Biology subjects

Frias-Anaya, E.

Publications and source records attributed to Frias-Anaya, E..

4 recordsLinked to original sources

bioRxiv2025

Persistent Activation of Endothelial Cells is Linked to Thrombosis and Inflammation in Cerebral Cavernous Malformation Disease

AbstractO_ST_ABSBACKGROUNDC_ST_ABSCerebral cavernous malformations (CCM) are neurovascular lesions that affect both children and adults, and morbidity often results from thrombosis, bleeding, and neurological dysfunction. Studies indicate that inflammation-related activation of endothelial cells contributes significantly to the worsening of CCM disease. This suggests that ongoing vascular inflammation and endothelial dysfunction are key factors associated with thrombosis and bleeding in CCM disease. However, the inflammatory mechanisms leading to altered brain endothelial cell function with a high propensity for thrombosis, inflammation, and dysfunction are not fully understood. METHODSMulti-omic analyses was conducted by performing simultaneous high-throughput single-nucleus RNA sequencing (snRNA-seq) and single-nucleus transposase-accessible chromatin sequencing (snATAC-seq) with the 10x Genomics multiome platform in combination with immunofluorescence to study CCM pathogenesis in both female and male mice with CCM (Slco1c1-CreERT2; Pdcd10fl/fl) disease. The analysis was complemented with bulk RNA-seq, bulk ATAC-seq, and ChIP-seq (Chromatin immunoprecipitation sequencing) using an in vitro human CCM model. An AAV-BR1 viral system selectively upregulates the activator protein-1 (AP-1) transcription factor JUNB in brain endothelial cells was used to evaluate its effectiveness in maintaining a persistent activated cell state during the pathogenesis of CCM. RESULTSWe found that epigenetics significantly influences the subtype identity and function of brain endothelial cells within the arteriovenous axis. Through multi-omic analyses, specific regulatory elements and enhancers (cis-Regulatory Elements, cCREs) in mouse brain endothelial cells were identified that influence subtype-specific transcriptional programs and the transcription factors responsible for establishing the various subtypes of brain endothelial cells. Additionally, large-scale epigenomic reprogramming of brain endothelial cell subtypes was observed during the pathogenesis of CCM disease. Among the most significant changes were alterations in the chromatin state of endothelial cells, along with transcriptional processes associated with a persistently activated endothelial cell state, which renders them susceptible to inflammation and thrombosis. The activator AP-1 transcription factor JUNB was identified as a key regulator of the persistently activated endothelial state during chronic neuroinflammation. Moreover, both trans- and cis-regulatory factors conserved between mice and humans were discovered and contribute to the progression of chronic CCM disease. CONCLUSIONSEpigenetics plays a crucial role in determining the transcription patterns and functions of brain arteriovenous endothelial cells. The activator JUNB is identified as a driver of chronic brain vascular inflammation by inducing a persistent activated endothelial cell state from epigenome reprogramming. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/662238v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1297edforg.highwire.dtl.DTLVardef@1488e27org.highwire.dtl.DTLVardef@6cae62org.highwire.dtl.DTLVardef@12cb282_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Silencing KRIT1 Partially Reverses the Effects of Disturbed Flow onThe Endothelial Cell Transcriptome

BackgroundEndothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow sheer stress are primarily mediated by the modulation of transcription factors Kruppel-like factors 2 and 4 (KLF2 and KLF4). Notably, disturbed flow patterns, which are found in vascular areas predisposed to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4, resulting in changes in the transcriptome that exacerbate inflammation and thrombosis. The endothelial CCM complex, comprising KRIT1, CCM2, and CCM3, suppresses the expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that silencing of KRIT1, the central scaffold of the CCM complex, can normalize the atherogenic effects of disturbed flow on the human endothelial transcriptome MethodsBulk RNA sequencing (RNA-seq) was conducted on human umbilical vein endothelial cells (HUVECs) after the expression of KRIT1 was silenced using specific siRNAs. The endothelial cells were exposed to three different conditions for 24 hours: pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), and static conditions (no flow). ResultsWe found that silencing KRIT1 expression in HUVECs restored the expression of the transcription factors KLF2 and KLF4 under oscillatory shear stress. This treatment resulted in a transcriptomic profile similar to that of endothelial cells under pulsatile shear stress. These findings suggest that inhibition of the CCM complex in endothelium plays a vasoprotective role by reactivating a protective gene program to help endothelial cells resist disturbed blood flow. ConclusionsTargeting CCM genes can activate well-known vasoprotective gene programs that enhance endothelial resilience to inflammation, hypoxia, and angiogenesis under disturbed flow conditions, providing a novel pathway for preventing atherosclerosis. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/642862v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@a20119org.highwire.dtl.DTLVardef@1f62f68org.highwire.dtl.DTLVardef@1a26d71org.highwire.dtl.DTLVardef@bf3c82_HPS_FORMAT_FIGEXP M_FIG C_FIG Endothelial cells respond to blood flow by expressing transcription factors such as Kruppel-Like Factors 2 and 4 (KLF2 and KLF4). Under oscillatory shear stress (OS) conditions, characterized by disturbed flow, KLF2 and KLF4 are suppressed in endothelial cells, which is linked to an atheroprone gene program that includes increased inflammation and risk of atherosclerosis (unhealthy endothelium). Our work shows that silencing of endothelial KRIT1 under OS increases expression of KLF2 and KLF4 mRNA levels as well as the expression of a protective gene program associated with resilience to inflammation, hypoxia, and angiogenesis, similar to those observed under pulsatile shear stress conditions (healthy endothelium).

pathology↗

bioRxiv2024

Behavioral impairments are linked to neuroinflammation in mice with Cerebral Cavernous Malformation disease

BackgroundCerebral Cavernous Malformations (CCMs) are neurovascular abnormalities in the central nervous system (CNS) caused by loss of function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3) genes. One of the most common symptoms in CCM patients is associated with motor disability, weakness, seizures, stress, and anxiety, and the extent of the symptom or symptoms may be due to the location of the lesion within the CNS or whether multiple lesions are present. Previous studies have primarily focused on understanding the pathology of CCM using animal models. However, more research has yet to explore the potential impact of CCM lesions on behavioral deficits in animal models, including effects on short-term and long-term memory, motor coordination, and function. MethodsWe used the accelerating RotaRod test to assess motor and coordination deficits. We also used the open field test to assess locomotor activity and pathology-related behavior and Pavlovian fear conditioning to assess short--and long-term memory deficits. Our behavioral studies were complemented by proteomics, histology, immunofluorescence, and imaging techniques. We found that neuroinflammation is crucial in behavioral deficits in male and female mice with neurovascular CCM lesions (Slco1c1-iCreERT2; Pdcd10fl/fl; Pdcd10BECKO). ResultsFunctional behavior tests in male and female Pdcd10BECKO mice revealed that CCM lesions cause sudden motor coordination deficits associated with the manifestation of profound neuroinflammatory lesions. Our findings indicate that maturation of CCM lesions in Pdcd10BECKO mice also experienced a significant change in short- and long-term memory compared to their littermate controls, Pdcd10fl/fl mice. Proteomic experiments reveal that as CCM lesions mature, there is an increase in pathways associated with inflammation, coagulation, and angiogenesis, and a decrease in pathways associated with learning and plasticity. Therefore, our study shows that Pdcd10BECKO mice display a wide range of behavioral deficits due to significant lesion formation in their central nervous system and that signaling pathways associated with neuroinflammation and learning impact behavioral outcomes. ConclusionsOur study found that CCM animal models exhibited behavioral impairments such as decreased motor coordination and amnesia. These impairments were associated with the maturation of CCM lesions that displayed a neuroinflammatory pattern.

animal behavior and cognition↗

bioRxiv2022

Neuroinflammation plays a critical role in cerebral cavernous malformation disease

BackgroundCerebral Cavernous Malformations (CCMs) are neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect [~]1/200 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-seq, histology, flow cytometry and imaging techniques to report the interaction between CCM-endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CALMN 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-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 astrogliosis. CCM reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-seq analysis on RNA isolated from brain endothelial cells (BECs) 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 significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA caspase-1) in BECs 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-kB activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in elevated number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-kB 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-kB activity may contribute to detrimental side effects.

neuroscience↗