Microvascular Endothelial Cell Line Research Articles

Healthy Serum-Derived Exosomes Improve Neurological Outcomes and Protect Blood-Brain Barrier by Inhibiting Endothelial Cell Apoptosis and Reversing Autophagy-Mediated Tight Junction Protein Reduction in Rat Stroke Model.

Blood–brain barrier (BBB) dysfunction causing edema and hemorrhagic transformation is one of the pathophysiological characteristics of stroke. Protection of BBB integrity has shown great potential in improving stroke outcome. Here, we assessed the efficacy of exosomes extracted from healthy rat serum in protection against ischemic stroke in vivo and in vitro. Exosomes were isolated by gradient centrifugation and ultracentrifugation and exosomes were characterized by transmission electron microscopy (TEM) and nanoparticle tracking video microscope. Exosomes were applied to middle cerebral artery occlusion (MCAO) rats or brain microvascular endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD) injury. Serum-derived exosomes were injected intravenously into adult male rats 2 h after transient MCAO. Infarct volume and gross cognitive function were assessed 24 h after reperfusion. Poststroke rats treated with serum-derived exosomes exhibited significantly reduced infarct volumes and enhanced neurological function. Apoptosis was assessed via terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining and the expression of B-cell lymphoma-2 (Bcl-2), Bax, and cleaved caspase-3 24 h after injury. Our data showed that serum exosomes treatment strikingly decreased TUNEL+ cells in the striatum, enhanced the ratio of Bcl-2 to Bax, and inhibited cleaved caspase-3 production in MCAO rats and OGD/reoxygenation insulted bEnd.3 cells. Under the consistent treatment, the expression of microtubule-associated protein 1 light chain 3B-II (LC3B-II), LC3B-I, and Sequestosome-1 (SQSTM1)/p62 was detected by Western blotting. Autolysosomes were observed via TEM. We found that serum exosomes reversed the ratio of LC3B-II to LC3B-I, prevented SQSTM1/p62 degradation, autolysosome formation, and autophagic flux. Together, these results indicated that exosomes isolated from healthy serum provided neuroprotection against experimental stroke partially via inhibition of endothelial cell apoptosis and autophagy-mediated BBB breakdown. Intravenous serum-derived exosome treatment may, therefore, provide a novel clinical therapeutic strategy for ischemic stroke.

Cerebral Biomarkers and Blood-Brain Barrier Integrity in Preeclampsia.

Cerebral complications in preeclampsia contribute substantially to maternal mortality and morbidity. There is a lack of reliable and accessible predictors for preeclampsia-related cerebral complications. In this study, plasma from women with preeclampsia (n = 28), women with normal pregnancies (n = 28) and non-pregnant women (n = 16) was analyzed for concentrations of the cerebral biomarkers neurofilament light (NfL), tau, neuron-specific enolase (NSE) and S100B. Then, an in vitro blood–brain barrier (BBB) model, based on the human cerebral microvascular endothelial cell line (hCMEC/D3), was employed to assess the effect of plasma from the three study groups. Transendothelial electrical resistance (TEER) was used as an estimation of BBB integrity. NfL and tau are proteins expressed in axons, NSE in neurons and S100B in glial cells and are used as biomarkers for neurological injury in other diseases such as dementia, traumatic brain injury and hypoxic brain injury. Plasma concentrations of NfL, tau, NSE and S100B were all higher in women with preeclampsia compared with women with normal pregnancies (8.85 vs. 5.25 ng/L, p < 0.001; 2.90 vs. 2.40 ng/L, p < 0.05; 3.50 vs. 2.37 µg/L, p < 0.001 and 0.08 vs. 0.05 µg/L, p < 0.01, respectively). Plasma concentrations of NfL were also higher in women with preeclampsia compared with non-pregnant women (p < 0.001). Higher plasma concentrations of the cerebral biomarker NfL were associated with decreased TEER (p = 0.002) in an in vitro model of the BBB, a finding which indicates that NfL could be a promising biomarker for BBB alterations in preeclampsia.

Secreted gingipains from Porphyromonas gingivalis increase permeability in human cerebral microvascular endothelial cells through intracellular degradation of tight junction proteins

Despite a clear correlation between the infiltration of periodontal pathogens in the brain and cognitive decline in Alzheimer's disease (AD), the precise mechanism underlying bacteria crossing the blood-brain barrier (BBB) remains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteases termed gingipains. Gingipains appear to be key virulence factors that exacerbate sporadic AD. We herein report that gingipains are involved in increasing permeability of hCMEC/D3 cell monolayer, human cerebral microvascular endothelial cell lines, through degradation of tight junction proteins including Zonula occludens-1 (ZO-1) and occludin. There was a significant decrease in the mean protein levels of ZO-1 and occludin after infection of hCMEC/D3 cells with wild-type (WT) P. gingivalis. However, infection of these cells with a gingipain-deficient P. gingivalis strain showed significantly lower reduction of the mean protein levels of either ZO-1 and occludin, compared to the WT strain. Similar results were obtained after treatment with culture supernatant from WT and gingipain-deficient P. gingivalis strains. In vitro digestion of human recombinant ZO-1 and occludin by WT P. gingivalis culture supernatant in the absence or presence of gingipain inhibitors indicated that gingipains directly degraded these tight junction proteins. A close immunohistochemical examination using anti-gingipain antibody further revealed that gingipains localized in the cytosol and nuclei of hCMEC/D3 cells after infection with WT P. gingivalis and treatment with its culture supernatant. Furthermore, intracellular localization of outer membrane vesicles (OMVs) bound gingipains from WT P. gingivalis and OMV-induced degradation of ZO-1 and occludin were also observed in hCMEC/D3 cells. Thus, the delivery of gingipains into the cerebral microvascular endothelial cells, probably through OMV, may be responsible for the BBB damage through intracellular degradation of ZO-1 and occludin.

A Hybrid Human Microvascular Endothelial Cell Line Created by Utilizing a Cell Fusion Technique and Its Advantage for Expressing High Endothelial Barrier Function

A Hybrid Human Microvascular Endothelial Cell Line Created by Utilizing a Cell Fusion Technique and Its Advantage for Expressing High Endothelial Barrier Function

Caveolin-1 Down-Regulation Reduces VEGF-A Secretion Induced by IGF-1 in ARPE-19 Cells.

The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.

Synthesis, Molecular Docking and Antiplasmodial Activities of New Tetrahydro-β-Carbolines.

Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-β-carbolines were designed, synthesized by the Pictet–Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified N-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxamide (7) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.

Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood\u2013brain barrier injury through the Wnt/\u03b2-catenin signalling pathway

BackgroundDisruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear.MethodsAdult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test.ResultsCerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.ConclusionThis study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

A Peptide/MicroRNA-31 nanomedicine within an electrospun biomaterial designed to regenerate wounds in vivo

microRNA-31 (miR-31) has been identified to be downregulated in pathologies associated with delayed wound repair. Thus, it was proposed that the delivery of a plasmid encoding miR-31 (pmiR-31) to the skin could hold potential in promoting wound healing. Effective delivery of pmiR-31 was potentiated by encapsulation with the CHAT peptide to form nanocomplexes, this improved cellular entry and elicited a potent increase in miR-31 expression in vitro in both skin human keratinocyte cell line (HaCaT) and human microvascular endothelial cell line (HMEC-1). Transfection efficiencies with CHAT/pEFGP-N1 were significant at 15.2 ± 8.1% in HMEC-1 cells and >40% in HaCaT cells. In this study, the CHAT/pmiR-31 nanocomplexes at a N:P ratio of 10 had an average particle size of 74.2 nm with a cationic zeta potential of 9.7 mV. Delivery of CHAT/pmiR-31 to HaCaT and HMEC-1 cells resulted in significant improvements in cell migration capacity and increased angiogenesis. In vivo studies were conducted in C57BL/6 J mice were CHAT/pmiR-31 was delivered via electrospun PVA nanofibres, demonstrating a significant increase in epidermal (increase of ∼38.2 µm) and stratum corneum (increase of 8.2 µm) layers compared to controls. Furthermore, treatment in vivo with CHAT/pmiR-31 increased angiogenesis in wounds compared to controls, with a significant increase in vessel diameter by ∼20.4 µm compared against a commercial dressing control (Durafiber™). Together, these data demonstrate that the delivery of CHAT/pmiR-31 nanocomplexes from electrospun PVA nanofibres represent an innovative therapy for wound repair, eliciting a positive therapeutic response across both stromal and epithelial tissue compartments of the skin. Statement of significanceThis study advances research regarding the development of our unique electrospun nanofibre patch to deliver genetic nanoparticles into wounds in vivo to promote healing. The genetic nanoparticles are comprised of: (a) plasmid micro-RNA31 that has been shown to be downregulated in pathologies with delayed wound repair and (b) a 15 amino acid linear peptide termed CHAT. The CHAT facilitates complexation of miR-31 and cellular uptake. Herein, we report for the first time on the use of CHAT to deliver a therapeutic cargo pmiR-31 for wound healing applications from a nanofibre patch. Application of the nanofibre patch resulted in the controlled delivery of the CHAT/pmiR-31 nanoparticles with a significant increase in both epidermal and stratum corneum layers compared to untreated and commercial controls.

Leishmania Promastigotes Enhance Neutrophil Recruitment through the Production of CXCL8 by Endothelial Cells

Endothelial cells represent one of the first cell types encountered by Leishmania promastigotes when inoculated into the skin of the human hosts by the bite of phlebotomine sand flies. However, little is known on their role in the early recruitment of phagocytic cells and in the establishment of the infection. Initially, neutrophils, rapidly recruited to the site of promastigotes deposition, phagocytize Leishmania promastigotes, which elude the killing mechanisms of the host cells, survive, and infect other phagocytic cells. Here, we show that Leishmania promastigotes co-incubated with HMEC-1, a microvascular endothelial cell line, exhibited significant morphological changes and loss of infectivity. Moreover, promastigotes of different Leishmania species stimulated the production of CXCL8 by HMEC-1 in a dose- and TLR4-dependent manner. Interestingly, we observed that the conditioned media from Leishmania-stimulated HMEC-1 cells attracted leukocytes, mostly neutrophils, after 2 h of incubation. After 24 h, a higher percentage of monocytes was detected in conditioned media of unstimulated HMEC-1 cells, whereas neutrophils still predominated in conditioned medium from Leishmania-stimulated cells. The same supernatants did not contain CCL5, a chemokine recruiting T cells and monocytes. On the contrary, inhibition of the production of CCL5 induced by TNF-α was seen. These data indicate that the interaction of Leishmania promastigotes with endothelial cells leads to the production of chemokines and the recruitment of neutrophils, which contribute to the establishment of Leishmania infection.

OC15.06: *Endothelial damage and inflammation cell signalling triggered by pre‐eclampsia versus COVID‐19 in pregnancy

To study endothelial damage and alteration in signalling pathways in endothelial cells exposed to sera from pregnant women with PE and COVID-19 in vitro. Sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) in addition to normotensive pregnancies as controls (n=10) and patients with PE (n=13). Endothelial cells were obtained from the human dermal microvascular endothelial cell line. Nuclei and deposits of ICAM-1and Von Willebrand factor (VWF) staining was performed, and micrographs were captured by fluorescent microscopy. The area covered by fluorescent ICAM-1 or VWF labelling was calculated and expressed as the average fold increase compared to controls. The effect of sera samples on the inflammation cell signalling pathways was assessed by the quantification of phospho-P38MAPK. Both COVID-19 and PE induced an overexpression of ICAM-1 and VWF on endothelial cells although the effect of pre-eclampsia was less pronounced than the one triggered by severe COVID-19 (p < 0.05). Exposure of endothelial cells to the sera of severe COVID-19 and PE had the capacity to activate inflammatory signalling pathways. Immunoblots showed an increase in the degree of phosphorylation of p38MAPK being both severe COVID-19 and PE statistically different from controls (p < 0.05) (figure 1). Both COVID-19 and PE trigger endothelial damage and induce the activation of inflammation cell signalling pathways in endothelial cells. The effect on cell adhesion and prothrombosis of severe COVID-19 is more pronounced than PE. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Mast cells-derived exosomes worsen the development of experimental cerebral malaria

Cerebral malaria (CM) is the most severe neurological complication caused by Plasmodium falciparum infection. The accumulating evidence demonstrated that mast cells (MCs) and its mediators played a critical role in mediating malaria severity. Earlier studies identified that exosomes were emerging as key mediators of intercellular communication and can be released from several kinds of MCs. However, the potential functions and pathological mechanisms of MCs-derived exosomes (MCs-Exo) impacting on CM pathogenesis remain largely unknown. Herein, we utilized an experimental CM (ECM) model (C57BL/6 mice infected with P. berghei ANKA strain), and then intravenously (i.v.) injected MCs-Exo into P. berghei ANKA-infected mice to unfold this mechanism and investigate the effect of MCs-Exo on ECM pathogenies. We also used an in vitro model by investigating the pathogenesis development of brain microvascular endothelial cells line (bEnd.3 cells) co-cultured with P. berghei ANKA blood-stage soluble antigen (PbAg) after MCs-Exo treatment. The higher numbers of MCs and levels of MCs degranulation were observed in skin, cervical lymph node, and brain of ECM mice than those of the uninfected mice. Exosomes were successfully isolated from culture supernatants of mouse MCs line (P815 cells) and characterized by spherical vesicles with the diameter of 30-150 nm, and expression of typical exosomal markers (e.g., CD9, CD63, and CD81). The i.v. injection of MCs-Exo dramatically elevated incidence of ECM in the P. berghei ANKA-infected mice, exacerbated liver and brain histopathological damage, promoted Th1 cytokine response, aggravated brain vascular endothelial activation and blood brain barrier breakdown in ECM mice. In addition, the treatment of MCs-Exo led to the decrease of cells viability and mRNA levels of Ang-1, ZO-1, and Claudin-5, but increase of mRNA levels of Ang-2, CCL2, CXCL1, and CXCL9 in bEnd.3 cells co-cultured with PbAg in vitro. Taken together, our data indicated that MCs-Exo could worsen pathogenesis of ECM in mice.

Glial cell line‐derived neurotrophic factor and basic fibroblast growth factor derived from skeletal muscle pericytes increase the barrier function of endothelial cells in the endomysium

AbstractObjectivesIn skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood‐brain barrier (BBB) and blood‐nerve barrier (BNB). Although the endomysial capillaries include endothelial cells and pericytes, the properties of the endomysial pericytes have not been well elucidated. The purpose of this study was to establish a human skeletal muscle pericyte cell (HSMPCT) line, and examine whether the established cell line has similar properties to pericytes from the BBB and BNB.MethodHSMPCTs were isolated and introduced with retroviruses harboring temperature‐sensitive SV40 T antigen and telomerase genes. Then, we examined whether this cell line modifies the barrier function of a human skeletal muscle microvascular endothelial cell line, TSM15.ResultsA new HSMPCT cell line was successfully established. The conditioned media (CM) from HSMPCTs increased the transendothelial electrical resistance of the TSM15 cells and decreased permeability to 10kD‐dextran. Glial cell line‐derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) increased the barrier function of TSM15 cells. The CM of HSMPCTs with anti‐GDNF or anti‐bFGF antibodies attenuated the effect of upregulating the barrier function of TSM15 cells. These results indicate that soluble factors, such as GDNF and bFGF, released from HSMPCTs strengthen the barrier function of TSM15 cells.ConclusionGDNF and bFGF released from HSMPCTs increase the barrier function of endomysial endothelium. These HSMPCTs and TSM15 cells, which are recognized as an in vitro model of the endomysial capillary, might facilitate the analysis of the pathophysiology of the microvasculature in human skeletal muscle.

NMDA receptors elicit flux-independent intracellular Ca2+ signals via metabotropic glutamate receptors and flux-dependent nitric oxide release in human brain microvascular endothelial cells

The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO and trigger neurovascular coupling (NVC). Neuronal and glial NMDARs may also operate in a flux-independent manner, although it is unclear whether their non-ionotropic mode of action is involved in NVC. Recently, endothelial NMDARs were found to trigger Ca2+-dependent NO production and induce NVC, but the underlying mode of signaling remains elusive. Herein, we report that GluN1 protein, as well as GluN2C and GluN3B transcripts and proteins, were expressed and that NMDA did not elicit inward currents, but induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in the human brain microvascular endothelial cell line, hCMEC/D3. A multidisciplinary approach, including live cell imaging, whole-cell patch-clamp recordings, pharmacological manipulation and gene targeting, revealed that NMDARs increase the [Ca2+]i in a flux-independent manner in hCMEC/D3 cells. The Ca2+ response to NMDA was triggered by endogenous Ca2+ release from the endoplasmic reticulum and the lysosomal Ca2+ stores and sustained by store-operated Ca2+ entry. Unexpectedly, pharmacological and genetic blockade of mGluR1 and mGluR5 dramatically impaired NMDARs-mediated Ca2+ signals. These findings indicate that NMDARs may increase the endothelial [Ca2+]i in a flux-independent manner via group 1 mGluRs. However, imaging of DAF-FM fluorescence revealed that NMDARs may also induce Ca2+-dependent NO release by signaling in a flux-dependent manner. These findings, therefore, shed novel light on the mechanisms whereby brain microvascular endothelium decodes glutamatergic signaling and regulates NVC.

An affordable and tunable continuous wrinkle micropattern for cell physical guidance study

Geometrical cues in the extracellular environment are essential for cells guiding. Conventional fabrications for well-defined microstructures are usually associated with expensive and time-consuming micro-electro-mechanical systems (MEMS), including lithography and etching. Additionally, the microstructures produced on silicon wafers are not amendable after molding, hindering the applicability. Here, we developed a low-cost and efficient fabrication process to generate highly ordered microstructures for cell culture by designing and fabricating a tunable wrinkled on biocompatible polydimethylsiloxane (PDMS) surfaces based on different mechanical properties of the biomaterials. The mechanical properties, including Young's modulus, tensile strength, and thickness ratio between the stiff and soft bilayers, can be determined by the thickness of PDMS film, curing temperature, UV exposure time, applied strain rates during the fabrication, leading to various amplitudes and wavelengths of the wrinkled topography on polymeric materials. Those wrinkled surfaces with micro-pattern are further applied to hCMEC/D cell culture, an immortalized human cerebral microvascular endothelial cell line, to induce cell proliferation and provide guidance for cell arrangement and migration. Therefore, this report sheds light on cell biology and biomedical tissue engineering by delivering an affordable and tunable fabrication process for wrinkles formations on the polymeric thin film.

Selenium attenuates ischemia/reperfusion injury‑induced damage to the blood‑brain barrier in hyperglycemia through PI3K/AKT/mTOR pathway‑mediated autophagy inhibition.

Ischemic stroke is a leading cause of mortality and disability. Diabetes mellitus, characterized by hyperglycemia, is a common concomitant disease of ischemic stroke, which is associated with autophagy dysfunction and blood-brain barrier (BBB) damage following cerebral ischemia/reperfusion (I/R) injury. At present, there is no effective treatment strategy for the disease. The purpose of the present study was to explore the molecular mechanisms underlying the protective effects of selenium on the BBB following I/R injury in hyperglycemic rats. Middle cerebral artery occlusion was performed in diabetic Sprague-Dawley rats. Treatment with selenium and the autophagy inhibitor 3-methyladenine significantly reduced cerebral infarct volume, brain water content and Evans blue leakage, while increasing the expression of tight junction (TJ) proteins and decreasing that of autophagy-related proteins (P<0.05). In addition, selenium increased the phosphorylation levels of PI3K, AKT and mTOR (P<0.05). A mouse bEnd.3 brain microvascular endothelial cell line was co-cultured in vitro with an MA-h mouse astrocyte-hippocampal cell line to simulate the BBB. The cells were then subjected to hyperglycemia, followed by oxygen-glucose deprivation for 1 h and reoxygenation for 24 h. It was revealed that selenium increased TJ protein levels, reduced BBB permeability, decreased autophagy levels and enhanced the expression of phosphorylated (p)-AKT/AKT and p-mTOR/mTOR proteins (P<0.05). Treatment with wortmannin (an inhibitor of PI3K) significantly prevented the beneficial effects of selenium on the BBB, whereas insulin-like growth factor 1 (a PI3K activator) mimicked the effects of selenium. In conclusion, the present findings indicated that selenium can inhibit autophagy by regulating the PI3K/AKT/mTOR signaling pathway, significantly preventing BBB damage following cerebral I/R injury in hyperglycemic conditions.

Establishment and characterization of a rat intestinal microvascular endothelial cell line

Intestinal microvascular endothelial cell (IMVEC) is a fundamental and essential component of gut-vascular barrier which is closely associated with intestinal disorders However, there is still a lack of established intestinal microvascular endothelial cell line. In the present study, a newly established rat intestinal microvascular endothelial cell line termed RIMVEC-11 was described and characterized which has been stably cultured for more than 90 passages so far. RIMVEC-11 was characterized by endothelial features with the cobblestone morphology under light microscopy, the Weibel-Palade body and rich vesicles in the cytoplasm on the ultrastructural level, and positive endothelial specific markers CD31 and von Willebrand factor by immunocytochemistry analysis. Meanwhile, RIMVEC-11 maintained the fundamental physiological function of the microvascular endothelial cells. Tube formation assay confirmed that RIMVEC-11 retained the potential for capillaries formation. Scratch assay confirmed the endothelial cell migration potential of RIMVEC-11. Thus, a novel IMVEC cell line RIMVEC-11 was established, which could be used as a promising model for the gut-vascular barrier research.

A novel rat model of chronic subdural hematoma: Induction of inflammation and angiogenesis in the subdural space mimicking human-like features of progressively expanding hematoma

Pathophysiological mechanisms of chronic subdural hematoma (CSDH) involve localized inflammation, angiogenesis, and dysregulated coagulation and fibrinolysis. The scarcity of reproducible and clinically relevant animal models of CSDH hinders further understanding the underlying pathophysiology and improving new treatment strategies. Here, we developed a novel rat model of CSDH using extracellular matrices (Matrigel) and brain microvascular endothelial cell line (bEnd.3 cells). One hundred-microliter of Matrigel-bEnd.3 cell (106 cells per milliliter) mixtures were injected into the virtual subdural space of elderly male Sprague-Dawley rats. This approach for the first time led to a spontaneous and expanding subdural hematoma, encapsulated by internal and external neomembranes, formed as early as 3 d, reached its peak at 7 d, and lasted for more than 14 d, mimicking the progressive hemorrhage observed in patients with CSDH. The external neomembrane and hematoma fluid involved numerous inflammatory cells, fibroblasts, and highly fragile neovessels. Furthermore, a localized pathophysiological process was validated as evidenced by the increased expressions of inflammatory and angiogenic mediators in external neomembrane and hematoma fluid rather than in peripheral blood. Notably, the specific expression profiles of these mediators were closely associated with the dynamic changes in hematoma volume and neurological outcome. In summary, the CSDH model described here replicated the characteristics of human CSDH, and might serve as an ideal translational platform for preclinical studies. Meanwhile, the crucial roles of angiogenesis and inflammation in CSDH formation were reaffirmed.

X‑irradiation induces acute and early term inflammatory responses in atherosclerosis‑prone ApoE‑/‑ mice and in endothelial cells.

Thoracic radiotherapy is an effective treatment for many types of cancer; however it is also associated with an increased risk of developing cardiovascular disease (CVD), appearing mainly ≥10 years after radiation exposure. The present study investigated acute and early term physiological and molecular changes in the cardiovascular system after ionizing radiation exposure. Female and male ApoE−/− mice received a single exposure of low or high dose X-ray thoracic irradiation (0.1 and 10 Gy). The level of cholesterol and triglycerides, as well as a large panel of inflammatory markers, were analyzed in serum samples obtained at 24 h and 1 month after irradiation. The secretion of inflammatory markers was further verified in vitro in coronary artery and microvascular endothelial cell lines after exposure to low and high dose of ionizing radiation (0.1 and 5 Gy). Local thoracic irradiation of ApoE−/− mice increased serum growth differentiation factor-15 (GDF-15) and C-X-C motif chemokine ligand 10 (CXCL10) levels in both female and male mice 24 h after high dose irradiation, which were also secreted from coronary artery and microvascular endothelial cells in vitro. Sex-specific responses were observed for triglyceride and cholesterol levels, and some of the assessed inflammatory markers as detailed below. Male ApoE−/− mice demonstrated elevated intercellular adhesion molecule-1 and P-selectin at 24 h, and adiponectin and plasminogen activator inhibitor-1 at 1 month after irradiation, while female ApoE−/− mice exhibited decreased monocyte chemoattractant protein-1 and urokinase-type plasminogen activator receptor at 24 h, and basic fibroblast growth factor 1 month after irradiation. The inflammatory responses were mainly significant following high dose irradiation, but certain markers showed significant changes after low dose exposure. The present study revealed that acute/early inflammatory responses occurred after low and high dose thoracic irradiation. However, further research is required to elucidate early asymptomatic changes in the cardiovascular system post thoracic X-irradiation and to investigate whether GDF-15 and CXCL10 could be considered as potential biomarkers for the early detection of CVD risk in thoracic radiotherapy-treated patients.

Methamphetamine and HIV-Tat Protein Synergistically Induce Oxidative Stress and Blood-Brain Barrier Damage via Transient Receptor Potential Melastatin 2 Channel.

Synergistic impairment of the blood-brain barrier (BBB) induced by methamphetamine (METH) and HIV-Tat protein increases the risk of HIV-associated neurocognitive disorders (HAND) in HIV-positive METH abusers. Studies have shown that oxidative stress plays a vital role in METH- and HIV-Tat-induced damage to the BBB but have not clarified the mechanism. This study uses the human brain microvascular endothelial cell line hCMEC/D3 and tree shrews to investigate whether the transient receptor potential melastatin 2 (TRPM2) channel, a cellular effector of the oxidative stress, might regulate synergistic damage to the BBB caused by METH and HIV-Tat. We showed that METH and HIV-Tat damaged the BBB in vitro, producing abnormal cell morphology, increased apoptosis, reduced protein expression of the tight junctions (TJ) including Junctional adhesion molecule A (JAMA) and Occludin, and a junctional associated protein Zonula occludens 1 (ZO1), and increased the flux of sodium fluorescein (NaF) across the hCMEC/D3 cells monolayer. METH and HIV-Tat co-induced the oxidative stress response, reducing catalase (CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) activity, as well as increased reactive oxygen species (ROS) and malonaldehyde (MDA) level. Pretreatment with n-acetylcysteine amide (NACA) alleviated the oxidative stress response and BBB damage characterized by improving cell morphology, viability, apoptosis levels, TJ protein expression levels, and NaF flux. METH and HIV-Tat co-induced the activation and high protein expression of the TRPM2 channel, however, early intervention using 8-Bromoadenosine-5′-O-diphosphoribose (8-Br-ADPR), an inhibitor of TPRM2 channel, or TRPM2 gene knockdown attenuated the BBB damage. Oxidative stress inhibition reduced the activation and high protein expression of the TRPM2 channel in the in vitro model, which in turn reduced the oxidative stress response. Further, 8-Br-ADPR attenuated the effects of METH and HIV-Tat on the BBB in tree shrews—namely, down-regulated TJ protein expression and increased BBB permeability to Evans blue (EB) and NaF. In summary, the TRPM2 channel can regulate METH- and HIV-Tat-induced oxidative stress and BBB injury, giving the channel potential for developing drug interventions to reduce BBB injury and neuropsychiatric symptoms in HIV-infected METH abusers.

Exosomal miR-155 from M1-polarized macrophages promotes EndoMT and impairs mitochondrial function via activating NF-κB signaling pathway in vascular endothelial cells after traumatic spinal cord injury

Pathologically, blood-spinal-cord-barrier (BSCB) disruption after spinal cord injury (SCI) leads to infiltration of numerous peripheral macrophages into injured areas and accumulation around newborn vessels. Among the leaked macrophages, M1-polarized macrophages are dominant and play a crucial role throughout the whole SCI process. The aim of our study was to investigate the effects of M1-polarized bone marrow-derived macrophages (M1-BMDMs) on vascular endothelial cells and their underlying mechanism. Microvascular endothelial cell line bEnd.3 cells were treated with conditioned medium or exosomes derived from M1-BMDMs, followed by evaluations of endothelial-to-mesenchymal transition (EndoMT) and mitochondrial function. After administration, we found conditioned medium or exosomes from M1-BMDMs significantly promoted EndoMT of vascular endothelial cells in vitro and in vivo, which aggravated BSCB disruption after SCI. In addition, significant dysfunction of mitochondria and accumulation of reactive oxygen species (ROS) were also detected. Furthermore, bioinformatics analysis demonstrated that miR-155 is upregulated in both M1-polarized macrophages and microglia. Experimentally, exosomal transfer of miR-155 participated in M1-BMDMs-induced EndoMT and mitochondrial ROS generation in bEnd.3 cells, and subsequently activated the NF-κB signaling pathway by targeting downstream suppressor of cytokine signaling 6 (SOCS6), and suppressing SOCS6-mediated p65 ubiquitination and degradation. Finally, a series of rescue assay further verified that exosomal miR155/SOCS6/p65 axis regulated the EndoMT process and mitochondrial function in vascular endothelial cells. In summary, our work revealed a potential mechanism describing the communications between macrophages and vascular endothelial cells after SCI which could benefit for future research and aid in the development of potential therapies for SCI.