Brain Endothelial Cell Line Research Articles

Eucalyptus Wood Smoke Extract Elicits a Dose-Dependent Effect in Brain Endothelial Cells

The frequency, duration, and size of wildfires have been increasing, and the inhalation of wildfire smoke particles poses a significant risk to human health. Epidemiological studies have shown that wildfire smoke exposure is positively associated with cognitive and neurological dysfunctions. However, there is a significant gap in knowledge on how wildfire smoke exposure can affect the blood–brain barrier and cause molecular and cellular changes in the brain. Our study aims to determine the acute effect of smoldering eucalyptus wood smoke extract (WSE) on brain endothelial cells for potential neurotoxicity in vitro. Primary human brain microvascular endothelial cells (HBMEC) and immortalized human brain endothelial cell line (hCMEC/D3) were treated with different doses of WSE for 24 h. WSE treatment resulted in a dose-dependent increase in IL-8 in both HBMEC and hCMEC/D3. RNA-seq analyses showed a dose-dependent upregulation of genes involved in aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways and a decrease in tight junction markers in both HBMEC and hCMEC/D3. When comparing untreated controls, RNA-seq analyses showed that HBMEC have a higher expression of tight junction markers compared to hCMEC/D3. In summary, our study found that 24 h WSE treatment increases IL-8 production dose-dependently and decreases tight junction markers in both HBMEC and hCMEC/D3 that may be mediated through the AhR and NRF2 pathways, and HBMEC could be a better in vitro model for studying the effect of wood smoke extract or particles on brain endothelial cells.

Colombian cyanobacteria with cytotoxic activity in cancer cell lines

In the last decade, cyanobacteria have emerged as significant reservoirs of bioactive molecule for the pharmaceutical, cosmetic, and alimentary industries, given the wide spectrum of new possibilities of different organic pigments, proteins, carbohydrates, lipids, and powerful antioxidant sources for specific and stronger cancer treatments, autoimmune syndromes, obesity, and inflammatory diseases. A bioactivity screening was executed for 12 strains of Colombian cyanobacteria (10 marine and 2 freshwater) representative of the orders present in the LAUN culture collection, performing methanol extracts per sample, which was fractionated by reverse phase HPLC protocol, obtaining 8 fractions for each crude extract. All these crude extracts were tested for antimicrobial activities through disk diffusion methodology, and fractions were tested for cytotoxic activity against cancer cell lines by cell viability detection with MTT. Cyanobacteria's extracts showed considerable cytotoxic activity for osteosarcoma (MG063) and colon cancer (HCT116) cell lines (61 and 66 % of reduced viability compared to untreated controls, respectively) and a surprising cell growth promotion for the control group fibroblast (3T3L1) and brain endothelial (HCMEC) cell lines (121 and 127 % growth, respectively), no bioactivity was observed in the antimicrobial tests. These findings underscore the expansive cytotoxic potential of Colombian cyanobacteria against cancer cell lines and, notably, their growth-promoting effects on healthy cell lines. This positions them as promising sources of bioactive compounds for future pharmaceutical developments.

Cellular uptake of allicin in the hCMEC/D3 human brain endothelial cells: exploring blood-brain barrier penetration in an in vitro model.

Allicin, a bioactive compound derived from garlic (Allium sativum), demonstrates antibacterial activity against a broad spectrum of bacteria including the most common meningitis pathogens. In order to advocate for allicin as a potential therapeutic candidate for bacterial meningitis, the present study aimed to assess the ability of allicin to cross the blood-brain barrier (BBB) using an in vitro model. The cell viability of the human brain endothelial cell line hCMEC/D3 after incubation with various concentrations of allicin was investigated using an MTT assay at 3 and 24 h. Additionally, reactive oxygen species (ROS) production of allicin-treated hCMEC/D3 cells was examined at 3 h. The concentrations of allicin that were not toxic to the cells, as determined by the MTT assay, and did not significantly increase ROS generation, were then used to investigate allicin's ability to traverse the in vitro BBB model for 3 h. High-performance liquid chromatography (HPLC) analysis was utilized to examine the allicin concentration capable of passing the in vitro BBB model. The cellular uptake experiments were subsequently performed to observe the uptake of allicin into hCMEC/D3 cells. The pkCSM online tool was used to predict the absorption, distribution, metabolism, excretion, and pharmacokinetic properties of allicin and S-allylmercaptoglutathione (GSSA). The results from MTT assay indicated that the highest non-toxicity concentration of allicin on hCMEC/D3 cells was 5 µg/ml at 3 h and 2 µg/ml at 24 h. Allicin significantly enhanced ROS production of hCMEC/D3 cells at 10 µg/ml at 3 h. After applying the non-toxicity concentrations of allicin (0.5-5 µg/ml) to the in vitro BBB model for 3 h, allicin was not detectable in both apical and basolateral chambers in the presence of hCMEC/D3 cells. On the contrary, allicin was detected in both chambers in the absence of the cells. The results from cellular uptake experiments at 3 h revealed that hCMEC/D3 cells at 1×104 cells could uptake allicin at concentrations of 0.5, 1, and 2 µg/ml. Moreover, allicin uptake of hCMEC/D3 cells was proportional to the cell number, and the cells at 5×104 could completely uptake allicin at a concentration of 5 µg/ml within 0.5 h. The topological polar surface area (TPSA) predicting for allicin was determined to be 62.082 Å2, indicating its potential ability to cross the BBB. Additionally, the calculated logBB value surpassing 0.3 suggests that the compound may exhibit ease of penetration through the BBB. The present results suggested that allicin was rapidly taken up by hCMEC/D3 cells in vitro BBB model. The prediction results of allicin's distribution patterns suggested that the compound possesses the capability to enter the brain.

Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase.

Recent research suggests that photobiomodulation therapy (PBMT) positively impacts the vascular function associated with various cerebrovascular diseases. Nevertheless, the specific mechanisms by which PBMT improves vascular function remain ambiguous. Since endothelial nitric oxide synthase (eNOS) is crucial in regulating vascular function following cerebral ischemia, we investigated whether eNOS is a key element controlling cerebrovascular function and the senescence of vascular endothelial cells following PBMT treatment. Both rat photothrombotic (PT) stroke and in vitro oxygen-glucose deprivation (OGD)-induced vascular endothelial injury models were utilized. We demonstrated that treatment with PBMT (808 nm, 350 mW/cm2, 2 min/day) for 7 days significantly reduced PT-stroke-induced vascular permeability. Additionally, PBMT inhibited the levels of endothelial senescence markers (senescence green and p21) and antiangiogenic factor (endostatin), while increasing the phospho-eNOS (Ser1177) in the peri-infarct region following PT stroke. In vitro study further indicated that OGD increased p21, endostatin, and DNA damage (γH2AX) levels in the brain endothelial cell line, but they were reversed by PBMT. Intriguingly, the beneficial effects of PBMT were attenuated by a NOS inhibitor. In summary, these findings provide novel insights into the role of eNOS in PBMT-mediated protection against cerebrovascular senescence and endothelial dysfunction following ischemia. The use of PBMT as a therapeutic is a promising strategy to improve endothelial function in cerebrovascular disease.

Abstract 2457: Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma

Abstract The median survival time for newly diagnosed patients with glioblastoma (GBM) is 15 months, highlighting the need for improved therapies. Current treatment for newly diagnosed and recurrent GBM consists of tumor resection and chemoradiation therapy. The immunosuppressive tumor microenvironment (TME) is a central obstacle in the treatment of GBM. Lack of NK cell and CD8+ T cell infiltration prevents efficacy with immunotherapies like immune checkpoint blockade (ICB).One approach our lab is pursuing to enhance tumor infiltrating lymphocytes is through harnessing the anti-viral cGAS-STING pathway. Upon cGAS activation by cytosolic double stranded DNA, the TBK1 kinase is phosphorylated, initiating a subsequent type I interferon (IFN) response and the recruitment of immune cells. This pathway is commonly altered in many cancer types to evade immune activation. Our lab has demonstrated survival benefits with STING agonist treatment in immunocompetent GBM mouse models. We have also shown strong STING localization in the vasculature, implicating endothelial cells as a potential treatment target for anti-tumor efficacy. Here we investigated the potential role of tumor vasculature in mediating the effects of STING agonists in GBM therapy.We have shown robust cGAS-STING activation in brain endothelial cell lines through enhanced secretion of CXCL10, IFN-β, and RANTES upon STING agonist treatment in vitro. Western blotting also shows pathway activation in response to treatment through increased phospho-TBK1 levels. Activation was further potentiated by manganese.To investigate the effects of STING activation in brain endothelial cells in detail, we performed RNA sequencing. This showed 262 differentially expressed genes in response to pharmacologic cGAS-STING activation, with 183 of these being upregulated. The top gene ontology terms enriched include type I interferon signaling pathway, defense response to virus, and antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent. In addition, gene expression of tight junction protein Claudin 5 was also enhanced. We have also shown increased resistance in response to activation, through transendothelial electrical resistance (TEER) assays. Taken together, these results suggest that cGAS-STING activation in endothelial cells leads to tight junction remodeling in addition to immunomodulatory changes, implicating roles for endothelial cells in barrier function and immune cell interactions respectively. Current research in our lab focuses on elucidating the mechanisms behind these changes. Our goal is to identify beneficial endothelial cell changes which promote anti-tumor immunity in order to inform future treatment strategies. We are currently performing studies aimed at demonstrating that cGAS-STING activation in endothelial cells is critical for the efficacy of STING agonist treatment. Citation Format: Andrea Schmidt, Sandra Remson, Philippa Vaughn-Beaucaire, Jorge L. Jimenez-Macias, Praveen Srinivasan, Maximillian Pinho-Schwermann, Sean Lawler. Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma [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 2457.

Abstract 3602: TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results

Abstract Introduction: Natural killer (NK) cell tumor infiltration and higher expression of activating receptors NKp30 and NKp46 are associated with improved clinical outcomes in prostate cancer (PC). Activation of NK cells by androgen receptor inhibitors enhanced the killing of PC cell lines (Schwermann 2023). These findings support the therapeutic potential of strategies inducing NK cell activation to treat CRPC. NK cells induce apoptosis of cancer cells via TNF-related apoptosis-inducing ligand) (TRAIL) binding of death receptor 5 (DR5), which is overexpressed in PC cells. This is the first report of adoptive therapy utilizing high expression of TRAIL as a novel therapeutic strategy. Methods: We transduced NK-92-MI and CD4+ (Jurkat) cell lines with human TRAIL (pLVX-EF1alpha-TRAIL-IRES-eGFP).Transduced cells were pre-treated with simvastatin or atorvastatin (4uM) for 36h, followed by stimulation (IL-2 100 IU/ml and IL-12 100 ng/ml) for 2h. The eGFP-expressing cells were then sorted by FACS. PC (PC3, DU145, LNCaP, and 22Rv1), fibroblast (IMR-90), and brain endothelial (hCMEC/D3) cell lines were used in co-culture experiments with non-transduced and transduced NK cells and CD4+ cells. These experiments were quantified using the ImageXpress Confocal HT instrument. Viability assays were performed using Annexin-PI and flow cytometry staining for caspases 3/7. Results: Transduced NK-92 MI cells with TRAIL displayed increased surface expression of TRAIL (control[C]: 4.38%±1; TRAIL-overexpression[NK-TRAIL]: 78.54%±3.76; p<0.001). Co-culture of PC cell lines with TRAIL-NK-92 MI cells resulted in significant tumor-induced apoptosis compared to co-culture with non-transduced NK cells after 48 hours (LNCaP + NK [C]: 16.52%±5, LNCaP + NK-TRAIL: 73.19%±6; PC [C]: 14.4%±3, PC3 + NK-TRAIL: 79%±8; 22Rv1 [C]: 16.78%±1.7, 22Rv1 + NK-TRAIL: 76%±8; DU145 [C]: 13%±4, DU145 + NK-TRAIL: 59.1%±10; p<0.001). This enhanced cytotoxic function was also observed in CD4+ cells expressing high levels of TRAIL. Annexin-PI staining as well as Caspase 3 and 7 showed increased apoptosis in PC co-cultured with NK-TRAIL. Co-culture of NK-TRAIL cells did not affect the viability of the non-malignant cells (IMR-90 and hCMEC/D3) over 48h (IMR-90 + NK: 4.6%±1, IMR-90 + NK-TRAIL: 4.47±2, p=0.9; hCMEC/D3 + NK: 2.7%±0.8, hCMEC/D3 + NK-TRAIL: 2.2±0.5, p>0.99). These results align with the lower expression of DR5 on non-malignant cells compared to PC cell lines (IMR-90: 1.46%±0.5, hCMEC/D3: 1.9%±0.8, LNCaP: 92.3%±4.5). Conclusion: Transduced NK-92 MI cells with human TRAIL-induced higher levels of apoptosis of PC cell lines compared with non-transduced NK cells in co-culture experiments. The results highlight the potential of TRAIL-expressing adoptive cell therapy for CRPC. Engaging adoptive therapies with specific targets (anti-PSMA CAR) might increase the therapeutic potential of this strategy. Citation Format: Maximilian Schwermann, Praveen Srinivasan, William MacDonald, Vida Tajiknia, Andrea Schmidt, Shengliang Zhang, Lindsey Carlsen, Andrew George, Connor Purcell, Shaolei Lu, Matthew Hadfield, Anthony Mega, Benedito Carneiro, Wafik El-Deiry. TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results [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 3602.

A Critical Review of Nanosponge Based Formulation System from The perspective of Brain Targeted Drug Delivery

Brain tumor causes of millions of life every year due to poor treatment options. The blood-brain barrier prevents most of the treatment molecules to reach the tumor region. Tight junctions within adjacent brain endothelial cell lines including other components make the brain highly impermeable to all the unwanted and foreign materials. The antineoplastic drug molecules which has a molecular weight of less than 400 daltons and have less than 8 hydrogen bonds are only able to access the brain without any hindrance. Hence, most of the small and large anti-cancer drug molecules hardly can cross the barrier. To overcome these problems formulation scientists have adopted various strategies and techniques so that the intended drug molecule can reach the target region of the brain tumor. Among them nanosponges drug delivery is highly appreciated as emerging brain tumor targeted drug delivery. Nanosponges are tiny sponge in the size of nano range with a vesicle filled with various types of drugs. These kinds of the formulation can circulate throughout the blood and reach the target region where drugs are released in a controlled manner. This review article highlights the unique features of blood-brain barrier and novel strategies based on drug formulation to access the core of the brain tumor by overcoming the resistance rendered by blood brain barrier. In addition, it also demonstrates how nanosponges is emerging as one of the best options to prevail over various challenges associated with penetration of the blood-brain barrier.

Activation of testosterone-androgen receptor mediates cerebrovascular protection by photobiomodulation treatment in photothrombosis-induced stroke rats.

Numerous epidemiological studies have reported a link between low testosterone levels and an increased risk of cerebrovascular disease in men. However, there is ongoing controversy surrounding testosterone replacement therapy due to potential side effects. PBMT has been demonstrated to improve cerebrovascular function and promote testosterone synthesis in peripheral tissues. Despite this, the molecular mechanisms that could connect PBMT with testosterone and vascular function in the brain of photothrombosis (PT)-induced stroke rats remain largely unknown. We measured behavioral performance, cerebral blood flow (CBF), vascular permeability, and the expression of vascular-associated and apoptotic proteins in PT-induced stroke rats treated with flutamide and seven consecutive days of PBM treatment (350 mW, 808 nM, 2 min/day). To gain further insights into the mechanism of PBM on testosterone synthesis, we used testosterone synthesis inhibitors to study their effects on bEND.3 cells. We showed that PT stroke caused a decrease in cerebrovascular testosterone concentration, which was significantly increased by 7-day PBMT (808 nm, 350 mW/cm2 , 42 J/cm2 ). Furthermore, PBMT significantly increased cerebral blood flow (CBF) and the expression of vascular-associated proteins, while inhibiting vascular permeability and reducing endothelial cell apoptosis. This ultimately mitigated behavioral deficits in PT stroke rats. Notably, treatment with the androgen receptor antagonist flutamide reversed the beneficial effects of PBMT. Cellular experiments confirmed that PBMT inhibited cell apoptosis and increased vascular-associated protein expression in brain endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD). However, these effects were inhibited by flutamide. Moreover, mechanistic studies revealed that PBMT-induced testosterone synthesis in bEnd.3 cells was partly mediated by 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5). Our study provides evidence that PBMT attenuates cerebrovascular injury and behavioral deficits associated with testosterone/AR following ischemic stroke. Our findings suggest that PBMT may be a promising alternative approach for managing cerebrovascular diseases.

Increased Expression of Transferrin Receptor 1 in the Brain Cortex of 5xFAD Mouse Model of Alzheimer's Disease Is Associated with Activation of HIF-1 Signaling Pathway.

Alzheimer's disease (AD) is the most common cause of dementia. Despite intensive research efforts, there are currently no effective treatments to cure and prevent AD. There is growing evidence that dysregulation of iron homeostasis may contribute to the pathogenesis of AD. Given the important role of the transferrin receptor 1 (TfR1) in regulating iron distribution in the brain, as well as in the drug delivery, we investigated its expression in the brain cortex and isolated brain microvessels from female 8-month-old 5xFAD mice mimicking advanced stage of AD. Moreover, we explored the association between the TfR1 expression and the activation of the HIF-1 signaling pathway, as well as oxidative stress and inflammation in 5xFAD mice. Finally, we studied the impact of Aβ1-40 and Aβ1-42 on TfR1 expression in the brain endothelial cell line hCMEC/D3. In the present study, we revealed that an increase in TfR1 protein levels observed in the brain cortex of 5xFAD mice was associated with activation of the HIF-1 signaling pathway as well as accompanied by oxidative stress and inflammation. Interestingly, incubation of Aβ peptides in hCMEC/D3 cells did not affect the expression of TfR1, which supported our findings of unaltered TfR1 expression in the isolated brain microvessels in 5xFAD mice. In conclusion, the study provides important information about the expression of TfR1 in the 5xFAD mouse model and the potential role of HIF-1 signaling pathway in the regulation of TfR1 in AD, which could represent a promising strategy for the development of therapies for AD.

Host-microbe interactions at the blood-brain barrier through the lens of induced pluripotent stem cell-derived brain-like endothelial cells.

Microbe-induced meningoencephalitis/meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when pathogens are able to cross the blood-brain barrier (BBB) and gain access to the CNS. The BBB consists of highly specialized brain endothelial cells that exhibit specific properties to allow tight regulation of CNS homeostasis and prevent pathogen crossing. However, during meningoencephalitis/meningitis, the BBB fails to protect the CNS. Modeling the BBB remains a challenge due to the specialized characteristics of these cells. In this review, we cover the induced pluripotent stem cell-derived, brain-like endothelial cell model during host-pathogen interaction, highlighting the strengths and recent work on various pathogens known to interact with the BBB. As stem cell technologies are becoming more prominent, the stem cell-derived, brain-like endothelial cell model has been able to reveal new insights in vitro, which remain challenging with other in vitro cell-based models consisting of primary human brain endothelial cells and immortalized human brain endothelial cell lines.

Activation of CB2R by synthetic CB2R agonist, PM289, improves brain endothelial barrier properties, decreases inflammatory response and enhances endothelial repair.

The Cannabinoid 2 Receptor (CB2R) has been found to provide immunological modulation in different cell types. More recently, detection of CB2R in the cerebral endothelium suggests a possible role in the resolution of inflammation at the level of the blood-brain-barrier (BBB). Here, the notion that CB2R upregulation in brain endothelial cells could be exploited to promote vascular protection and BBB integrity was evaluated. Targeting and activation of CB2R was accomplished by a novel and highly specific chromenopyrazole based CB2R agonist, PM289. This study demonstrates that CB2R upregulation is induced as early as 8 h in the cortical vasculature in an experimental mouse model of TBI. Unlike CB2R, CB1R was marginally detected and not significantly induced. In the human brain endothelial cell line, hCMEC/D3 cells, similar induction of CB2R was observed upon stimulation with TNFα. Analysis of transendothelial electrical resistance shows that PM289 markedly prevented the barrier-leakiness induced by TNFα. The BBB is also responsible for maintaining an immunological barrier. The five-fold increase in ICAM1 expression in stimulated endothelial cells was significantly diminished due to CB2R activation. Utilizing wounding assays, results showed that wound repair could be accomplished in nearly half the time when the novel CB2R agonist is present compared to the untreated control. Lastly, mechanistically, the effects of CB2R may be explained by the observed inhibition of the p65 NFκB subunit. Overall, these studies support the notion that targeting and activating CB2R in the brain vasculature could aid in BBB and vascular protection in the context of neuroinflammation.

TRPM7 Mediates BSCB Disruption After Spinal Cord Injury by Regulating the mTOR/JMJD3 Axis in Rats.

After spinal cord injury (SCI), secondary injuries including blood cells infiltration followed by the production of inflammatory mediators are led by blood-spinal cord barrier (BSCB) breakdown. Therefore, preventing BSCB damage could alleviate the secondary injury progresses after SCI. Recently, we reported that transient receptor potential melastatin 7 channel (TRPM7) expression is increased in vascular endothelial cells after injury and thereby mediates BSCB disruption. However, the mechanism by which TRPM7 regulates BSCB disruption has not been examined yet. In current research, we show that TRPM7 mediates BSCB disruption via mammalian target of rapamycin (mTOR) pathway after SCI in rats. After contusion injury at T9 level of spinal cord, mTOR pathway was activated in the endothelial cells of blood vessels and TRPM7 was involved in the activation of mTOR pathway. BSCB disruption, MMP-2/9 activation, and blood cell infiltration after injury were alleviated by rapamycin, a mTOR signaling inhibitor. Rapamycin also conserved the level of tight junction proteins, which were decreased after SCI. Furthermore, mTOR pathway regulated the expression and activation of histone H3K27 demethylase JMJD3, known as a key epigenetic regulator mediating BSCB damage after SCI. In addition, rapamycin inhibited JMJD3 expression, the loss of tight junction molecules, and MMP-2/9 expression in bEnd.3, a brain endothelial cell line, after oxygen-glucose deprivation/reoxygenation. Thus, our results suggest that TRPM7 contributes to the BSCB disruption by regulating JMJD3 expression through the mTOR pathway after SCI.

High-throughput sequencing-based Detection of Japanese encephalitis virus and its effect on micro ribonucleic acid

it was to explore the mechanism of Japanese encephalitis virus (JEV) and micro ribonucleic acid (miRNA) under high-throughput sequencing. 20 experimental mice, with good growth status and no disease infection, were selected. The cells used in the experiment included mouse microglial cell line (BV2), mouse neuroblastoma cell line (NA), and mouse brain endothelial cell line (bEnd.3). JEV titration was performed with JEV-infected cells, ribonucleic acid (RNA) in the cells was extracted, and finally the miRNA high-throughput sequencing data was analyzed. Agarose gel electrophoresis showed that the 28S and 18S electrophoresis bands were bright. Among the miRNAs detected in mouse brain tissues, 2986 were down-regulated and 1251 were up-regulated. Among miRNAs detected in NA cells, 4238 the decreasing expression and 2356 were expressed increasingly. In reducing miRNA expression, 1 multiplicity of infection (MOI) of P3 strain infection was more significant than 0.1 MOI. 10 miRNAs with significantly decreasing expression were miR-466d-3p, miR-381–3p, miR-540–3p, miR-466a-3p, miR-467a-3p, miR-574–5p, miR-199a-5p, miR-467a-5p, miR-674–5p, and miR-376b-3p. These were all obviously down-regulated in JEV-infected BV2, NA, and bEnd.3 neurons. High-throughput sequencing of JEV-infected mouse brain tissues and mouse neuronal cells found that JEV infection led to down-regulation of overall miRNA expression in host cells.

Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor.

The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.

Abstract TP209: Role Of Endoglin In The Regulation Of Organic Anion Transporting Polypeptide 1a4 At The Blood-brain Barrier: Relevance To The Treatment Of Ischemic Stroke

Objectives: We have shown that transforming growth factor-β (TGF-β)/activin-like kinase 1 (ALK1) signaling at the blood-brain barrier (BBB) can regulate organic anion transporting polypeptide 1a4 (Oatp1a4), a transporter involved in brain uptake of drugs relevant to stroke pharmacotherapy (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins)). To date, the involvement of endoglin (ENG), a co-receptor that can modulate the TGF-β/ALK1 pathway, in regulation of Oatp1a4 expression/activity has not been elucidated. The objective of this study is to evaluate whether ENG plays a role in the control of BBB transport mechanisms for stroke therapeutics. Methods: Our studies were conducted using the immortalized mouse brain endothelial cell line bEnd.3. Endoglin knockdown mutants were generated through lentiviral transfection of ENG-shRNA and validated using confocal microscopy and western blot analysis. Cells were then treated with bone morphogenetic protein-9 (BMP-9; 1 nM), a known TGF-β/ALK1 agonist. Oatp1a4 protein expression and transport activity were measured using western blot analysis and in vitro uptake assays using the known Oatp substrate drug [ 3 H]atorvastatin. Additionally, ENG expression in bEnd.3 cells was assessed under normoxic and oxygen/glucose deprivation (OGD) conditions (i.e., 1% O 2 in glucose-free medium). Results: BMP-9 treatment in non-transfected bEnd.3 cells resulted in increased Oatp1a4 protein expression and increased cellular uptake of [ 3 H]atorvastatin. These enhancements in Oatp1a4 functional expression were attenuated in ENG-shRNA knockdown bEnd.3 cells treated with BMP-9. Confocal microscopy and western blot analysis confirmed decreased ENG protein expression following shRNA transfection. Under OGD conditions, expression of ENG in bEnd.3 cells was significantly increased. Conclusions: Our data provide the first evidence for involvement of ENG in regulation of Oatp1a4 by TGF-β/ALK1 signaling in brain microvessel endothelial cells. These observations have clear implications for CNS delivery of drugs relevant to treatment of stroke. Studies are ongoing in our laboratory to fully evaluate ENG regulation of BBB transport mechanisms in the setting of ischemic stroke.

Morphological and Functional Effects of Ultrasound on Blood–Brain Barrier Transitory Opening: An In Vitro Study on Rat Brain Endothelial Cells

With the recent advances in medicine, human life expectancy is increasing; however, the extra years of life are not necessarily spent in good health or free from disability, resulting in a significantly higher incidence of age-associated pathologies. Among these disorders, neurodegenerative diseases have a significant impact. To this end, the presence of the protective blood-brain barrier (BBB) represents a formidable obstacle to the delivery of therapeutics. Thus, this makes it imperative to define strategies to bypass the BBB in order to successfully target the brain with the appropriate drugs. It has been demonstrated that targeting the BBB by ultrasound (US) can transiently make this anatomical barrier permeable and in so doing, allow the delivery of therapeutics. Thus, our aim was to carry out an in depth in vitro molecular and morphological study on the effects of US treatment on the BBB. The rat brain endothelial (RBE4) cell line was challenged with exposure to 12 MHz diagnostic US treatment for 10, 20, and 30 min. Cell viability assays, Western blotting analysis on the endoplasmic reticulum (ER), and oxidative stress marker evaluation were then performed, along with cytological and immunofluorescence staining, in order to evaluate the effects of US on the intercellular spaces and tight junction distribution of the brain endothelial cells. We observed that the US treatment exerted no toxic effects on either RBE4 cell viability or the upregulation/dislocation of the ER and oxidative stress marker (GRP78 and cytochrome C, respectively). Further, we observed that the application of US induced an increase in the intercellular spaces, as shown by Papanicolaou staining, mainly due to the altered distribution of the tight junction protein zonula occludens-1 (ZO-1). This latter US-dependent effect was transient and disappeared 20 min after the removal of the stimulus. In conclusion, our results show that US induces a transient alteration of the BBB, without altering the intracellular signaling pathways such as the ER and oxidative stress that could potentially be toxic for endothelial cells. These results suggested that US treatment could represent a potential strategy for improving drug delivery to the brain.

Role of soy lecithin combined with soy isoflavone on cerebral blood flow in rats of cognitive impairment and the primary screening of its optimum combination.

Soy isoflavone (SIF) and soy lecithin (SL) have beneficial effects on many chronic diseases, including neurodegenerative diseases. Regretfully, there is little evidence to show the combined effects of these soy extractives on the impairment of cognition and abnormal cerebral blood flow (CBF). This study examined the optimal combination dose of SIF + SL to provide evidence for improving CBF and protecting cerebrovascular endothelial cells. In vivo study, SIF50 + SL40, SIF50 + SL80 and SIF50 + SL160 groups were obtained. Morris water maze, laser speckle contrast imaging (LSCI), and hematoxylin-eosin staining were used to detect learning and memory impairment, CBF, and damage to the cerebrovascular tissue in rat. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) and the oxidized glutathione (GSSG) were detected. The anti-oxidative damage index of superoxide dismutase (SOD) and glutathione (GSH) in the serum of an animal model was also tested. In vitro study, an immortalized mouse brain endothelial cell line (bEND.3 cells) was used to confirm the cerebrovascular endothelial cell protection of SIF + SL. In this study, 50 μM of Gen were used, while the 25, 50, or 100 μM of SL for different incubation times were selected first. The intracellular levels of 8-OHdG, SOD, GSH, and GSSG were also detected in the cells. In vivo study, SIF + SL could increase the target crossing times significantly and shorten the total swimming distance of rats. The CBF in the rats of the SIF50 + SL40 group and SIF50 + SL160 group was enhanced. Pathological changes, such as attenuation of the endothelium in cerebral vessels were much less in the SIF50 + SL40 group and SIF50 + SL160 group. The 8-OHdG was reduced in the SIF50 + SL40 group. The GSSG showed a significant decrease in all SIF + SL pretreatment groups, but the GSH showed an opposite result. SOD was upregulated by SIF + SL pretreatment. Different combinations of Genistein (Gen)+SL, the secondary proof of health benefits found in vivo study, showed they have effective anti-oxidation and less side reaction on protecting cerebrovascular endothelial cell. SIF50 + SL40 in rats experiment and Gen50 + SL25 in cell test were the optimum joint doses on alleviating cognitive impairment and regulating CBF through protecting cerebrovascular tissue by its antioxidant activity. SIF+SL could significantly prevent cognitive defect induced by β-Amyloid through regulating CBF. This kind of effect might be attributed to its antioxidant activity on protecting cerebral vessels.

Endothelial cell activation by interleukin-1 and extracellular matrix laminin-10 occurs via the YAP signalling pathway

Laminin-10 (LM-10) is a key regulator of blood-brain barrier (BBB) repair after hypoxia and inflammation. Here we investigated the signalling mechanisms regulated by LM-10 in human brain endothelial cell line hCMEC/D3 in response to interleukin(IL)-1beta(β) in vitro. LM-10 promoted endothelial proliferation and repair of an endothelial monolayer after scratch injury, and upregulated IL-1β-induced ICAM-1 and VCAM-1 expression. IL-1β and LM-10 regulated YAP signalling pathway in endothelial cells leading to differential expression of YAP target genes, ctgf and serpine-1, providing evidence that the YAP signalling pathway could be a new therapeutic target for the treatment of BBB dysfunction in CNS diseases.

Exosomes derived from M2 Macrophages Improve Angiogenesis and Functional Recovery after Spinal Cord Injury through HIF-1α/VEGF Axis.

Exosomes are nano-sized vesicles that contain a variety of mRNAs, miRNAs, and proteins. They are capable of being released by a variety of cells and are essential for cell–cell communication. The exosomes produced by cells have shown protective benefits against spinal cord damage (SCI). Recently, it was discovered that M2 macrophages aid in the angiogenesis of numerous illnesses. However, the functional role of M2 macrophage-derived exosomes on SCI is unclear. Here, we investigate the pro-angiogenesis of M2 macrophage-derived exosomes on SCI. We founded that M2 macrophage exosomes alleviated tissue damage and enhanced functional recovery post-SCI. We discovered that M2 macrophage exosome administration increased angiogenesis after SCI in vivo using immunohistochemistry, immunofluorescence labeling, and Western blot analysis. Additionally, the expression of the pro-angiogenesis factors, HIF-1α and VEGF, were enhanced with the treatment of the M2 macrophage exosomes. Furthermore, we found that M2 macrophage exosomes enhanced neurogenesis after SCI in vivo. In vitro, we found that M2 macrophage exosomes can be taken up by the brain endothelial cell line (bEnd.3) and that they enhanced the tube formation, migration, and proliferation of bEnd.3 cells. Furthermore, by using special siRNA to inhibit HIF-1α expression, we observed that the expression of VEGF decreased, and the tube formation, migration, and proliferation of bEnd.3 cells were attenuated with the treatment of HIF-1α-siRNA. In conclusion, our findings reveal that M2 macrophage exosomes improve neurological functional recovery and angiogenesis post-SCI, and this process is partially associated with the activation of the HIF-1/VEGF signaling pathway.

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load.

Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs ("mito-EVs") may increase the recipient BEC ATP levels to a greater extent than naïve MVs. We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs-suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases. The online version contains supplementary material available at 10.1007/s12195-022-00738-8.