Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A ) The experimental plan for lectins binding to ihBMECs at the end of hypoxia or after re-oxygenation in absence/presence/. B ) Sensorgrams obtained with Quartz Crystal Microbalance showing the binding of ConA (upper panels) and WGA (lower) injected at four different concentration (0.7-2-6-18 µg/mL) over chip-adherent ihBMECs. The data show decreased binding at the end of the 16h of hypoxia and increased binding after the 4h of re-oxygenation either in presence or absence of MBL compared to normoxic ihBMECs. C ) Microphotographs of MBL (red) deposited on normoxic (left) or hypoxic (right) ihBMEC after re-oxygenation. Nuclei in blue (DAPI), scale bars 10 µm.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Binding Assay, Injection, Concentration Assay

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A ) MBL detection on the soft and hard corona samples obtained after preincubation of GNPs with human serum. The MBL signal decreased in the soft corona concomitantly with the three washes (Soft C1-3) and no signal was captured in the fourth wash (Soft C.4). The presence of MBL in the Hard Corona (Hard C., i.e. the proteins remaining after the washings because of their high affinity for the GNPs,) was strong for Man-GNPs (black arrow), and much less for Glc-GNPs (white arrow). B ) The experimental plan for testing sugar-GNPs localization on ihBMEC. C ) 3D microphotographs of Man-GNPs (red, reflectance microscopy) and F-actin (phalloidin, green) in normoxic (CTRL) or hypoxic (HYP) ihBMECs undergone re-oxygenation in the presence of 40 µg/mL Man-GNPs in 30% human serum. Man-GNPs were internalized in the cytoplasm of ihBMECs. Nuclei in blue (DAPI), scale bar 10 µm. D ) Normoxic (CTRL) or hypoxic (HYPOXIA) ihBMECs undergone re-oxygenation in the presence of 5, 20 or 40 µg/mL Man-GNPs in 30% HS were analyzed by reflectance confocal microscopy for Man-GNPs and MBL co-localization. Microphotographs show that Man-GNPs (white, reflectance microscopy) and hMBL (red) did not co-localize (as seen in magnification of white frame, scale bar 1 µm). Phalloidin in green, nuclei in blue (DAPI), scale bar 10 µm.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A) Microphotographs of MBL (red) deposited on normoxic (CTRL) or hypoxic (HYPOXIA) ihBMECs undergone re-oxygenation in the presence of 5, 20 or 40 µg/mL Man-GNPs in 30% HS (w/Man-GNPs). Nuclei in blue (DAPI), scale bar 200 µm. B ) MBL deposition, measured as fluorescence intensity, was greater on hypoxic than normoxic cells exposed to 30% HS. This increase was significantly reduced when ihBMECs were exposed to 5 and 20 µg/mL of Man-GNPs. Data as mean with individual values ± SD (n= 4). Two-way ANOVA followed by Tukey’s multiple comparisons, **p<0.001, *p<0.05. C ) Overexpression of ICAM-1 in hypoxic ihBMECs was significantly reduced when the cells were exposed to 20 µg/mL of Man-GNPs, to a similar extent than exposure to MBL depleted HS 30% (Δ MBL). D ) Overexpression of MMP-2 in hypoxic ihBMECs was partially counteracted by 20 µg/mL of Man-GNPs. E ) Expression of IL-1α was not significantly changed in presence of Man-GNPs with or without hypoxia. Data from 3 independent experiments, presented as mean with individual values ± SD (n= 4-12). Two-way ANOVA followed by Tukey’s multiple comparisons, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Fluorescence, Over Expression, Expressing

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A) The experimental plan to generate ihBMECs’ normoxic or hypoxic conditioned medium (NORM CM, HYP CM, respectively) and co-cultures of hIPSC-derived neurons, astrocytes and microglia. B ) Microphotographs of GFAP (astrocytes, green), MAP-2 (neurons, red) exposed for 24h to NORM CM or HYP CM +/− Man-GNPs. White arrows point to damaged neurons, i.e. circular cells without dendrites. Nuclei in blue (DAPI), scale bar 10 µm. C ) The quantification of stained volumes (in µm 3 ) showed a decrease of MAP-2 volumes in co-cultures exposed to HYP CM, which was counteracted by Man-GNPs. Data as mean ± SD. Each value is a random field of view (FOV) selected automatically from the overview image. Two-way ANOVA for repeated measures followed by Sidak’s multiple comparisons, ****p<0.0001 (n= 16 FOVs from two experimental replicates, empty rectangles indicate the mean of each replicate). D ) Microphotographs of GFAP (green) and nuclei (DAPI, blue) with a yellow line along which we calculated the FWHM reported in the graph. Width of the first ramification emerging from astrocytic soma was calculated at gray level’s half maximum (HM) and was larger in HYP CM compared to NORM CM or HYP CM + Man-GNPs. Data as mean gray levels of 8 cells per group ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons, ***p<0.001. Scale bars 10 µm. E ) Microphotographs of GFAP (green), β3-tubulin (neurons, red) and Iba1 (microglia, purple) exposed for 24h to NORM CM or HYP CM +/− Man-GNPs. Dashed squares indicate the magnified views of microglia on the right panels. Nuclei in blue (DAPI), scale bar 100 µm in full images, 20 µm in magnifications. White traces in the magnifications correspond to the Iba1 skeletonized signal. F ) The quantification of microglia morphological parameters showed increased number of branches and junctions after HYP CM exposure, which was counteracted by Man-GNPs. Data as violin plot. Each dot is individual microglia. Kruskal-Wallis test, **p<0.01, ***p<0.001 (n= 25-40 cells from 3 FOVs placed in one well). G ) Histograms of frequency distributions of the morphological parameters in E, shown with automatically chosen bin size.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Derivative Assay, Staining

Journal: Cancer research

Article Title: Targeting brain-adaptive cancer stem cells prohibits brain metastatic colonization of triple-negative breast cancer

doi: 10.1158/0008-5472.CAN-17-2994

Figure Lengend Snippet: A. Oncomine database analysis of PCDH7 expression in normal human tissue (Neurogenetics 2006 7:67–80). B. Western Blot analysis of PCDH7 in independent brain metastasis-derived tumorspheres (TS1 and TS2) and corresponding brain-seeking (Br) cell lines. C. Protein expression of PCDH7 in TNBC patient brain metastasis tumorspheres (BM-TS1 and 2) and various cell models. MCF7, SUM159, SKBR3: human breast cancer cell lines; BoM1833: MB231 bone-seeking cell line; LM4175: MB231 lung seeking cell line; NMA: primary normal mouse astrocytes; NHA: normal human astrocytes; HBVEC: human brain microvascular endothelial cells. D. Receiver Operating Characteristic (ROC) curve for PCDH7 expression in primary breast tumor samples of brain metastatic patients using the combined 368 microarray data (MSK-82 and EMC-286 cohort). E. Kaplan–Meier curves showing the brain metastasis-free survival of patients with positive or negative PCDH7 expression in the combined cohort of 368 breast cancer patients, P=1.21×10−5 determined by log rank test. F. Representative PCDH7 immunohistochemistry staining of matched patient tissue sections of brain metastasis, lung metastasis and primary breast tumors. Scale bar: 20µm.

Article Snippet: Normal human astrocytes and human brain microvascular endothelial cells were purchased from Lonza Group Ltd (Allendale, NJ).

Techniques: Expressing, Western Blot, Derivative Assay, Microarray, Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Knockdown of Atg7 inhibited angiogenesis of brain microvascular endothelial cells. ( A ) Human brain microvascular endothelial cells (HBMEC) were stably transfected with Atg7-specific shRNA construct, Atg7 shRNA1, and Atg7 shRNA2, respectively. HBMEC stably transfected with non-silencing shRNA were served as the control. Then the protein levels of Atg7 were examined by western blot, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. The relative expression level of Atg7 and Atg7/GAPDH were calculated by measuring the band intensity using ImageJ software. ** p < 0.01; ( B ) Tube formation assays were performed with HBMEC stably transfected with Atg7 shRNA1 and Atg7 shRNA2, respectively, with non-silencing shRNA as the control. Then the images were captured under an inverted microscope at indicated times. The representative images from three independent experiments were shown. Scale, 200 μm; ( C , D ) To quantify the results of tube formation assays in ( B ), the number of branch points were counted and the tube length were calculated. ** p < 0.01.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown, Stable Transfection, Transfection, shRNA, Construct, Control, Western Blot, Expressing, Software, Inverted Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Atg7 knockdown reduced IL-6 production in brain endothelial cells. ( A ) The mRNA levels of IL-6 in the HBMEC transfected with Atg7 shRNA1 were determined by real time RT-PCR. HBMEC transfected with non-silencing shRNA were used as control. ** p < 0.01; ( B ) The concentration of IL-6 and vascular endothelial growth factor (VEGF) in the supernatant of HBMEC transfected with Atg7 shRNA1 were determined by ELISA. ** p < 0.01; ( C ) The mRNA levels of IL-6 and VEGF in the brain cortex from the three-month-old Atg7 endothelial-specific knockout mice were determined by real time RT-PCR, with littermate wild-type mice as control. * p < 0.05.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, shRNA, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Exogenous addition of IL-6 restored the impaired angiogenesis in brain endothelial cells with Atg7 knockdown. ( A ) Tube formation assays were performed with the indicated HBMEC in the absence (vehicle) or presence of IL-6 (10 ng/mL). The representative images from three independent experiments were presented. Scale, 200 μm; ( B , C ) To quantify the results in ( A ), the number of branch points ( B ) and the tube lengths were calculated ( C ). * p < 0.05, ** p < 0.01.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Knockdown of Atg7 inhibited migration of brain microvascular endothelial cell, which was restored by exogenous IL-6. ( A ) The scratch wound assays were performed using the indicated transfected HBMEC in the absence (vehicle) or presence of IL-6 (10 ng/mL). The representative images at indicated times from three independent experiments were shown. Scale, 200 μm; ( B ) To quantify the results in ( A ), the wound recover rates were calculated by ImageJ software. * p < 0.05, ** p < 0.01.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown, Migration, Transfection, Software

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Interleukin-6 promoted cell migration to rescue angiogenesis in the Atg7-knockdown brain microvascular endothelial cells. ( A ) The nuclear and cytoplasmic extracts were obtained in HBMEC stably transfected with Atg7 shRNA1 (Atg7 KD). Then the expression of the NF-κB p65 subunit was analyzed by western blot. β-tubulin and 38F3 were detected as marker proteins for cytoplasm and nuclear, respectively. HBMEC transfected with non-silencing shRNA were used as control. The representative images were from three independent experiments; ( B ) To quantify the results in ( A ), the band intensities of p65 in nuclear and cytoplasm fractions were measured by ImageJ software and the nuclear to cytoplasm ratios of p65 was calculated. * p < 0.05; ( C ) HBMEC stably transfected with Atg7 shRNA1 were seeded on coverslips and immunofluorescence was conducted with antibody against p65 (green). DAPI (blue) was used for counterstaining. HBMEC transfected with non-silencing shRNA were served as a control. Scale, 20 μm; ( D ) The mRNA levels of IL-6 in the HBMEC transfected with Atg7 shRNA1 were determined by real time RT-PCR, with HBMEC transfected with non-silencing shRNA as a control. When indicated, the cells were incubated with NF-κB agonist and betulinic acid (10 μg/mL) for 2 h. *** p < 0.001.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Migration, Knockdown, Stable Transfection, Transfection, Expressing, Western Blot, Marker, shRNA, Control, Software, Immunofluorescence, Quantitative RT-PCR, Incubation

Journal: Scientific Reports

Article Title: Key role of 15-LO/15-HETE in angiogenesis and functional recovery in later stages of post-stroke mice

doi: 10.1038/srep46698

Figure Lengend Snippet: ( a ) Immunohistochemical staining of 15-LO in brain arteries from WT mice suffered MCAO for different days and 12/15-LO −/− mice (n = 10, * p < 0.05, ** p < 0.01, ## p < 0.01). Values are represented as the mean ± S.E.M. ( b ) BMVECs were fixed and stained with anti-15-LO (green), anti-CD31 (red) and DAPI to stain nuclei (blue). Merged images show 15-LO colocalizes to CD31 (a marker of ECs). Scale bars are 25 μm. Images shown are representative of at least three independent experiments. ( c ) Quantification of 15-LO protein levels in BMVECs under OGD for different time points (0, 4, 8, 12, 24, 48 hours; n = 4, * p < 0.05). Values are represented as the mean ± S.E.M. ( d ) The endogenous level of 15-HETE was measured by15-HETE EIA kit in BMVECs. OGD increases the endogenous15-HETE production compared with normoxic conditions (n = 4, * p < 0.05).

Article Snippet: Brain microvascular endothelial cells (BMVECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and maintained in RPMI 1640 medium supplemented with 15% foetal bovine serum (FBS).

Techniques: Immunohistochemical staining, Staining, Marker

Journal: Scientific Reports

Article Title: Key role of 15-LO/15-HETE in angiogenesis and functional recovery in later stages of post-stroke mice

doi: 10.1038/srep46698

Figure Lengend Snippet: ( a ) 15-LO protein expression was down-regulated after transfecting the 15-LO siRNA sequence into BMVECs. ( b ) BMVECs were subjected to the tube formulation assay. 15-LO siRNA inhibited cell tube formation induced by OGD, which was attenuated by exogenous 15-HETE (1 μmol/mL). ( c ) OGD and exogenous 15-HETE upregulated proliferating cell nuclear antigen (PCNA). ( d ) 15-LO siRNA decreased 5-bromodeoxyuridine (BrdU) incorporation compared with the OGD group; however, the incorporation was significantly enhanced by both endogenous and exogenous 15-HETE. (n = 4, * p < 0.05, ** p < 0.01). Values are represented as the mean ± S.E.M. ( e ) Cells accumulated in S and G 2 /M under OGD. This was reversed by 15-LO inhibition. ( f ) OGD increased cyclin An expression and this was reversed by siRNA for 15-LO. ( g ) Expression of cyclin D in BMVECs was similarly affected by OGD or 15-HETE. (n = 4, * p < 0.05, ** p < 0.01). Values are represented as the mean ± S.E.M.

Article Snippet: Brain microvascular endothelial cells (BMVECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and maintained in RPMI 1640 medium supplemented with 15% foetal bovine serum (FBS).

Techniques: Expressing, Sequencing, Formulation, BrdU Incorporation Assay, Inhibition

Journal: Scientific Reports

Article Title: Key role of 15-LO/15-HETE in angiogenesis and functional recovery in later stages of post-stroke mice

doi: 10.1038/srep46698

Figure Lengend Snippet: ( a ) In BMVECs under OGD conditions, both endogenous and exogenous 15-HETE (1μmol/L) increased PI3K/Akt expression. (n = 4, * p < 0.05, ** p < 0.01). Values are represented as the mean ± S.E.M. ( b ) Pretreatment with LY-294002 blocked the effects of 15-HETE on cell migration. ( c ) The increased PCNA expression after 15-HETE under OGD was blocked by the PI3K/Akt inhibitor. ( d ) Pretreatment of LY-294002 blocked the exogenous 15-HETE-induced BrdU incorporation. (n = 4, * p < 0.05, ** p < 0.01). Values are represented as the mean ± S.E.M. ( e ) Flow cytometry for cell-cycle analysis indicated that 15-HETE stimulated BMVEC progression into G 2 /M + S phase, and this effect was blocked by LY-294002. ( f ) LY-294002 also blocked the increase in cyclin A or ( g ) cyclin D, induced by 15-HETE. (n = 4, * p < 0.05, ** p < 0.01). Values are represented as the mean ± S.E.M.

Article Snippet: Brain microvascular endothelial cells (BMVECs) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and maintained in RPMI 1640 medium supplemented with 15% foetal bovine serum (FBS).

Techniques: Expressing, Migration, BrdU Incorporation Assay, Flow Cytometry, Cell Cycle Assay

Journal: PLoS Pathogens

Article Title: Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis

doi: 10.1371/journal.ppat.1002947

Figure Lengend Snippet: (A) Fibrinogen on the surface of hBMEC pretreated with or without exogenous fibrinogen (Fg; 20 µg/ml). Nuclei were stained with DAPI (blue) and fibrinogen was detected with anti-fibrinogen IgG, followed by Alexa Fluor 488 conjugated anti-rabbit IgG (red). (B) NCTC 10/84 (WT) or PS2645 (Δ srr1 ) incubated with hBMEC, with or without fibrinogen pretreatment (20 µg/ml). Unbound bacteria were washed out and bound bacteria were counted. Values represent percent (mean ± S.D.) of total GBS inoculum bound to the monolayers. * = P<0.01.

Article Snippet: The human brain microvascular endothelial cell line (hBMEC) was developed and kindly provided by Kwang Sik Kim (Johns Hopkins University) , and cultured as previously described .

Techniques: Staining, Incubation, Bacteria

Journal: PLoS Pathogens

Article Title: Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis

doi: 10.1371/journal.ppat.1002947

Figure Lengend Snippet: (A) Binding of FLAG Srr1-BR and FLAG Srr1-BRΔlatch proteins to immobilized fibrinogen. Indicated concentration of FLAG Srr1-BR and FLAG Srr1-BRΔlatch were added to wells coated with fibrinogen or casein blocking reagent. (B) Binding of FLAG Srr1-BR and FLAG Srr1-BRΔlatch proteins to hBMEC monolayers pretreated with PBS (left panels) or fibrinogen (20 µg/ml, right panels). After washing out unbound proteins, bound proteins were detected with anti-FLAG mAb, followed by Alexa Fluor 488 conjugated anti-mouse IgG (red). Nuclei were stained with DAPI (blue). (C) Expression of Srr1-WT and Srr1Δ latch on the cell surface. Isolated cell wall proteins were probed by Western blotting with anti-Srr1 IgG. (D) GBS NCTC 10/84 WT, Δ srr1 and Δlatch variant binding to immobilized fibrinogen. Values represent percent of WT GBS binding to fibrinogen. (E) GBS NCTC 10/84 WT, Δ srr1 and Δ latch isogenic variant adherence to hBMEC monolayers. * = P<0.01.

Article Snippet: The human brain microvascular endothelial cell line (hBMEC) was developed and kindly provided by Kwang Sik Kim (Johns Hopkins University) , and cultured as previously described .

Techniques: Binding Assay, Concentration Assay, Blocking Assay, Staining, Expressing, Isolation, Western Blot, Variant Assay