleosome remodeling and decreased cytosine methylation, leading to increased expression of nuclear genes encoding factors involved in mitochondrial biogenesis and function, such as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), transcription factor A (Tfam), and uncoupling proteins 2 and 3 (UCP2 and UCP3). Similar effects were seen in the aortas of mice given pharmacological AMPK activators, and these effects required AMPK2α. These results enhance our understanding of AMPK-mediated mitochondrial gene expression through nucleosome remodeling.

Sterol regulatory element binding protein 2 activation of NLRP3 inflammasome in endothelium mediates hemodynamic-induced atherosclerosis susceptibility.

BACKGROUND: The molecular basis for the focal nature of atherosclerotic lesions is poorly understood. Here, we explored whether disturbed flow patterns activate an innate immune response to form the NLRP3 inflammasome scaffold in vascular endothelial cells via sterol regulatory element binding protein 2 (SREBP2). METHODS AND RESULTS: Oscillatory flow activates SREBP2 and induces NLRP3 inflammasome in endothelial cells. The underlying mechanisms involve SREBP2 transactivating NADPH oxidase 2 and NLRP3. Consistently, SREBP2, NADPH oxidase 2, and NLRP3 levels were elevated in atheroprone areas of mouse aortas, suggesting that the SREBP2-activated NLRP3 inflammasome causes functionally disturbed endothelium with increased inflammation. Mimicking the effect of atheroprone flow, endothelial cell-specific overexpression of the activated form of SREBP2 synergized with hyperlipidemia to increase atherosclerosis in the atheroresistant areas of mouse aortas. CONCLUSIONS: Atheroprone flow induces NLRP3 inflammasome in endothelium through SREBP2 activation. This increased innate immunity in endothelium synergizes with hyperlipidemia to cause topographical distribution of atherosclerotic lesions.

AMPKα2 exerts its anti-inflammatory effects through PARP-1 and Bcl-6.

B-cell lymphoma-6 protein (Bcl-6) is a corepressor for inflammatory mediators such as vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and -3, which function to recruit monocytes to vascular endothelial cells upon inflammation. Poly [ADP ribose] polymerase 1 (PARP-1) is proinflammatory, in part through its binding at the Bcl-6 intron 1 to suppress Bcl-6 expression. We investigated the mechanisms by which PARP-1 dissociates from the Bcl-6 intron 1, ultimately leading to attenuation of endothelial inflammation. Analysis of the PARP-1 primary sequence suggested that phosphorylation of PARP-1 Serine 177 (Ser-177) by AMP-activated protein kinase (AMPK) is responsible for the induction of Bcl-6. Our results show that AMPK activation with treatment of 5-aminoimidazole-4-carboxamide ribonucleotide, metformin, or pulsatile shear stress induces PARP-1 dissociation from the Bcl-6 intron 1, increases Bcl-6 expression, and inhibits expression of inflammatory mediators. Conversely, AMPKα suppression or knockdown produces the opposite effects. The results demonstrate an anti-infamatory pathway linking AMPK, PARP-1, and Bcl-6 in endothelial cells.