Journal: Science Advances

Article Title: Biomimetic human small muscular pulmonary arteries

doi: 10.1126/sciadv.aaz2598

Figure Lengend Snippet: ( A ) Color photomicrograph of an adult hSMPA at the level of the respiratory bronchiole shown in cross section with hematoxylin and eosin staining. Black arrows indicate the EC of the intimal layer (E) and the VSMCs of the muscularis (M). Scale bar, 20 μm. Original magnification, ×200. This native artery exhibits several important structural characteristics including multicellular layering, curvature, and patterning. ( B ) Schematic illustration of the biomimetic hSMPA featuring patterning of cells and layering of VSMCs (M), laminin, and ECs (E). ( C ) Schematic illustration of the highly parallel, multistep patterning and assembly process for biomimetic hSMPA. Germanium (Ge) and bilayers of optically transparent silicon oxide and silicon dioxide (SiO/SiO 2 ) were deposited on silicon wafers using electron-beam evaporation, followed by adhesive protein patterning and cell layering, which were all achieved in 2D. Upon dissolution of the sacrificial germanium layer in cell culture medium, the 2D bilayer films were released and then self-folded into tubes. Additional fabrication details are shown in the schematic in fig. S1, and snapshots of the roll-up process are shown in fig. S2. Fn, fibronectin; Lm, laminin. ( D ) Confocal microscope images of tubular constructs with tunable 1 and 2 mm length and protein pattern. Luminal surfaces of the tubular constructs were patterned with fluorescently labeled fibronectin (red) or bovine serum albumin (green). The distribution of protein fluorescence intensity is shown in fig. S8A. For cell culture, fibronectin without fluorescence labeling was used. Scale bar, 500 μm. ( E ) Epifluorescence images of rhodamine-phalloidin–labeled ECs growing on the luminal surfaces of biomimetic microvessels. Scale bar, 500 μm.

Article Snippet: A confluent monolayer of HPMEC, with tightly contiguous vascular endothelial (VE)–cadherin–mediated junctions, was visualized on the luminal surface of the biomimetic microvessel 48 hours after cell seeding ( ; movie S1; and figs. S3A, S4A, and S6).

Techniques: Staining, Evaporation, Adhesive, Dissolution, Cell Culture, Microscopy, Construct, Labeling, Fluorescence

Journal: Science Advances

Article Title: Biomimetic human small muscular pulmonary arteries

doi: 10.1126/sciadv.aaz2598

Figure Lengend Snippet: Reconstructions of confocal Z -stacks of biomimetic microvessels populated by HPMECs, featuring the following: ( A ) VE-cadherin at endothelial adherens junctions (antibody labeling, green), ( B ) F-actin (phalloidin, red), and ( C ) merged images that include nuclei [4′,6-diamidino-2-phenylindole(DAPI), gray scale]. Insets in (A) and (B) show the cross-sectional views in the corresponding color channel and demonstrate the amount of overlap due to the roll-up process. Scale bars, 100 μm. A green-magenta rendering of this figure is shown in fig. S6.

Article Snippet: A confluent monolayer of HPMEC, with tightly contiguous vascular endothelial (VE)–cadherin–mediated junctions, was visualized on the luminal surface of the biomimetic microvessel 48 hours after cell seeding ( ; movie S1; and figs. S3A, S4A, and S6).

Techniques: Antibody Labeling

Journal: Science Advances

Article Title: Biomimetic human small muscular pulmonary arteries

doi: 10.1126/sciadv.aaz2598

Figure Lengend Snippet: ( A ) Confocal image (side view) of HPASMCs on an unpatterned tubular construct, stained for F-actin (phalloidin, red), smooth muscle α-actin (antibody labeling, green), and nuclei (DAPI, gray scale). The polar plot is based on image analysis of the gray scale F-actin image and shows that without patterning, HPASMCs attached and spread with random orientation. ( B ) Confocal image (side view) of HPASMCs grown on a patterned tubular construct, stained for F-actin (phalloidin, red), smooth muscle α-actin (antibody labeling, green), and nuclei (DAPI, gray scale). On the basis of image analysis of the gray scale F-actin image, the polar plot shows that F-actin filaments demonstrated alignment in parallel helical structures on the fibronectin-patterned scaffold. Binning in the polar plots is 10°. ( C ) 3D views of biomimetic microvessels demonstrating tunable variations in orientation angles and patterning periodicity, with labeled F-actin (phalloidin, red), smooth muscle α-actin (antibody labeling, green), and nuclei (DAPI, gray scale). Scale bars in (A to C), 100 μm.

Article Snippet: A confluent monolayer of HPMEC, with tightly contiguous vascular endothelial (VE)–cadherin–mediated junctions, was visualized on the luminal surface of the biomimetic microvessel 48 hours after cell seeding ( ; movie S1; and figs. S3A, S4A, and S6).

Techniques: Construct, Staining, Antibody Labeling, Labeling

Journal: Science Advances

Article Title: Biomimetic human small muscular pulmonary arteries

doi: 10.1126/sciadv.aaz2598

Figure Lengend Snippet: ( A ) Cell viability was calculated as the percentage of live cells compared with baseline (day 3) using the CyQUANT assay. Coculture of HPMEC and HPASMC populations were assayed. Data from flat (red) and tubular constructs (green) were compared. Data are displayed as means ± SEM. ( B ) HPMECs in the biomimetic microvessels exhibited a fourfold rise in nitric oxide production over the levels observed in HPMEC monolayers in flat culture. Nitrite levels in cell culture medium were determined by using a Sievers bioluminescence nitric oxide analyzer. Nitrite levels were normalized to total protein content for each sample. The numbers in the y axis indicate picomoles of nitrite per micrograms of protein. Medium was collected 48 hours after confluency and medium replacement. Data are displayed as means ± SEM (** P < 0.01). ( C ) Phosphorylation of eNOS (at Ser 1177 ) was found to be greater in HPMECs that were seeded and cultured on biomimetic microvessels than that in cells on flat SiO/SiO 2 films. HPMECs were grown at each condition for 48 hours. Western blots of cell lysates from each population were analyzed with antibodies against phosphorylated eNOS (p-eNOS), total eNOS, and β-tubulin (protein loading control). Data are displayed as means ± SEM (** P < 0.01).

Article Snippet: A confluent monolayer of HPMEC, with tightly contiguous vascular endothelial (VE)–cadherin–mediated junctions, was visualized on the luminal surface of the biomimetic microvessel 48 hours after cell seeding ( ; movie S1; and figs. S3A, S4A, and S6).

Techniques: CyQUANT Assay, Construct, Cell Culture, Phospho-proteomics, Western Blot, Control

Journal: Brain Sciences

Article Title: Histopathological Investigation of Dura-like Membrane in Vestibular Schwannomas

doi: 10.3390/brainsci11121649

Figure Lengend Snippet: Immunohistopathological analysis of CD34 staining. ( A ) Microvessel density (MVD) and diameter analyzed using CD34 staining are shown. Large CD34(+) vessels are observed in the tumors with DLM. Few CD34(+) vessels are identified in DLM (scale bar= 100 μm). ( B ) No significant difference is observed in the MVD in the tumors between DLM group and non-DLM groups ( p = 0.19). Vessel diameters are significantly larger in DLM group than that in non-DLM group ( p < 0.01). ( C ) The correlation between tumor volume and MVD are shown. The larger-sized VSs demonstrate higher MVD in the tumor with DLM ( p < 0.05, r = 0.89).

Article Snippet: To assess microvessel density (MVD) and vessel diameter, tissue sections were screened using CD34 immunohistochemistry in low-power fields and the three most vascularized regions (hot spots) were selected for automatic microvessel counting under high-power magnification microscopy (0.95 mm 2 ) (Biorevo BZ-9000, KEYENCE, Osaka, Japan), as previously described [ ].

Techniques: Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: BACE-1 is expressed in the blood–brain barrier endothelium and is upregulated in a murine model of Alzheimer’s disease

doi: 10.1177/0271678X15606463

Figure Lengend Snippet: BACE-1 mRNA expression analysis in mouse brain microvessels and cultured endothelial cells. In freshly isolated mouse brain microvessels (MBMVs) and primary cultured mouse brain microvascular endothelial cells (MBMECs) mRNA expression analysis by qRT-PCR showed significant expression of BACE-1 using two different primer pairs. Whole brain mRNA served as a positive control for BACE-1 expression as neurons are known to express high levels of BACE-1. BACE-1 expression was present even in pure cultured endothelial cells that have no contamination from neurons thus indicating a specific expression of BACE-1. Cldn-5 served as a marker for endothelium, which was at much higher levels in cultured and freshly isolated brain microvascular endothelial cells. The Ct range for BACE-1 qRT-PCR was in the range 23–26 cycles with non-template control about 35 cycles indicating specificity of expression. Statistical significance was by One-way ANOVA followed by TUKEY-HSD test for multiple groups (n = 4 experiments, *p < 0.05, **p < 0.01, and ***p < 0.001 with MBMV set as 1 with 2–4 mice in each experiment).

Article Snippet: Nylon mesh cell strainers for microvessel preparation were obtained from BD Biosciences (Germany).

Techniques: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, Positive Control, Marker

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: BACE-1 is expressed in the blood–brain barrier endothelium and is upregulated in a murine model of Alzheimer’s disease

doi: 10.1177/0271678X15606463

Figure Lengend Snippet: BACE-1 protein expression analysis in mouse brain microvessels. (a) Western Blots for BACE-1 using a highly specific antibody (B0681, Sigma) made against the N-terminus of BACE-1 (AA 46-62 of BACE, N-terminal) show the staining of the 75 and 50 kDa isoforms of BACE-1 (lane 1) in purified microvessels from wild-type mouse brains; the staining is specific as a blocking peptide (lane 2) completely abolished the staining of mouse brain microvessels. Lanes 3 and 4 show silver-stained 1D gels of recombinant BACE-1 (Invitrogen P2947 and Sigma S4195) as positive controls; lanes 5 and 6 show Western blots of the recombinant BACE-1 proteins with B0681. The blot is representative of three independent preparations of brain microvessels using 8–10 mice each time. The recombinant proteins were made to the extracellular domain of BACE-1 fused to Fc region of human IgG1 (Invitrogen P2947) or with a C-terminal FLAG-tag (Sigma S4195), hence they migrate at a lower molecular weight than the endogenous full-length BACE-1, which runs at 75 kDa. (b) In addition to B0681 (N-terminal epitope, Sigma) ab2077 (C-terminal epitope, Abcam) and 2882-1 (C-terminal, Epitomics) were also used to confirm the expression of BACE-1 in mouse brain microvessels. Competing peptides (for the antibody epitopes) from the vendor were used to obtain the specificity of the bands. For the Epitomics antibody for which the vendor competing peptides were not available only the C-terminal peptide for ab2077 but not the N-terminal one corresponding to B0681 abolished the specific band. The N-terminal antibody from Sigma recognizes two isoforms (full length 75 kDa, soluble 50 kDa) but the C-terminal antibodies (Abcam, Epitomics) recognize only the higher molecular weight full-length isoform. The red arrowheads indicate the specific bands and the red cross marks point the loss of these specific bands.

Article Snippet: Nylon mesh cell strainers for microvessel preparation were obtained from BD Biosciences (Germany).

Techniques: Expressing, Western Blot, Staining, Purification, Blocking Assay, Recombinant, FLAG-tag, Molecular Weight

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: BACE-1 is expressed in the blood–brain barrier endothelium and is upregulated in a murine model of Alzheimer’s disease

doi: 10.1177/0271678X15606463

Figure Lengend Snippet: Localization and activity of BACE-1 in brain microvessels and cultured endothelial cells. Immunofluorescence for BACE-1 showing specific staining for BACE-1 at the PM and intracellular vesicles in (a) freshly isolated MBMVs (representative pictures from 3 preparations using 3–4 mice) and in (b) primary cultured MBMECs (representative of three preparations using 4–6 mice each time). Claudin-5 (Cldn5) served as an endothelial marker. (c) Secondary antibody control for BACE-1, showing no staining for BACE-1 when the primary antibody was omitted. (d) Localization analysis in fractionated freshly isolated bovine brain capillary endothelial cells shows the predominant expression of BACE-1 in the abluminal membranes (*p < 0.05, 2-tailed paired t-test). PgP and EAAT-2 served as markers of luminal and abluminal membranes, respectively. GLUT-1 served as a marker expressed equally on both membranes. Quantitation was performed using blots for BACE-1 from three preparations utilizing 10 bovine brains each time. (e) Activity of BACE-1 is shown by inhibition of BACE-1 (Merck IV, 24 h) in MBMECs in medium with 20% bovine serum that is reported to contain full-length APP. The blots show an increase in the mature form of APP (mAPP) in the medium indicating reduced cleavage by BACE-1 and a concomitant increase in α-secretase activity resulting in increased levels of sAPPα. Loading differences accounted for by taking equal volume fraction from cells seeded at the same density along with a Ponceau S protein stain (not shown). Quantitation of the blots was performed from three experiments utilizing two animals in each preparation. Significance was by 2-tailed paired t-tests (*p < 0.05, **p < 0.01).

Article Snippet: Nylon mesh cell strainers for microvessel preparation were obtained from BD Biosciences (Germany).

Techniques: Activity Assay, Cell Culture, Immunofluorescence, Staining, Isolation, Marker, Expressing, Quantitation Assay, Inhibition

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: BACE-1 is expressed in the blood–brain barrier endothelium and is upregulated in a murine model of Alzheimer’s disease

doi: 10.1177/0271678X15606463

Figure Lengend Snippet: Expression analysis in brain microvessels from an AD mouse model and schematic for APP/Aβ processing at the BBB. (a) MBMVs from transgenic mice (hAPPSL) over-expressing the 751 amino acid form of human amyloid precursor protein (hAPP) with London (V717I) and Swedish (KM670/671NL) mutations under the control of the murine Thy-1 promoter were compared with wild-type mice. We observed a four-fold increase of BACE-1 expression in the BBB microvessels isolated from hAPPSL mice when compared to age-matched wild-type mice suggesting an increase in the APP cleavage activity of BACE-1 at the BBB in the mutant animals. Endothelial marker genes such as VE-cadherin and tight junction molecules ZO-1, claudin-5 were unchanged whereas GLUT-1, the primary glucose transporter at the BBB was downregulated. Interestingly, the luminal Aβ transporter RAGE was upregulated in the AD mice suggesting that circulating Aβ could potentially contribute to the brain amyloidosis. Furthermore the luminally expressed p-glycoprotein (PgP) known to be involved in efflux of Aβ into the circulation was downregulated suggesting a decreased clearance of amyloid peptides from the brain. The abluminally located LRP-1 was upregulated, which is known to endocytose APP, also supporting an increase in the BBB BACE-1 activity in this AD model. Abluminal Aβ antibody transporter FcRN was unchanged. Statistical significance was obtained from three qRT-PCR experiments using six transgenic mice (10 months age) or age-matched wild-type animals (***p < 0.001 using 2-tailed paired t-tests). (b) APP internalized from circulation via LRP2 or from brain parenchyma via LRP-1 or from the ECs is cleaved by BACE-1 to form Aβ. The predominant localization of BACE-1 at the abluminal membrane supports neuronal APP processing within the BBB endothelium. This Aβ is deposited as cerebrovascular plaques and/or is cleared from the brain parenchyma into circulation via LRP-1, FcRn from the abluminal side, and PgP from the luminal side. Additionally Aβ influx via RAGE can potentially regulate the Aβ transport across the BBB.

Article Snippet: Nylon mesh cell strainers for microvessel preparation were obtained from BD Biosciences (Germany).

Techniques: Expressing, Transgenic Assay, Isolation, Activity Assay, Mutagenesis, Marker, Quantitative RT-PCR