Human Brain Microvascular Endothelial Cells Research Articles

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

BackgroundTriptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp).MethodsWe investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice.ResultsWe demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice.ConclusionsWe have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.Graphical

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Genomic characterization of Cronobacter sakazakii strains in the neonatal meningitis clonal complex 4

AbstractCronobacter sakazakii clonal complex 4 (CC4) is strongly associated with neonatal meningitis. However, C. sakazakii CC4‐specific pathogenicity traits have not been determined. In this study, the comparative genomic analysis of 144 genomes of C. sakazakii CC4 strains and 376 non‐CC4 C. sakazakii strains was undertaken. Twenty‐four CC4 strains were previously undescribed clinical and nonclinical strains that had been collected from various regions in China (2006–2017). The remaining genomes were of multiple sequence types of C. sakazakii strains, which had been isolated worldwide (1973–2021). The pan‐genome of C. sakazakii comprised 32,332 genes, of which 2.58% (832 genes) constituted the core genome. More C. sakazakii CC4 strains have a complete cusABCFR efflux system with a significant difference compared with non‐CC4 strains, and the ibeB‐homologous cusC gene in the cusABCFR efflux system is probably associated with the invasion of human brain microvascular endothelial cells (BMEC). In addition, the isolates with K2:CA2 capsule or type IV pili‐associated genes associated with neonatal meningitis were statistically more present in CC4 strains than non‐CC4 strains (p < .0001). The thorough description of VirB/VirD4 gene cluster for the type IV secretion system and the impB/impF and vasL/vasJ gene clusters for the type VI secretion system in C. sakazakii were provided. Overall, the specific and diverse virulence factors and genes may have led to C. sakazakii CC4 clone increasing its ability to invade human BMEC and leading to neonatal meningitis. Our findings should facilitate the development of novel strategies to prevent, diagnose, and treat C. sakazakii CC4 infections.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

CircRNA-PTPN4 mediated regulation of FOXO3 and ZO-1 expression: implications for blood–brain barrier integrity and cognitive function in uremic encephalopathy

Uremic encephalopathy (UE) poses a significant challenge in neurology, leading to the need to investigate the involvement of non-coding RNA (ncRNA) in its development. This study employed ncRNA-seq and RNA-seq approaches to identify fundamental ncRNAs, specifically circRNA and miRNA, in the pathogenesis of UE using a mouse model. In vitro and in vivo experiments were conducted to explore the circRNA-PTPN4/miR-301a-3p/FOXO3 axis and its effects on blood–brain barrier (BBB) function and cognitive abilities. The research revealed that circRNA-PTPN4 binds to and inhibits miR-301a-3p, leading to an increase in FOXO3 expression. This upregulation results in alterations in the transcriptional regulation of ZO-1, affecting the permeability of human brain microvascular endothelial cells (HBMECs). The axis also influences the growth, proliferation, and migration of HBMECs. Mice with UE exhibited cognitive deficits, which were reversed by overexpression of circRNA-PTPN4, whereas silencing FOXO3 exacerbated these deficits. Furthermore, the uremic mice showed neuronal loss, inflammation, and dysfunction in the BBB, with the expression of circRNA-PTPN4 demonstrating therapeutic effects. In conclusion, circRNA-PTPN4 plays a role in promoting FOXO3 expression by sequestering miR-301a-3p, ultimately leading to the upregulation of ZO-1 expression and restoration of BBB function in mice with UE. This process contributes to the restoration of cognitive abilities.Graphical 1. The circRNA-PTPN4/miR-301a-3p/FOXO3 axis is identified as a key regulator of blood–brain barrier integrity and cognitive function in uremic encephalopathy.2. circRNA-PTPN4 sequestration of miR-301a-3p enhances FOXO3 expression, leading to upregulation of ZO-1 and improved endothelial permeability.3. Overexpression of circRNA-PTPN4 in uremic mice restores cognitive abilities and reduces neuronal loss and inflammatory infiltration.

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4-NOTCH-CLDN5 pathway and is vulnerable to neonatal hyperoxia.

The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4 regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.

High glutamine increases stroke risk by inducing the endothelial-to-mesenchymal transition in moyamoya disease.

At present, there is limited research on the mechanisms underlying moyamoya disease (MMD). Herein, we aimed to determine the role of glutamine in MMD pathogenesis, and 360 adult patients were prospectively enrolled. Human brain microvascular endothelial cells (HBMECs) were subjected to Integrin Subunit Beta 4 (ITGB4) overexpression or knockdown and atorvastatin. We assessed factors associated with various signaling pathways in the context of the endothelial-to-mesenchymal transition (EndMT), and the expression level of related proteins was validated in the superficial temporal arteries of patients. We found glutamine levels were positively associated with a greater risk of stroke (OR=1.599, p=0.022). After treatment with glutamine, HBMECs exhibited enhanced proliferation, migration, and EndMT, all reversed by ITGB4 knockdown. In ITGB4-transfected HBMECs, the MAPK-ERK-TGF-β/BMP pathway was activated, with Smad4 knockdown reversing the EndMT. Furthermore, atorvastatin suppressed the EndMT by inhibiting Smad1/5 phosphorylation and promoting Smad4 ubiquitination in ITGB4-transfected HBMECs. We also found the protein level of ITGB4 was upregulated in the superficial temporal arteries of patients with MMD. In conclusion, our study suggests that glutamine may be an independent risk factor for hemorrhage or infarction in patients with MMD and targeting ITGB4 could potentially be therapeutic approaches for MMD.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Mast cell activation triggered by SARS-CoV-2 causes inflammation in brain microvascular endothelial cells and microglia.

SARS-CoV-2-induced excessive inflammation in brain leads to damage of blood-brain barrier, hypoxic-ischemic injury, and neuron degeneration. The production of inflammatory cytokines by brain microvascular endothelial cells and microglia is reported to be critically associated with the brain pathology of COVID-19 patients. However, the cellular mechanisms for SARS-CoV-2-inducing activation of brain cells and the subsequent neuroinflammation remain to be fully delineated. Our research, along with others', has recently demonstrated that SARS-CoV-2-induced accumulation and activation of mast cells (MCs) in mouse lung could further induce inflammatory cytokines and consequent lung damages. Intracerebral MCs activation and their cross talk with other brain cells could induce neuroinflammation that play important roles in neurodegenerative diseases including virus-induced neuro-pathophysiology. In this study, we investigated the role of MC activation in SARS-CoV-2-induced neuroinflammation. We found that (1) SARS-CoV-2 infection triggered MC accumulation in the cerebrovascular region of mice; (2) spike/RBD (receptor-binding domain) protein-triggered MC activation induced inflammatory factors in human brain microvascular endothelial cells and microglia; (3) MC activation and degranulation destroyed the tight junction proteins in brain microvascular endothelial cells and induced the activation and proliferation of microglia. These findings reveal a cellular mechanism of SARS-CoV-2-induced neuroinflammation.

Inhibition of DAPK3 Suppresses Radiation-Induced Cellular Senescence by Activation of a PGC1α-Dependent Metabolism Pathway in Brain Endothelial Cells.

In the brain, environmental changes, such as neuroinflammation, can induce senescence, characterized by the decreased proliferation of neurons and dendrites and synaptic and vascular damage, resulting in cognitive decline. Senescence promotes neuroinflammatory disorders by senescence-associated secretory phenotypes and reactive oxygen species. In human brain microvascular endothelial cells (HBMVECs), we demonstrate that chronological aging and irradiation increase death-associated protein kinase 3 (DAPK3) expression. To confirm the role of DAPK3 in HBMVEC senescence, we disrupted DAPK3 activity using small interfering RNA (siRNA) or a dominant-negative mutant (DAPK3-P216S), which reduced cellular senescence phenotypes, as assessed by changes in tube formation, senescence-associated beta-galactosidase activity, and cell proliferation. In endothelial cells, DAPK3 promotes cellular senescence by regulating the phosphorylation and inactivation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) via the protein kinase B pathway, resulting in the decreased expression of mitochondrial metabolism-associated genes, such as ATP5G1, BDNF, and COX5A. Our studies show that DAPK3 is involved in cellular senescence and PGC1α regulation, suggesting that DAPK3 regulation may be important for treating aging-related brain diseases or the response to radiation therapy.

309 High Glutamine Associated With the Risk of Stroke by Inducing Endothelial to Mesenchymal Transition Through ITGB4 MAPK-ERK Smad in Moyamoya Disease

INTRODUCTION: Previous studies have suggested potential association between glutamine and stroke. The role of glutamine in moyamoya disease (MMD) manifestation and pathological process remains unknown. METHODS: A prospective cohort of 360 adult MMD patients between September 2020 to December 2021 was established. Human brain microvascular endothelial cells (HBMECs) were utilized for rt-qPCR, western blot, immunofluorescence, and functional assays including CCK8, Edu for proliferation and wound healing for migration. HBMECs were transfected with ITGB4 adenovirus, ITGB4 siRNA or Smad4 siRNA to explore the downstream alterations. Atorvastatin was used to treat HBMECs and the mechanism was explored. Finally, the expression of ITGB4 was validated in databases and immunofluorescence staining of superficial temporal artery in MMD patients. RESULTS: After fully adjusted, the risk of stroke increased with each increment of glutamine level (OR = 1.599, 95% CI = 1.071 – 2.387, p = 0.022). Consistently, compared with patients in the lower tertiles of serum glutamine levels, a significantly higher risk of complete stroke was found in those in highest glutamine tertile (OR = 2.752, 95% CI = 1.556 – 4.868, p = 0.001). After treated with glutamine, HBMECs showed increased proliferation, migration, and endothelial-mesenchymal transition (EndMT) which was reversed by ITGB4 knockdown. In ITGB4-transfected HBMECs, MAPK-ERK-TGF-β/BMP pathway was activated and Smad4 knockdown could reverse the EndMT. Atorvastatin alleviated the EndMT by inhibiting Smad2/3 phosphorylation in ITGB4-transfected HBMECs. The upregulated ITGB4 expression was validated in the superficial temporal artery of MMD patients. CONCLUSIONS: High glutamine was independently associated with the risk of stroke in MMD. Glutamine promotes the proliferation, migration, and EndMT by ITGB4 through MAPK-ERK-TGFβ/BMP-Smad pathway. Atorvastatin could alleviate the EndMT by inhibiting Smad2/3 phosphorylation and promoting Smad4 ubiquitination and thus may be a potential drug for MMD patients.

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease

BackgroundMoyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. MethodsOur study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. ResultsThe results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. ConclusionThese findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

ADAMTS5 Promotes Permeability of the Blood-Brain Barrier during Treponema pallidum Subspecies pallidum Invading the Central Nervous System.

The pathogenesis of neurosyphilis remains unclear. A previous study found a noteworthy up-regulation of a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 (ADAMTS5) gene in human brain microvascular endothelial cells cocultured with Treponema pallidum subspecies pallidum (Tp). To investigate the ADAMTS5 role in Tp invading the central nervous system (CNS), we conducted relevant experiments. Our study revealed that Tp caused an increase in human cortical microvascular endothelial cell/D3 (hCMEC/D3) barrier permeability and significantly enhanced ADAMTS5 expression. The heightened permeability of the hCMEC/D3 barrier was effectively mitigated by inhibiting ADAMTS5. During this process, Tp promoted interleukin-1β production, which, in turn, facilitated ADAMTS5 expression. Furthermore, Tp significantly reduced the glycocalyx on the surface of hCMEC/D3 cells, which was also ameliorated by inhibiting ADAMTS5. Additionally, ADAMTS5 and endothelial glycocalyx components notably increased in the cerebrospinal fluid of HIV-negative neurosyphilis patients. This research provided the first demonstration of the ADAMTS5 role in Tp invading the CNS and offered new insight into neurosyphilis pathogenesis.

CsiR-mediated signal transduction pathway in response to low iron conditions promotes Escherichia coli K1 invasion and penetration of the blood-brain barrier.

Escherichia coli K1 is the leading cause of neonatal Gram-negative bacterial meningitis, but the pathogenesis of E. coli K1 meningitis remains unclear. Blood-brain barrier (BBB) penetration is a crucial step in E. coli meningitis development. Here, we uncovered the crucial role of CsiR, a GntR family regulator, in E. coli K1 virulence. During infection, csiR expression was induced due to the derepression by Fur in the blood and human brain microvascular endothelial cells (HBMECs). CsiR positively regulated ilvB expression, which is associated with branched chain amino acid synthesis. Furthermore, we revealed that IlvB activated the FAK/PI3 K pathway of HBMECs to induce actin cytoskeleton rearrangements, thereby promoting the bacterial invasion and penetration of the BBB. Overall, this study reveals a CsiR-mediated virulence regulation pathway in E. coli K1, which may provide a useful target for the prevention or therapy of E. coli meningitis.

Abstract 5803: Tumor treating fields alter PDGFR-β localization in immortalized human pericytes in an in vitro model of the blood-brain barrier

Abstract Background: Tumor Treating Fields (TTFields) are alternating electric fields of intermediate frequency (100-500 kHz) and low intensity (1-3 V/cm). TTFields at 200 kHz, together with adjuvant chemotherapy, increased progression-free and overall survival of newly diagnosed Isocitrate dehydrogenase (IDH) wildtype and IDH mutant glioblastoma (GBM, WHO CNS 2016) patients in a randomized controlled trial and were therefore approved for the treatment of these patients. We recently demonstrated that TTFields at lower frequencies transiently increase the permeability of blood-brain barrier (BBB) models of murine and human origin by delocalizing junctional proteins such as claudin-5, ZO-1, and occludin in endothelial cells. The interaction between pericytes and endothelial cells contributes to the rigorous integrity of the BBB and is driven by endothelial cells releasing the platelet-derived growth factor β-subunit (PDGF-β), which binds to the receptor PDGFR-β on the surface of pericytes. Here, we investigated the effects of TTFields on pericytes in a human cell-based in vitro BBB model in order to gain a better understanding of the effects of TTFields on BBB integrity. Methods: We prepared an in vitro BBB model composed of human brain microvascular endothelial cells and human pericytes. The model was subjected to TTFields at 100-300 kHz for 24 to 72 h. Afterwards, pericytes were incubated with Alexa Fluor 488-labeled anti-PDGFR-β antibody in order to visualize PDGFR-β localization and cell morphology under the microscope. To make sure that no cell loss occurred due to TTFields treatment, the nuclei of pericytes were counted. Results: TTFields treatment altered the distribution of PDGFR-β staining after treatment, with fewer, less distinct and more punctate PDGFR-β foci. These changes were most prominent after 72 h of TTFields treatment at 300 kHz. We also observed larger nuclei in the pericytes after treatment but no significant loss of cells could be observed as per the nuclei count. Conclusion: Together with the previously observed delocalization of tight junctional proteins in endothelial cells, the altered distribution of PDGFR-β pericytes could reveal new mechanistic insights into the effects of TTFields on BBB integrity. Citation Format: Ellaine Salvador, Theresa Koeppl, Almuth F. Kessler, Malgorzata Burek, Ralf-Ingo Ernestus, Mario Löhr, Carsten Hagemann. Tumor treating fields alter PDGFR-β localization in immortalized human pericytes in an in vitro model of the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5803.

Adipokine vaspin maintains angiogenesis and neurological function during cerebral ischemia-reperfusion via suppressing endoplasmic reticulum stress.

Visceral adipose tissue-derived serine protease inhibitor (vaspin) is an adipokine. It has been reported that decreased serum vaspin levels are significantly associated with stroke severity and prognosis. This article aims to explore the theoretical feasibility of vaspin supplementation for cerebral ischemia-reperfusion (I/R) injury. The I/R mouse models were constructed by the middle cerebral artery occlusion (MCAO) method, and the effects of vaspin on cerebral infarction, neurological function, angiogenesis and endoplasmic reticulum (ER) stress were explored. To verify the mediation of ER stress in the regulation of vaspin, human brain microvascular endothelial cells (HBMECs) were subjected to ER stress agonist tunicamycin in vitro. The impacts of vaspin and tunicamycin on oxygen glucose deprivation/ recovery (OGD/R)-induced cell viability, apoptosis, and angiogenesis were examined. Vaspin inhibited blood-brain barrier breakdown and infarction occurred in the brain tissue of the I/R mice. Vaspin also enhanced cerebral neovascularization and reduced the apoptosis. Additional tunicamycin increased the apoptosis of HBMECs and inhibited angiogenesis, reversing the protective effect of vaspin on cells. Together, this study reveals that vaspin supplementation reduces cerebral infarction and works against neurological dysfunction. It maintains the survival and angiogenesis capacity of HBMECs by inhibiting ER stress.

LOX-1 and MMP-9 Inhibition Attenuates the Detrimental Effects of Delayed rt-PA Therapy and Improves Outcomes After Acute Ischemic Stroke.

Acute ischemic stroke triggers endothelial activation that disrupts vascular integrity and increases hemorrhagic transformation leading to worsened stroke outcomes. rt-PA (recombinant tissue-type plasminogen activator) is an effective treatment; however, its use is limited due to a restricted time window and hemorrhagic transformation risk, which in part may involve activation of MMPs (matrix metalloproteinases) mediated through LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1). This study's overall aim was to evaluate the therapeutic potential of novel MMP-9 (matrix metalloproteinase 9) and LOX-1 inhibitors in combination with rt-PA to improve stroke outcomes. A rat thromboembolic stroke model was utilized to investigate the impact of rt-PA delivered 4 hours poststroke onset as well as selective LOX-1 (BI-0115) and MMP-9 (JNJ0966) inhibitors given before rt-PA administration. Infarct size, perfusion, and hemorrhagic transformation were evaluated by 9.4-T magnetic resonance imaging, vascular and parenchymal MMP-9 activity via zymography, and neurological function was assessed using sensorimotor functioning testing. Human brain microvascular endothelial cells were exposed to hypoxia plus glucose deprivation/reperfusion (hypoxia plus glucose deprivation 3 hours/R 24 hours) and treated with ±rt-PA, ±MMP-9, and LOX-1 inhibitors. Barrier function was assessed via transendothelial electrical resistance, MMP-9 activity was determined with zymography, and LOX-1 and barrier gene expression/levels were measured using qRT-PCR and Western blot. Stroke and subsequent rt-PA treatment increased edema, hemorrhage, MMP-9 activity, LOX-1 expression, and worsened neurological outcomes. LOX-1 inhibition improved neurological function, reduced edema, and improved endothelial barrier integrity. Elevated MMP-9 activity correlated with increased edema, infarct volume, and decreased neurological function. MMP-9 inhibition reduced MMP-9 activity and LOX-1 expression. In human brain microvascular endothelial cells, LOX-1/MMP-9 inhibition differentially attenuated MMP-9 levels, inflammation, and activation following hypoxia plus glucose deprivation/R. Our findings indicate that MMP-9 and LOX-1 inhibition attenuates negative aspects of ischemic stroke with an rt-PA therapy, thus resulting in improved neurological function. While no synergistic effect was observed with simultaneous LOX-1 and MMP-9 inhibition, a distinct interaction is evident.

Geraniol attenuates oxygen-glucose deprivation/reoxygenation-induced ROS-dependent apoptosis and permeability of human brain microvascular endothelial cells by activating the Nrf-2/HO-1 pathway.

Blood-brain barrier breakdown and ROS overproduction are important events during the progression of ischemic stroke aggravating brain damage. Geraniol, a natural monoterpenoid, possesses anti-apoptotic, cytoprotective, anti-oxidant, and anti-inflammatory activities. Our study aimed to investigate the effect and underlying mechanisms of geraniol in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human brain microvascular endothelial cells (HBMECs). Apoptosis, caspase-3 activity, and cytotoxicity of HBMECs were evaluated using TUNEL, caspase-3 activity, and CCK-8 assays, respectively. The permeability of HBMECs was examined using FITC-dextran assay. Reactive oxygen species (ROS) production was measured using the fluorescent probe DCFH-DA. The protein levels of zonula occludens-1 (ZO-1), occludin, claudin-5, β-catenin, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were determined by western blotting. Geraniol showed no cytotoxicity in HBMECs. Geraniol and ROS scavenger N-acetylcysteine (NAC) both attenuated OGD/R-induced apoptosis and increase of caspase-3 activity and the permeability to FITC-dextran in HBMECs. Geraniol relieved OGD/R-induced ROS accumulation and decrease of expression of ZO-1, occludin, claudin-5, and β-catenin in HBMECs. Furthermore, we found that geraniol activated Nrf2/HO-1 pathway to inhibit ROS in HBMECs. In conclusion, geraniol attenuated OGD/R-induced ROS-dependent apoptosis and permeability in HBMECs through activating the Nrf2/HO-1 pathway.