Journal: Advanced Science

Article Title: Pericytes Contribute to Dysfunction in a Human 3D Model of Placental Microvasculature through VEGF‐Ang‐Tie2 Signaling

doi: 10.1002/advs.201900878

Figure Lengend Snippet: Placental pericytes reduce microvessel growth and connectivity. a) Schematic diagram showing pericyte location in relation to microvessels in vivo. b) Confocal image of HPP‐cocultured with HUVEC fixed at day 5. Shown is a single XY plane and orthogonal projections demonstrating lumen (red) wrapped by HPPs (green), as indicated by white arrows. Nuclei were labeled with Dapi (blue). Scale bar is 200 µm. c) Schematic showing the various geometric measurements using binary projection images. d) Comparison of mean vessel area (EC coverage), branch length, and microvessel connectivity between HLF and HPP cocultures. Significant differences between parameters appear early on. e) Parameters are compared for HPP cocultures with (green) and without (gray) added VEGF+FGF. Shown is mean ± s.e.m. * P > 0.05 with t ‐test.

Article Snippet: GFP‐labeled HPP (microvascular) were acquired from Angioproteomie and were cultured in pericyte growth medium according to manufacturer's protocols.

Techniques: In Vivo, Labeling, Comparison

Journal: Advanced Science

Article Title: Pericytes Contribute to Dysfunction in a Human 3D Model of Placental Microvasculature through VEGF‐Ang‐Tie2 Signaling

doi: 10.1002/advs.201900878

Figure Lengend Snippet: A triculture model for increased microvessel connectivity. a) A triculture microvascular system perfused with fluorescently labeled beads. HUVEC—red, pericytes—green, 10 µm beads—magenta. b) Binary images from maximum intensity projections for co‐ and tricultures, as shown at day 5. c) Schedule for media change from full growth endothelial growth medium (EGM) to reduced serum basal medium (EBM). d) Representative flow cytometry density plots for HPP, HLF, and tricultures. e) Mean population of ECs and stromal cells for the co‐ and tricultures at day 5, as measured by flow cytometry. Three separate devices for each culture condition were used for measurement and repeated in n = 3 separate experiments. f) Microvessel parameters are compared between co‐ and tricultures. Shown is mean ± s.e.m. Significance is indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one‐way ANOVA and Tukey test.

Article Snippet: GFP‐labeled HPP (microvascular) were acquired from Angioproteomie and were cultured in pericyte growth medium according to manufacturer's protocols.

Techniques: Labeling, Flow Cytometry

Journal: Advanced Science

Article Title: Pericytes Contribute to Dysfunction in a Human 3D Model of Placental Microvasculature through VEGF‐Ang‐Tie2 Signaling

doi: 10.1002/advs.201900878

Figure Lengend Snippet: Pericytes influence PE‐affiliated cytokine expression and endothelial barrier function. a) Cytokine expression is shown for HPP, HLF, and triculture microvessel supernatants collected at day 5. HPPs result in increased PE‐associated cytokine expression, as indicated by the last row demonstrating those that are up (+) and down (−) regulated in PE. ND—no‐data in found. Here red values are high, blue low, and white are mid‐level (0.5). All cytokines were normalized to numbers between 1 and 0 based on maximum and minimum intensities from the cytokine array (Figure S4b, Supporting Information). b) Ang1/2 expression analyzed by ELISA for co‐ and tricultures, measured from pooled samples ( n = 5). c) Permeability of microvessels perfused with 10 kDa dextran (blue) at day 7 for co‐ and tricultures. Shown is mean ± s.e.m. Significance is indicated by * P < 0.05, ** P < 0.01, using t ‐test. d) Confocal images demonstrating perfusability of HLF cocultures and Tricultures, and lack of perfusability in HPP cocultures. HUVEC—red, 10 kDa dextran–blue. Scale bar is 200 µm.

Article Snippet: GFP‐labeled HPP (microvascular) were acquired from Angioproteomie and were cultured in pericyte growth medium according to manufacturer's protocols.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Permeability

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a-d , Ifnar-/- mice were infected with ZIKV as above, and the choroid plexuses were isolated at 4 DPI. a , Choroid plexuses were stained with antibodies against TTR (choroid plexus epithelial cell maker, green) and ZIKV-E protein (red). left : three layers of confocal images (labeled with Top, Middle, and Bottom) along the Z-axis. right : a three-dimensional reconstruction of the z-stacked image, highlighting the ZIKV-infected cells in the stroma layer of the choroid plexus. The top, middle, and bottom layers showed TTR+/ZIKV-, TTR-/ZIKV+, and TTR+/ZIKV-, respectively. A representative image is shown. b , Choroid plexuses were costained with FITC-IB4 for capillaries (green), anti-PDGFβR antibody for pericytes (purple), and anti-ZIKV-E antibody for the Zika envelope protein (red). Nuclei were stained with Hoechst 3342 stain (blue). The ZIKV-E signal colocalized with PDGFβR. For a and b , scale bars = 20 μm. c-d , ChP (n = 6, two from three mice) were harvested following cardiac perfusion and stained with anti-PDGFβR antibody for pericytes, anti-ZIKV-E antibody for the Zika envelope protein, and Hoechst 33342 for nuclei. (See for detail). The percentage of ZIKV-infected cells in the total ChP stromal cells(c) and PDGFβR (+) populations in the ZIKV (+) cells (d) were enumerated by using Imaris image analysis software. Each symbol represents an individual choroid plexus** P <0.01 (Student’s t-test). e , An electron micrograph showing a pericyte infected with ZIKV on a choroid plexus capillary. Pseudocolors (green and blue) indicate the cytoplasm and the nucleus, respectively, of a choroid plexus endothelial cell (EC) facing the lumen of a capillary (LUMEN). Patches of electron-dense particles (arrowheads) were found in a pericyte next to the EC. f , A higher magnification of the image from e shows that the particles have characteristics typical of flavivirus nucleocapsids. fi , A higher magnification image of the inset area of Fig e , Scale bars: 800 nm in e and 100 nm in f and fi .

Article Snippet: Pericytes were selected and cultured in pericyte growth medium (Pericytes Medium-mouse, Sciencell) for four passages prior to experiments.

Techniques: Infection, Isolation, Staining, Labeling, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a , ZIKV replication in primary pericytes from mouse choroid plexus (IFNAR +/- ). Cells (three independent wells) were infected with ZIKV, PLCal_ZV (MOI = 0.1) and grown in the presence of 5 μg/mL mouse anti-IFNAR-1 neutralizing antibody (clone MAR 5A3, ɑ-IFNAR) or isotype control antibody (clone MOPC-21). The cell culture supernatant was harvested at 5 DPI, and the virus titer was enumerated. * P <0.05 (Student’s t-test) b , Primary human brain vascular pericytes (five independent wells) were infected with ZIKV as in a and then incubated in the presence/absence of a human type 1 IFN neutralizing antibody mixture (ɑ-IFN Aby). The supernatants were harvested every 24 hours for five days, and the virus titers were enumerated with a virus titration assay. ** P < 0.005 (two-way ANOVA). c , Primary human brain vascular pericytes were infected with ZIKV and ZIKV-infected cells were enumerated by using FACS with anti ZIKV-E (clone 4G2). Each dot represents cells from single well. d , Mock- or ZIKV-infected HBVP were stained with antibodies against PDGFβR (red) and ZIKV-E protein (green). Two consecutive confocal layers were projected into a single layer with the Z-project function of ImageJ software (version 2.0.0). e , HBVP infected with ZIKV were stained with antibodies against AXL (green), PDGFβR (magenta) and ZIKV-E protein (red). Scale bars = 20 μm. f and g , HBVP were pre-treated with antibody for three hours then infected with ZIKV (m.o.i. = 1). Three days later, viral RNA ( f ) and progeny virus titers in the supernatants ( g ) were analyzed. **** P < 0.001 (one-way ANOVA).

Article Snippet: Pericytes were selected and cultured in pericyte growth medium (Pericytes Medium-mouse, Sciencell) for four passages prior to experiments.

Techniques: Infection, Control, Cell Culture, Virus, Incubation, Titration, Staining, Software

Journal: Journal of Molecular Neuroscience

Article Title: Microtubule Stabilization Promotes Microcirculation Reconstruction After Spinal Cord Injury

doi: 10.1007/s12031-020-01679-5

Figure Lengend Snippet: Microtubule stabilization inhibits the migration of endothelial cells and pericytes. a Crystal violet staining of migrated endothelial cells. Scale bar: 25μm. b Statistical analysis of migrated numbers of endothelial cells. c Crystal violet staining of migrated pericytes. Scale bar: 25 μm. d Statistical analysis migrated numbers of pericytes. Results were expressed as mean ± SD ( n = 6). ** p < 0.01. Significance was determined by student t-tests.

Article Snippet: Pericytes were cultured in Pericyte Medium (2% fetal bovine serum and 1% pericyte growth supplement, PriMed-iCELL-015) and kept in a humidified incubator (37 °C, 5% CO 2 , and 95% O 2 ).

Techniques: Migration, Staining

Journal: Journal of Molecular Neuroscience

Article Title: Microtubule Stabilization Promotes Microcirculation Reconstruction After Spinal Cord Injury

doi: 10.1007/s12031-020-01679-5

Figure Lengend Snippet: Microtubule stabilization upregulates the expression of angiogenesis related proteins. a Immunoblotting analysis of the expressions of VEGFA, VEGFR2, PDGFB, PDGFRβ, Ang-1 and Tie-2 in three groups of SCI rats. b–g Statistical analysis of the relative intensity of VEGFA, VEGFR2, PDGFB, PDGFRβ, Ang-1, and Tie-2 in SCI rats. h–1 Immunoblotting analysis of the expressions of VEGFR2, PDGFB, and Tie-2 in endothelial cells in four groups. h–2 Immunoblotting analysis of the expressions of VEGFA, PDGFRβ, and Ang-1 in pericytes in four groups. i–k Statistical analysis of the relative intensity of VEGFR2, PDGFB, and Tie-2 in endothelial cells in four groups. l-n Statistical analysis of the relative intensity of VEGFA, PDGFRβ, and Ang-1 in pericytes in four groups. Results are expressed as mean ± SD ( n = 6). ** p < 0.01. Significance is determined by one-way ANOVA followed by LSD comparison tests.

Article Snippet: Pericytes were cultured in Pericyte Medium (2% fetal bovine serum and 1% pericyte growth supplement, PriMed-iCELL-015) and kept in a humidified incubator (37 °C, 5% CO 2 , and 95% O 2 ).

Techniques: Expressing, Western Blot, Comparison