Age-related Alterations on Ultrastructure and Gene Expression Profile of the Female Blood-brain Barrier

Blood-Brain Barrier (BBB) breakdown occurs in ageing and neurodegenerative disorders and affects several brain regions including cortex and hippocampus. During ageing, structural and functional changes affecting the main BBB components (brain endothelial cells (BECs), pericytes and astrocytes), appear to be associated with altered expression of genes and microRNAs (miRNAs) potentially related to development, protein synthesis or longevity pathways. However, little is known about the age-related BBB dysfunction in females. In this study, we aimed to assess the relation between ultrastructural and transcriptional changes in the ageing female BBB. A combination of transmission electron microscopy (TEM) and 3D reconstruction was used to study microvessel ultrastructure in 6- and 24-month-old female C57BL/6J mice. According to our results, the ageing female BBB shows a significant increase in basement membrane (BM) thickness, volume and number of BEC pseudopods, pericyte mitochondrial volume, pericyte – BEC contact and tight junction (TJ) tortuosity. Also, cortical capillaries appeared to be prominently more affected during ageing than hippocampal capillaries. These results suggest a higher impact of ageing on the cortical BBB in females, promoting changes that lead to a pro-inflammatory state, among other processes. In addition, sequencing results showed that the majority of upregulated genes in the ageing female BBB were involved in inflammation and immune response pathways, whereas the downregulated genes were mostly related to metabolism and signalling pathways. Amongst the age-deregulated mRNAs and miRNAs, miR-144-3p (upregulated) and Dnmt3a (downregulated) were selected for functional analysis in a human BEC line (hCMEC/D3), where their inverse correlation was confirmed. However, DNMT3A, not miR-144-3p, was shown to influence BEC function when deregulated, thereby promoting higher leukocyte adhesion and mRNA levels of adhesion molecules (ICAM-1, VCAM-1) and the chemokine CCL5. Age-induced increase of miR-144-3p appears to modulate DNMT3A expression, but DNMT3A might be independently contributing more to switching BECs into a pro-inflammatory state

Age-related ultrastructural neurovascular changes in the female mouse cortex and hippocampus

Blood-Brain Barrier (BBB) breakdown occurs in ageing and neurodegenerative diseases. Although age-associated alterations have previously been described, most studies focused in male brains; hence, little is known about BBB breakdown in females. This study measured ultrastructural features in the ageing female BBB using transmission electron microscopy (TEM) and 3D reconstruction of cortical and hippocampal capillaries from 6- and 24-month-old female C57BL/6J mice. Aged cortical capillaries showed more changes than hippocampal capillaries. Specifically, aged cortex showed thicker basement membrane (BM), higher number and volume of endothelial pseudopods, decreased endothelial mitochondrial number, larger pericyte mitochondria, higher pericyte – endothelial cell contact and increased tight junction tortuosity compared to young animals. Only increased BM thickness and pericyte mitochondrial volume were observed in aged hippocampus. Regional comparison revealed significant differences in endothelial pseudopods and tight junctions between cortex and hippocampus of 24-month-old mice. Therefore, the ageing female BBB shows region-specific ultrastructural alterations that may lead to oxidative stress and abnormal capillary blood flow and barrier stability, potentially contributing to cerebrovascular diseases, particularly in post-menopausal women

Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease

BackgroundCerebral 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.MethodsWe 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.ResultsExpression 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.ConclusionsOur 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