Microvascular Endothelial Cell Line Research Articles

Discovery of marine ent-eudesmane sesquiterpenoids as angiogenic inhibitors via suppressing VEGF-A/VEGFR2 signaling pathway.

Increasing evidence underscores the pivotal role of tumor angiogenesis for tumorigenesis and tumor metastasis. Inhibiting the tumor angiogenesis process is a promising therapeutic approach for cancer. In order to search for natural angiogenic inhibitors, the chemical constitutes of a marine-derived fungus Eutypella sp. F0219 were investigated, leading to the isolation and identification of twelve new ent-eudesmane sesquiterpenoids named eutypenes A-L (1-12). Their structures including absolute configurations were determined by extensive spectroscopic investigations, single crystal X-ray diffraction analyses, modified Mosher's method, and ECD calculations. Notably, eutypene A (1) represents a rare rearranged ent-eudesmane sesquiterpenoid with 5/7 fused ring system. Tube formation assay was performed to evaluate the antiangiogenic effect of all compounds. The results showed that compounds 4, 6, 7, 9, and 10 obviously suppressed the tube formation of human microvascular endothelial cell line (HMEC-1) cells in a dose-dependent manner. Moreover, the most bioactive and less toxic compound 9 displayed significant antiangiogenic effect in vitro and ex vivo. Further mechanistic investigation revealed that compound 9 restrained tumor angiogenesis by reducing the VEGF-A level and suppressing the VEGF-A/VEGFR2 signaling pathway. Our findings give insight into the application of marine ent-eudesmane sesquiterpenoids as potential angiogenesis inhibitor.

Designing and evaluating a novel blood-brain barrier in vitro model of teleost for reproducing alterations in brain infecting

A new blood-brain barrier (BBB) in vitro model of tilapia (Oreochromis niloticus) was successfully established by co-culture with brain microvascular endothelial cell line TVEC-01 and brain astrocyte cell line TA-02 of tilapia. Experiments with the expression levels of BBB-related genes, TEER value detection and four-hour water-leaking test have shown that the BBB in vitro model has an excellent barrier effect. In bacterial penetration experiments, the pathogenic strain of Streptococcus agalactiae was able to pass through the BBB in vitro model and initiate a series of immune responses, among which TA-02 contributed to the expression of pro-inflammatory cytokines and TVEC-01 contributed to the expression of anti-inflammatory cytokines, providing an excellent research tool and theoretical basis for further study of meningitis. Moreover, the qRT-PCR and transmission electron microscopy revealed that the pathogenic strain of S. agalactiae effectively penetrated the BBB in vitro model of tilapia during early-stage infection without destroying tight junction integrity. This suggested that, in the initial phases of infection, the pathogenic strain of S. agalactiae may breach the BBB via a transcellular pathway rather than a paracellular pathway. Summarily, a novel BBB in vitro model of tilapia was successfully designed and evaluated for reproducing alterations in brain infecting.

In vitro characterization of taurine transport using the human brain microvascular endothelial cell line as a human blood-brain barrier model

Taurine, a sulfur-containing β-amino acid, has various roles in the brain including cellular osmoregulation and neuroprotection. For adequate supply to the brain, taurine has to pass through the blood-brain barrier (BBB); however, the associated mechanism behind crossing the human BBB is not fully understood. Therefore, we characterized taurine transport in vitro using the human brain microvascular endothelial (hCMEC/D3) cell line, a model of human BBB function. [3H]Taurine uptake by hCMEC/D3 cells exhibited time-, as well as extracellular Na+- and Cl−-dependence. The uptake was saturable with a Km of 19 μM and was inhibited by GABA at an IC50 of 328 μM, which were similar to Km values of taurine transporter (TauT)-mediated transport of taurine and GABA, respectively, suggesting that TauT is a major contributor to taurine uptake. For distribution to the brain, taurine must undergo cellular efflux after uptake. Taurine efflux from hCMEC/D3 cells increased for at least 60 min, and monocarboxylate transporter 7 (MCT7)-targeted siRNA significantly reduced MCT7 mRNA levels and [3H]taurine efflux by 93% and 12%, respectively, suggesting that MCT7 partly contributes to taurine efflux from hCMEC/D3 cells. Taken together, these results suggest that TauT and MCT7 function cooperatively in the human BBB.

Unlocking the potential of biocompatible chitosan-hyaluronic acid nanogels labeled with fluorochromes: A promising step toward enhanced FRET bioimaging

Chitosan is a natural polysaccharide widely used in medical formulations as nanoparticles due to their special properties. Our work aimed to assess the biocompatibility of chitosan-hyaluronic acid nanogels labeled with fluorochromes for use in biomedical applications, based on the FRET effect. The preparation method included the ionic gelation, grafting rhodamine or fluorescein isothiocyanate molecules onto the chitosan backbone. To assess the potential applications as fluorescence imaging tools of chitosan-fluorophores conjugates in diagnostics and therapies, SVEC4-10 cells (simian virus 40-transformed mouse microvascular endothelial cell line) and RAW264.7 murine macrophages were used within this study. Good biocompatibility was observed after 6 and 24 h of incubation with nanogels, with no increase in cell death or membrane damage for concentrations up to 120 μg/mL. Both types of fluorescent nanogels presented the tendency to agglomerate on the cell membrane's surface, and few cells were internalized, especially at the periphery of cells. Molecular dynamics simulations showed that distances between fluorophores fitted at values close to those calculated based on FRET experiments. These formulations can further incorporate gadolinium for better nanomedicine tools.

Involvement of Proton-Coupled Organic Cation Antiporter in Human Blood-Brain Barrier Transport of Mesoridazine and Metoclopramide.

Mesoridazine and metoclopramide are cationic drugs that are distributed in the human brain despite being substrates of multidrug resistance protein 1 (MDR1), an efflux transporter expressed at the blood-brain barrier (BBB). We investigated their transport mechanisms at the BBB using hCMEC/D3, a human cerebral microvascular endothelial cell line often used as an in vitro BBB model. The cells exhibited time- and concentration-dependent uptake of mesoridazine and metoclopramide, with Km values of 34 and 277 µM, respectively. The uptake of both drugs significantly decreased in the presence of typical inhibitors and/or substrates of the H+-coupled organic cation (H+/OC) antiporter but not in the presence of inhibitors or substrates of organic cation transporters (OCTs), OCTN2, OATPs, SLC35F2, or the plasma membrane monoamine transporter (PMAT). Furthermore, metoclopramide uptake by hCMEC/D3 cells was pH- and energy-dependent, whereas mesoridazine uptake was unaffected by intracellular acidification and treatment with metabolic inhibitors. These results suggest that the H+/OC antiporter is involved in the influx of mesoridazine and metoclopramide into the brain across the BBB.

The transcription factor PPARA mediates SIRT1 regulation of NCOR1 to protect damaged heart cells.

Heart failure (HF) is a clinical syndrome with a high risk. Our previous research showed a regulatory relationship between Sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor α (PPARA) and nuclear receptor co-repressor 1 (NCOR1). This study aimed to investigate the regulatory mechanism of SIRT1/PPARA/NCOR1 axis in HF. HF models in vitro were established by doxorubicin (DOX)-induced AC16 and human cardiac microvascular endothelial cell (HCMEC) lines. The contents of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), interleukin-1β (IL-1β), and IL-18 were detected using enzyme-linked immunosorbent assay. Then, we assessed the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and adenosine triphosphate (ATP). Moreover, the relationship between SIRT1 and PPARA was detected using the co-immunoprecipitation (Co-IP) analysis. The connection between PPARA and NCOR1 was analyzed using chromatin immunoprecipitation (ChIP). Overexpression of SIRT1 or PPARA could reduce apoptosis in DOX-induced AC16 and HCMEC cells, the levels of IL-1β, IL-18, ANP, BNP, ROS and MDA, while increasing the levels of SOD and ATP. In addition, overexpression of PPARA could increase the viability of DOX-induced cells and the levels of myosin heavy chain 6 (Myh6) and Myh7. Co-IP showed that SIRT1 interacted with PPARA. Silencing PPARA could reverse the effect of SIRT1 overexpression on DOX-induced AC16 and HCMEC cells. ChIP assay demonstrated that PPARA could bind to the promoter region of NCOR1. Silencing NCOR1 could reverse the effect of PPARA overexpression on DOX-induced AC16 and HCMEC cells. This study revealed that PPARA could mediate SIRT1 to promote NCOR1 expression and thus protect damaged heart cells. The finding provided an important reference for the treatment of HF.

The effects of the activation of TLR2/TLR1 on in vitro angiogenesis in an immortalized ovine luteal endothelial cell line.

Activation of TLR2/TLR1 alters in vitro formation of capillary-like structures and induces inflammatory processes in ovine luteal endothelial (OLENDO) cells. Postpartum bacterial infections of the uterus affect uterine physiology and ovarian activity, causing fertility problems. The outer membrane component of Gram-negative bacteria, lipopolysaccharide, is involved in the initiation of the local inflammatory processes, and other bacterial toxins, particularly lipopeptides, have also been shown to be potent cytokine inducers, acting via Toll-like receptor-2 (TLR2). However, the possible adverse effects of TLR2 on ovarian and luteal activities have not yet been investigated in depth. The strong expression of TLR2 in the blood vessels of the corpus luteum led us to hypothesize that TLR2 activation might participate in the disruption of luteal vascular functionality. Therefore, we analyzed the effects of Pam3CSK4 (Pam3CysSerLys4), a synthetic triacylated lipopeptide and TLR2/TLR1 ligand, on the functionality of gap junctional intercellular communication (GJIC), endothelial cell invasion, and in vitro capillary-like network formation in an immortalized ovine luteal endothelial (OLENDO) cell line. Pam3CSK4 treatment of OLENDO cells disrupted in vitro tube formation but had no effect on GJIC or migration of OLENDO cells. Furthermore, Pam3CSK4 induced the expression of NFKB, IL6, and IL8 in OLENDO cells. Additionally, the basal availability of TLRs (TLR1-10) and TLR co-receptors (MYD88, LY96/MD2, and CD14) in OLENDO cells was confirmed by conventional PCR. Finally, the activation of TLR2/TLR1 appears to alter in vitro formation of capillary-like structures and induce inflammatory processes in OLENDO cells.

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

Intranasal delivery of small extracellular vesicles from specific subpopulation of mesenchymal stem cells mitigates traumatic spinal cord injury

Vascular injury following spinal cord injury (SCI) can significantly exacerbate secondary SCI and result in neurological dysfunction. Strategies targeting angiogenesis have demonstrated potential in enhancing functional recovery post-SCI. In the context of angiogenesis, the CD146+ and CD271+ subpopulations of mesenchymal stem cells (MSCs) have been recognized for their angiogenic capabilities in tissue repair. Small extracellular vesicles (sEVs) derived from MSCs are nanoscale vesicles containing rich bioactive components that play a crucial role in tissue regeneration. However, the precise role of sEVs derived from CD146+CD271+ UCMSCs (CD146+CD271+ UCMSC-sEVs) in SCI remain unclear. In this study, CD146+CD271+ UCMSC-sEVs were non-invasively administered via intranasal delivery, demonstrating a significant capacity to stimulate angiogenesis and improve functional recovery in mice following SCI. Furthermore, in vitro assessments revealed the effective enhancement of migration and tube formation capabilities of the murine brain microvascular endothelial cell line (bEnd.3) by CD146+CD271+UCMSC-sEVs. MicroRNA array analysis confirmed significant enrichment of multiple microRNAs within CD146+CD271+ UCMSC-sEVs. Subsequent in vivo and in vitro experiments demonstrated that CD146+CD271+ UCMSC-sEVs promote enhanced angiogenesis and improved functional recovery mediated by miR-27a-3p. Further mechanistic studies revealed that miR-27a-3p sourced from CD146+CD271+ UCMSC-sEVs enhances migration and tube formation of bEnd.3 cells in vitro by suppressing the expression of Delta Like Canonical Notch Ligand 4 (DLL4), thereby promoting angiogenesis in vivo. Collectively, our results demonstrate that a crucial role of CD146+CD271+ UCMSC-sEVs in inhibiting DLL4 through the transfer of miR-27a-3p, which leads to the promotion of angiogenesis and improved functional recovery after SCI.

Novel simulation of the blood–brain barrier using the polyacrylonitrile electrospun nanofibrous membrane on 3D‐printed Transwell

AbstractThe blood–brain barrier (BBB) possesses an intricate structure and set of characteristics that effectively restricts the transportation of substances to the central nervous system. Consequently, the development of pharmaceuticals for brain disorders poses significant hurdles. Numerous attempts have been made to produce innovative simulates of BBB that exhibit a striking resemblance to BBB and are suitable for efficient drug screening, as well as being user‐friendly and easily reproducible. One of the models available is the commercial Transwell® system. In this system, chosen endothelial cells form a single layer on the rigid membrane of the upper chamber. In the present study, the two‐dimensional structure of this membrane was exchanged with a three‐dimensional membrane using the 3D printing method and developed a novel approach to fabricate the scaffold in one step on the fabricated Transwell. This novel method of horizontal electrospinning has reduced the mechanical damage associated with conventional vertical electrospinning, resulting in suitable mechanical properties for cell culture. The lower diameter of the nanofibers, the thickness of the mesh, bulk Young's modulus and hydrophobicity compared to conventional mesh‐based counterparts are characteristics which make it more similar to the natural basement membrane and improve cell culture necessities on the scaffold. The designed BBB model was characterized via cell morphology, trans‐endothelial electrical resistance, and permeability value. This human BBB model formed robust cell integrity and achieved the acceptable range of TEER and low permeability that confirm the polyacrylonitrile (PAN) scaffold as an in vitro cell culture platform with promising cell culture results of the brain microvascular endothelial cell line hCMEC/D3.

Glucose Promotes EMMPRIN/CD147 and the Secretion of Pro-Angiogenic Factors in a Co-Culture System of Endothelial Cells and Monocytes.

Vascular complications in Type 2 diabetes mellitus (T2DM) patients increase morbidity and mortality. In T2DM, angiogenesis is impaired and can be enhanced or reduced in different tissues ("angiogenic paradox"). The present study aimed to delineate differences between macrovascular and microvascular endothelial cells that might explain this paradox. In a monoculture system of human macrovascular (EaHy926) or microvascular (HMEC-1) endothelial cell lines and a monocytic cell line (U937), high glucose concentrations (25 mmole/L) increased the secretion of the pro-angiogenic factors CD147/EMMPRIN, VEGF, and MMP-9 from both endothelial cells, but not from monocytes. Co-cultures of EaHy926/HMEC-1 with U937 enhanced EMMPRIN and MMP-9 secretion, even in low glucose concentrations (5.5 mmole/L), while in high glucose HMEC-1 co-cultures enhanced all three factors. EMMPRIN mediated these effects, as the addition of anti-EMMPRIN antibody decreased VEGF and MMP-9 secretion, and inhibited the angiogenic potential assessed through the wound assay. Thus, the minor differences between the macrovascular and microvascular endothelial cells cannot explain the angiogenic paradox. Metformin, a widely used drug for the treatment of T2DM, inhibited EMMPRIN, VEGF, and MMP-9 secretion in high glucose concentration, and the AMPK inhibitor dorsomorphin enhanced it. Thus, AMPK regulates EMMPRIN, a key factor in diabetic angiogenesis, suggesting that targeting EMMPRIN may help in the treatment of diabetic vascular complications.

Contribution of platelets to disruption of the blood-brain barrier during arterial baroreflex dysfunction

BackgroundArterial baroreflex dysfunction, like many other central nervous system disorders, involves disruption of the blood-brain barrier, but what causes such disruption in ABR dysfunction is unclear. Here we explored the potential role of platelets in this disruption. MethodsABR dysfunction was induced in rats using sinoaortic denervation, and the effects on integrity of the blood-brain barrier were explored based on leakage of Evans blue or FITC-dextran, while the effects on expression of CD40L in platelets and of key proteins in microvascular endothelial cells were explored using immunohistochemistry, western blotting and enzyme-linked immunosorbent assay. Similar experiments were carried out in rat brain microvascular endothelial cell line, which we exposed to platelets taken from rats with ABR dysfunction. ResultsSinoaortic denervation permeabilized the blood-brain barrier and downregulated zonula occludens-1 and occludin in rat brain, while upregulating expression of CD40L on the surface of platelets and stimulating platelet aggregation. Similar effects of permeabilization and downregulation were observed in healthy rats that received platelets from animals with ABR dysfunction, and in rat brain microvascular endothelial cells, but only in the presence of lipopolysaccharide. These effects were associated with activation of NF-κB signaling and upregulation of matrix metalloprotease-9. These effects of platelets from animals with ABR dysfunction were partially blocked by neutralizing antibody against CD40L or the platelet inhibitor clopidogrel. ConclusionDuring ABR dysfunction, platelets may disrupt the blood-brain barrier when CD40L on their surface activates NF-kB signaling within cerebral microvascular endothelial cells, leading to upregulation of matrix metalloprotease-9. Our findings imply that targeting CD40L may be effective against cerebral diseases involving ABR dysfunction.

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1.

Retinoblastoma (RB) is a prevalent type of eye cancer in youngsters. Prospero homeobox 1 (Prox1) is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic, hepatocyte, pancreatic, heart, lens, retinal, and cancer cells. The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance, as well as to explore the underlying Notch1 mechanism. Human RB cell lines (SO-RB50 and Y79) and a primary human retinal microvascular endothelial cell line (ACBRI-181) were used in this study. The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay. Drug-resistant cell lines (SO-RB50/vincristine) were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance. We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1. Finally, a xenograft model was constructed to assess the effect of Prox1 on RB in vivo. Prox1 was significantly downregulated in RB cells. Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine. Notch1 was involved in Prox1's regulatory effects. Notch1 was identified as a target gene of Prox1, which was found to be upregulated in RB cells and repressed by increased Prox1 expression. When pcDNA-Notch1 was transfected, the effect of Prox1 overexpression on RB was removed. Furthermore, by downregulating Notch1, Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo. These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1, implying that Prox1 could be a potential therapeutic target for RB.

Modeling Shiga toxin-induced human renal-specific microvascular injury.

Shiga toxin (Stx) causes significant renal microvascular injury and kidney failure in the pediatric population, and an effective targeted therapy has yet to be demonstrated. Here we established a human kidney microvascular endothelial cell line for the study of Stx mediated injuries with respect to their morphologic, phenotypic, and transcriptional changes, and modeled Stx induced thrombotic microangiopathy (TMA) in flow-mediated 3D microvessels. Distinct from other endothelial cell lines, both isolated primary and immortalized human kidney microvascular endothelial cells demonstrate robust cell-surface expression of the Stx receptor Gb3, and concomitant dose-dependent toxicity to Stx, with significant contributions from caspase-dependent cell death. Use of a glucosylceramide synthase inhibitor (GCSi) to target disruption of the synthetic pathway of Gb3 resulted in remarkable protection of kidney microvascular cells from Stx injury, shown in both cellular morphologies, caspase activation and transcriptional analysis from RNA sequencing. Importantly, these findings are recapitulated in 3D engineered kidney microvessels under flow. Moreover, whole blood perfusion through Stx-treated microvessels led to marked platelet binding on the vessel wall, which was significantly reduced with the treatment of GCSi. These results validate the feasibility and utility of a bioengineered ex vivo human microvascular model under flow to recapitulate relevant blood-endothelial interactions in STEC-HUS. The profound protection afforded by GCSi demonstrates a preclinical opportunity for investigation in human tissue approximating physiologic conditions. Moreover, this work provides a broad foundation for novel investigation into TMA injury pathogenesis and treatment. Insight Box: Shiga toxin (Stx) causes endothelial injury that results in significant morbidity and mortality in the pediatric population, with no effective targeted therapy. This paper utilizes human kidney microvascular cells to examine Stx mediated cell death in both 2D culture and flow-mediated 3D microvessels, with injured microvessels also developing marked platelet binding and thrombi formation when perfused with blood, consistent with the clinical picture of HUS. This injury is abrogated with a small molecule inhibitor targeting the synthetic pathway of the Shiga toxin receptor. Our findings shed light onto Stx-induced vascular injuries and pave a way for broad investigation into thrombotic microangiopathies.

β-asarone induces viability and angiogenesis and suppresses apoptosis of human vascular endothelial cells after ischemic stroke by upregulating vascular endothelial growth factor A.

Ischemic stroke (IS) is a disease with a high mortality and disability rate worldwide, and its incidence is increasing per year. Angiogenesis after IS improves blood supply to ischemic areas, accelerating neurological recovery. β-asarone has been reported to exhibit a significant protective effect against hypoxia injury. The ability of β-asarone to improve IS injury by inducing angiogenesis has not been distinctly clarified. The experimental rats were induced with middle cerebral artery occlusion (MCAO), and oxygen-glucose deprivation (OGD) model cells were constructed using human microvascular endothelial cell line (HMEC-1) cells. Cerebral infarction and pathological damage were first determined via triphenyl tetrazolium chloride (TTC) and hematoxylin and eosin (H&E) staining. Then, cell viability, apoptosis, and angiogenesis were assessed by utilizing cell counting kit-8 (CCK-8), flow cytometry, spheroid-based angiogenesis, and tube formation assays in OGD HMEC-1 cells. Besides, angiogenesis and other related proteins were identified with western blot. The study confirms that β-asarone, like nimodipine, can ameliorate cerebral infarction and pathological damage. β-asarone can also upregulate vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) and induce phosphorylation of p38. Besides, the study proves that β-asarone can protect against IS injury by increasing the expression of VEGFA. In vitro experiments affirmed that β-asarone can induce viability and suppress apoptosis in OGD-mediated HMEC-1 cells and promote angiogenesis of OGD HMEC-1 cells by upregulating VEGFA. This establishes the potential for β-asarone to be a latent drug for IS therapy.

Lysosomal TRPML1 triggers global Ca2+ signals and nitric oxide release in human cerebrovascular endothelial cells.

Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.

Investigation of the Binding Interaction of Mfsd2a with NEDD4-2 via Molecular Dynamics Simulations.

Major facilitator superfamily domain-containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine cotransporter that plays an important role in maintaining the integrity of the blood-brain barrier and neurological function. Abnormal degradation of Mfsd2a often leads to dysfunction of the blood-brain barrier, while upregulation of Mfsd2a can retrieve neurological damage. It has been reported that Mfsd2a can be specifically recognized and ubiquitinated by neural precursor cell-expressed developmentally downregulated gene 4 type 2 (NEDD4-2) ubiquitin ligase and finally degraded through the proteasome pathway. However, the structural basis for the specific binding of Mfsd2a to NEDD4-2 is unclear. In this work, we combined deep learning and molecular dynamics simulations to obtain a Mfsd2a structure with high quality and a stable Mfsd2a/NEDD4-2-WW3 interaction model. Moreover, molecular mechanics generalized Born surface area (MM-GBSA) methods coupled with per-residue energy decomposition studies were carried out to analyze the key residues that dominate the binding interaction. Based on these results, we designed three peptides containing the key residues by truncating the Mfsd2a sequences. One of them was found to significantly inhibit Mfsd2a ubiquitination, which was further validated in an oxygen-glucose deprivation (OGD) model in a human microvascular endothelial cell line. This work provides some new insights into the understanding of Mfsd2a and NEDD4-2 interaction and might promote further development of drugs targeting Mfsd2a ubiquitination.

The Ohm-azing custom-made Transendothelial Electrical Resistance measuring device (and why is it a current sensation?)

The endothelial and epithelial cells form biological barriers, the unique anatomical structures that control substance movement between circulation and i.e., organs. Measuring trans-endothelial electrical resistance (TEER) is a standard method that enables the evaluation of barrier integrity. The capability of a novel measuring system (MS) that combines innovative components, customized electrodes, and user-friendly software tailored to provide accurate and adjustable measurements of endothelial barrier integrity. We demonstrated MS usage in TEER measurement of rat brain microvascular endothelial cell line (RBE-4), primary rat brain microvascular endothelial cells (PBMEC), rat brain microvascular endothelial cells (BMEC), and human intestinal epithelial cell line (HIEC-6). The MS was successfully applied to measure the TEER of cultured cell monolayers, finding that i) the device records stable values; ii) cells treated with ammonium chloride had lower TEER values compared to untreated cells, which was verified with permeability measurements with fluorescein dye and confocal microscopy. In conclusion, we invented a low-cost MS capable of accurate TEER measures in relevant biological ranges. The MS instrument facilitates long-time, real-time monitoring of the cellular barriers, giving vital insights into cell barrier stability, permeability, and the effects of pharmacological substances. The customized measurement duration and electrode configuration meet a broad range of experimental designs.

Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development.