Journal: Advanced Science

Article Title: Hyperglycemic Neurovasculature‐On‐A‐Chip to Study the Effect of SIRT1‐Targeted Therapy for the Type 3 Diabetes “Alzheimer's Disease”

doi: 10.1002/advs.202201882

Figure Lengend Snippet: Effect of hyperglycemic conditions on the brain microvasculature and neurons determined using the NV chip. A) Schematics of the hyperglycemic NV chip. Glucose concentrations under normal and hyperglycemic conditions are 5.5 and 15.5 m m , respectively. B) Immunofluorescence images of hBMECs on the NV chip under normal (NG) and high‐glucose (HG) conditions. VE‐cadherin (green) and F‐actin (red) were stained. The nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. C) Blood vessel diameter calculated on the NV chip under NG and HG ( n = 12 for NG, n = 20 for HG; ****, p < 0.0001). D) VE‐cadherin, CD31, ZO‐1, and SIRT1 expression in hBMECs on the NV chip under NG and HG conditions. Loading control is GAPDH. E) Ratio of normalized SIRT1 expression in hBMECs on the NV chip under NG and HG conditions. SIRT1 expression determined via western blotting was normalized to GAPDH, and the ratio was calculated based on NG conditions ( n = 4; ****, p < 0.0001). F) SIRT1 immunofluorescence (green) of hBMECs on the NV chip. The nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. G) Permeability of brain microvasculature on the NV chip under NG and HG conditions. Permeability to 4.4 kDa TRITC‐dextran within 5 min. Images were captured every 1 min. Scale bar = 500 µm. H) Accumulation and transport of amyloid‐beta (Aß, red) under NG and HG conditions. The nucleus was counterstained with DAPI (blue). Scale bar = 200 µm. I) Immunofluorescence of ReN cells on the NV chip with (w/) (+) or without (w/o) (−) Aß treatment under NG and HG conditions. Aß (red), pTau (green), and MAP2 (yellow) were stained. The nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. J) Protein expression in ReN cells cultured using the Transwell method under NG and HG conditions w/ (+) or w/o (−) Aß treatment. ß‐actin was used as the loading control. K) Viability of ReN cells determined using the Transwell method w/ (+) or w/o (−) Aß treatment under NG and HG conditions ( n = 4; *, p < 0.05; ****, p < 0.0001). L) SIRT1 immunofluorescence staining (green) of ReN cells on the NV chip under NG and HG conditions. Tuj1 (red) and nucleus (blue, DAPI) were co‐stained. Scale bar = 100 µm. M–P) Expression of total (M,N), cytoplasmic and nuclear (O,P) SIRT1 in ReN cells under NG and HG conditions. GAPDH was used as the loading control for total proteins, and ß‐tubulin and lamin A/C were used as loading controls for cytoplasmic and nuclear proteins, respectively. The ratio of normalized values (N,P) of SIRT1 expression in ReN cells under NG and HG conditions. SIRT1 expression determined using western blotting was normalized to GAPDH (N), ß‐tubulin, and lamin A/C (P), and the ratio was calculated based on NG ( n = 3; **, p < 0.01; n.s., no significance). Uncropped blotting images in (D), (J), (M), and (O) with the exact protein size represented in Figure , Supporting Information. Box and whiskers plots in (C) represent the median (horizontal bars), 25 to 75 percentiles (box edges), and minimum to maximum values (whiskers) including all points. The scatter dot plot in (E), (K), (N), and (P) represents the mean ± standard deviation (SD) with bars and error bars showing all points. Significance in (C), (E), (N), and (P) was calculated using an unpaired t ‐test. Significance in (K) was calculated using an ordinary one‐way ANOVA Tukey's multiple comparisons test.

Article Snippet: hBMECs (#1000, ScienCell, United States) were cultured to 90% confluency on 0.1 mg mL −1 collagen type I‐coated T75 flasks using endothelial cell culture media kit (ECM, #1001, ScienCell, United States) supplemented with 1% endothelial cell growth supplement (ECGS, #1052, ScienCell, United States), 1% penicillin/streptomycin (P/S, #0503, ScienCell, United States), and 5% fetal bovine serum (FBS, #0025, ScienCell, United States). hBMECs were sub‐cultured using 0.05% trypsin/EDTA (T/E, 25200056, Gibco, United States, 1/5 dilution of 0.25% T/E in Dulbecco's phosphate‐buffered saline (DPBS) following the manufacturer's protocol.

Techniques: Immunofluorescence, Staining, Expressing, Control, Western Blot, Permeability, Cell Culture, Standard Deviation

Journal: Advanced Science

Article Title: Hyperglycemic Neurovasculature‐On‐A‐Chip to Study the Effect of SIRT1‐Targeted Therapy for the Type 3 Diabetes “Alzheimer's Disease”

doi: 10.1002/advs.202201882

Figure Lengend Snippet: Effect of glucose re‐stabilization for the functional and protein‐level recovery of the brain microvasculature and neurons on the NV chip. A) Immunofluorescence images of hBMECs on the NV chip under normal (NG), high (HG), and recovered glucose (HG→NG) conditions w/ (+TNF α ) or w/o TNF α (−TNF α ) treatment. ICAM‐1 (green) and F‐actin (red) were stained, and the nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. B) Protein expression in hBMECs on the NV chip under NG, HG, and HG→NG conditions w/ or w/o TNF α (± TNF α ) treatment. GAPDH was used as a loading control. C) Ratio of normalized SIRT1 expression in hBMECs on the NV chip under NG, HG, and HG→NG conditions w/ or w/o TNF α (± TNF α ) treatment. SIRT1 expression determined via western blotting was normalized to GAPDH, and the ratio was calculated based on NG ( n = 5 in −TNF α , n = 3 in +TNF α ; **, p < 0.01). D) Blood vessel diameter calculated on the NV chip depending on NG, HG, and HG→NG conditions w/ or w/o TNF α (± TNF α ) treatment ( n = 8, **, p < 0.01; ****, p < 0.0001). E) Permeability of hBMECs to 4.4 kDa TRITC‐dextran determined using the Transwell assay under NG, HG, and HG→NG conditions with or without TNF α (± TNF α ) treatment ( n ≥ 12, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; n.s.; no significance). F) Transportation of Aß (red) in each condition. The nucleus was counterstained using DAPI (blue). Scale bar = 200 µm. G) Immunofluorescence staining of ReN cells on the NV chip under NG, HG, and HG→NG conditions w/ or w/o TNF α (± TNF α ) treatment. Aß (red), pTau (green) and MAP2 (yellow) were stained, and the nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. H) Protein expression in ReN cells cultured using the Transwell assay under NG, HG, and HG→NG conditions w/ or w/o TNF α (± TNF α ) treatment. ß‐actin was used as the loading control. I) Expression of cytoplasmic and nuclear SIRT1 in ReN cells under NG, HG, and HG→NG conditions. ß‐tubulin and Lamin A/C were used as loading controls for cytoplasmic and nuclear proteins, respectively. J) Ratio of normalized SIRT1 expression in ReN cells under NG, HG, and HG→NG conditions w/ or w/o TNF α (± TNF α ) treatment. Cytoplasmic and nuclear SIRT1 expressions determined using western blotting were normalized to ß‐tubulin and Lamin A/C, respectively, and the ratio was calculated based on NG ( n = 3; ****, p < 0.0001; n.s., no significance). Uncropped blotting images in (B), (H), and (I) with the exact protein size represented in Figure , Supporting Information. Box and whiskers plots in (D) and (E) represent the median (horizontal bars), 25 to 75 percentiles (box edges), and minimum to maximum values (whiskers) including all points. The scatter dot plot in (C) and (J) represents the mean ± SD with bars and error bars showing all points. Significance in (C), (D), (E), and (J) was calculated using an ordinary one‐way ANOVA Tukey's multiple comparisons test.

Article Snippet: hBMECs (#1000, ScienCell, United States) were cultured to 90% confluency on 0.1 mg mL −1 collagen type I‐coated T75 flasks using endothelial cell culture media kit (ECM, #1001, ScienCell, United States) supplemented with 1% endothelial cell growth supplement (ECGS, #1052, ScienCell, United States), 1% penicillin/streptomycin (P/S, #0503, ScienCell, United States), and 5% fetal bovine serum (FBS, #0025, ScienCell, United States). hBMECs were sub‐cultured using 0.05% trypsin/EDTA (T/E, 25200056, Gibco, United States, 1/5 dilution of 0.25% T/E in Dulbecco's phosphate‐buffered saline (DPBS) following the manufacturer's protocol.

Techniques: Functional Assay, Immunofluorescence, Staining, Expressing, Control, Western Blot, Permeability, Transwell Assay, Cell Culture

Journal: Advanced Science

Article Title: Hyperglycemic Neurovasculature‐On‐A‐Chip to Study the Effect of SIRT1‐Targeted Therapy for the Type 3 Diabetes “Alzheimer's Disease”

doi: 10.1002/advs.202201882

Figure Lengend Snippet: Inhibition of NV recovery through glucose stabilization in SIRT1‐depleted cells. A) Immunofluorescence images of wild‐type (WT) and SIRT1‐depleted (shSIRT1) hBMECs on the NV chip under NG, HG, and HG→NG conditions. CD31 (green) and nucleus (blue) were stained. Scale bar = 100 µm. B) SIRT1, VE‐cadherin and CD31 expression in WT and shSIRT1 hBMECs on the NV chip under NG, HG and HG→NG conditions. GAPDH was used as a loading control. C) Ratio of normalized SIRT1 expression in WT and shSIRT1 hBMECs on the NV chip under NG, HG, and HG→NG conditions. SIRT1 expression determined using western blotting was normalized to GAPDH, and the ratio was calculated based on NG ( n = 3; *, p < 0.05, **, p < 0.01, n.s., no significance). D) Blood vessel diameter of WT and shSIRT1 brain microvasculature on the NV chip under NG, HG, and HG→NG conditions (n ≥ 6, **, p < 0.01, ****, p < 0.0001). E) Permeability of WT and shSIRT1 hBMECs to 4.4 kDa TRITC‐dextran determined using the Transwell assay under NG, HG, and HG→NG conditions ( n = 8; ****, p < 0.0001, n.s., no significance). F) Transportation of Aß (red) under each condition. The nucleus was counterstained with DAPI (blue). Scale bar = 200 µm. G) Immunofluorescence staining of ReN cells on the NV chip co‐cultured with WT and shSIRT1 hBMECs under NG, HG, and HG→NG conditions w/ Aß treatment (+Aß). Aß (red), pTau (green), and MAP2 (yellow) were stained, and the nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. H) Expression of Aß, pTau and Tau in ReN cells co‐cultured with WT and shSIRT1 hBMECs using the Transwell assay under NG, HG and HG→NG conditions. ß‐actin was used as a loading control. Uncropped blotting images in (B) and (H) with the exact protein size represented in Figure , Supporting Information. Box and whiskers plots in (D) and (E) represent the median (horizontal bars), 25 to 75 percentiles (box edges), and minimum to maximum values (whiskers) including all points. The scatter dot plot in (C) represents the mean ± SD with bars and error bars showing all points. Significance in (C), (D) and (E) was calculated using an ordinary one‐way ANOVA Tukey's multiple comparisons test.

Article Snippet: hBMECs (#1000, ScienCell, United States) were cultured to 90% confluency on 0.1 mg mL −1 collagen type I‐coated T75 flasks using endothelial cell culture media kit (ECM, #1001, ScienCell, United States) supplemented with 1% endothelial cell growth supplement (ECGS, #1052, ScienCell, United States), 1% penicillin/streptomycin (P/S, #0503, ScienCell, United States), and 5% fetal bovine serum (FBS, #0025, ScienCell, United States). hBMECs were sub‐cultured using 0.05% trypsin/EDTA (T/E, 25200056, Gibco, United States, 1/5 dilution of 0.25% T/E in Dulbecco's phosphate‐buffered saline (DPBS) following the manufacturer's protocol.

Techniques: Inhibition, Immunofluorescence, Staining, Expressing, Control, Western Blot, Permeability, Transwell Assay, Cell Culture

Journal: Advanced Science

Article Title: Hyperglycemic Neurovasculature‐On‐A‐Chip to Study the Effect of SIRT1‐Targeted Therapy for the Type 3 Diabetes “Alzheimer's Disease”

doi: 10.1002/advs.202201882

Figure Lengend Snippet: Effect of the SIRT activator or inhibitor determined using the NV chip. A) Immunofluorescence images of hBMECs on the NV chip treated with resveratrol or sirtinol under NG and HG conditions. SIRT1 (green) and nucleus (blue) were stained. Scale bar = 100 µm. B) Protein expression in hBMECs on the NV chip treated with resveratrol (R) or sirtinol (S) under NG and HG conditions. GAPDH was used as a loading control. C) Ratio of normalized expression values for hBMECs on the NV chip treated with resveratrol or sirtinol under NG and HG conditions. SIRT1 expression determined using western blotting was normalized to GAPDH, and the ratio was calculated based on NG ( n = 3; *, p < 0.05; n.s., no significance). D) Blood vessel diameter calculated on the NV chip treated with resveratrol (Res) or sirtinol (Sir) under NG and HG conditions ( n ≥ 10; ****, p < 0.0001; n.s., no significance). E) Permeability of hBMECs to 4.4 kDa TRITC‐dextran determined using the Transwell assay upon treatment with resveratrol (Res) or sirtinol (Sir) under NG and HG conditions ( n = 8; ****, p < 0.0001). F) Transportation of Aß (red) under each condition. The nucleus was counterstained with DAPI (blue). Scale bar = 200 µm. G) Immunofluorescence of Aß (red), pTau (green), and MAP2 (yellow) in ReN cells treated with resveratrol (Res) or sirtinol (Sir) under NG and HG conditions. Scale bar = 100 µm. H) Protein expression in ReN cells cultured using the Transwell assay treated with resveratrol (Res) or sirtinol (Sir) under NG and HG conditions upon Aß treatment. ß‐actin was used as a loading control. I) Immunofluorescence staining of SIRT1 (green) in ReN cells on the NV chip treated with resveratrol (Res) or sirtinol (Sir) under NG and HG conditions. Nuclei (blue, DAPI) were counterstained. Scale bar = 100 µm. J–M) Expression of total (J,K) cytoplasmic and nuclear (L,M) SIRT1 in ReN cells treated with resveratrol or sirtinol under NG and HG conditions. GAPDH was used as a loading control for total proteins, and ß‐tubulin and Lamin A/C were used as loading controls for cytoplasmic and nuclear proteins, respectively. The ratio of normalized SIRT1 expression (K,M) in ReN cells upon treatment with resveratrol or sirtinol under NG and HG conditions. SIRT1 expression determined using western blotting was normalized to GAPDH (K), ß‐tubulin and Lamin A/C (M), and the ratio was calculated based on NG ( n = 3 in (K), and +Sir group in (M), n = 4 for Ctrl and +Res groups in (M); *, p < 0.05; **, p < 0.01). Uncropped blotting images in (B), (H), (J) and (L) with the exact protein size represented in Figure , Supporting Information. Box and whiskers plots in (D) and (E) represent the median (horizontal bars), 25 to 75 percentiles (box edges), and minimum to maximum values (whiskers) including all points. The scatter dot plot in (C), (K), and (M) represents the mean ± SD with bars and error bars showing all points. Significance in (C), (D), (E), (K) and (M) was calculated using an ordinary one‐way ANOVA Tukey's multiple comparisons test.

Article Snippet: hBMECs (#1000, ScienCell, United States) were cultured to 90% confluency on 0.1 mg mL −1 collagen type I‐coated T75 flasks using endothelial cell culture media kit (ECM, #1001, ScienCell, United States) supplemented with 1% endothelial cell growth supplement (ECGS, #1052, ScienCell, United States), 1% penicillin/streptomycin (P/S, #0503, ScienCell, United States), and 5% fetal bovine serum (FBS, #0025, ScienCell, United States). hBMECs were sub‐cultured using 0.05% trypsin/EDTA (T/E, 25200056, Gibco, United States, 1/5 dilution of 0.25% T/E in Dulbecco's phosphate‐buffered saline (DPBS) following the manufacturer's protocol.

Techniques: Immunofluorescence, Staining, Expressing, Control, Western Blot, Permeability, Transwell Assay, Cell Culture

Journal: Basic Research in Cardiology

Article Title: Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles

doi: 10.1007/s00395-022-00950-7

Figure Lengend Snippet: Amitriptyline inhibits ASM activity in vivo and promotes angiogenesis after I/R in an Asm dependent way. A Asm activity in the reperfused ischemic striatum (labeled I/R) and contralateral non-ischemic striatum (labeled C), measured using BODIPY-labeled sphingomyelin in wildtype mice exposed to transient MCAO, which were intraperitoneally treated with vehicle or amitriptyline (2 or 12 mg/kg b.w., b.i.d.) immediately after MCAO or with 24 h delay, followed by animal sacrifice after 24 h or after 14 days. B Ceramide content, measured by LC–MS/MS in I/R and C of wildtype MCAO mice treated with vehicle or amitriptyline for 14 days as above. C Total microvascular length, D branching point density and E mean microvascular branch length, evaluated by LSM in I/R and C of wildtype MCAO mice treated with vehicle or amitriptyline for 14 days. F Microvascular length, G branching point density and ( H ) mean branch length in C and I/R of Smpd1 + / + (wildtype) and Smpd1 −/− (that is, ASM-deficient) MCAO mice treated with vehicle or amitriptyline for 14 days. Note that amitriptyline increases angiogenesis in wildtype but not Smpd1 −/− mice. Representative 3D stacks post-I/R are shown in ( I ), ROIs for the evaluation of microvascular networks in ( J ), and maximum projection images inside these ROIs in ( K ). Data are means ± SD values. * p ≤ 0.05/** p ≤ 0.01/*** p ≤ 0.001 compared with non-ischemic C; † p ≤ 0.05/ †† p ≤ 0.01 compared with corresponding vehicle; ‡ p ≤ 0.05/ ‡‡ p ≤ 0.01 compared with corresponding Smpd1 + / + (n = 4–7 animals/group [in ( A )]; n = 7–9 animals/group [in ( B )]; n = 7–8 animals/group [in ( C – E )]; n = 5–7 animals/group [in ( F – H )]; analyzed by one-way ANOVA followed by LSD tests). Scale bars in 3D reconstructions in ( I ), 500 µm; in horizontal sections in ( I ), 1000 µm

Article Snippet: For comparative studies on sphingomyelinase expression and activities, primary human brain microvascular endothelial cells (HBMEC; catalog #1000; ScienCell™ Research Laboratories, Carlsbad, CA, U.S.A.) cultured in endothelial cell growth medium MV (ECGM-MV, PromoCell, Heidelberg, Germany) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium MV supplement mix (PromoCell) and human umbilical vein endothelial cells (HUVEC) cultured in endothelial cell growth medium (ECGM, PromoCell) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium supplement mix (PromoCell) were used.

Techniques: Activity Assay, In Vivo, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: Basic Research in Cardiology

Article Title: Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles

doi: 10.1007/s00395-022-00950-7

Figure Lengend Snippet: Amitriptyline, fluoxetine and desipramine promote cerebral angiogenesis in vitro in an ASM dependent way. A – C Matrigel-based tube formation evaluated for the number of closed tubes, microvascular length and branching point density, D transwell migration, E , F VEGFR2 abundance examined by Western blot and G , H VEGF concentration in supernatants measured by enzyme-linked immunosorbent assay (ELISA) of hCMEC/D3 exposed to vehicle or amitriptyline (0–50 µM). In ( F , H ), analyses were made after 4 and 24 h amitriptyline exposure, respectively. I Tube formation and J transwell migration of hCMEC/D3 exposed to vehicle or fluoxetine (0–20 µM). K Tube formation and L transwell migration of hCMEC/D3 exposed to vehicle or desipramine (0–50 µM). Note that all three ASM inhibitors increase angiogenesis. M Tube formation, N transwell migration, O VEGFR2 abundance and P VEGF concentration in supernatants of hCMEC/D3 transfected with scrambled siRNA (used as control) or SMPD1 siRNA which were exposed to vehicle or amitriptyline (50 µM). In ( O , P ), measurements were made after 4 and 24 h amitriptyline exposure, respectively. Data are means ± SD values. * p ≤ 0.05/** p ≤ 0.01/*** p ≤ 0.001 compared with corresponding vehicle; ‡ p ≤ 0.05/ ‡‡ p ≤ 0.01/ ‡‡‡ p ≤ 0.001 compared with corresponding scrambled siRNA ( n = 3–7 independent samples/group [in ( A – L )]; n = 5–8 independent samples/group [in ( M,N,O )]; n = 3 independent samples/group [in ( P )]; analyzed by one-way ANOVA [in ( A – D, G–L )] or two-way ANOVA [in ( M , N , P )] followed by LSD tests [in ( A – D , G – N , P )] or paired two-tailed t tests [in ( E , F , O )])

Article Snippet: For comparative studies on sphingomyelinase expression and activities, primary human brain microvascular endothelial cells (HBMEC; catalog #1000; ScienCell™ Research Laboratories, Carlsbad, CA, U.S.A.) cultured in endothelial cell growth medium MV (ECGM-MV, PromoCell, Heidelberg, Germany) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium MV supplement mix (PromoCell) and human umbilical vein endothelial cells (HUVEC) cultured in endothelial cell growth medium (ECGM, PromoCell) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium supplement mix (PromoCell) were used.

Techniques: In Vitro, Migration, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: The PKCε activator prevents an increase in reactive oxygen species (ROS) and a decrease in MnSOD mRNA expression and increases PKCε mRNA expression in human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). (A) TBHP was used to induce mitochondrial dysfunction and an increase in reactive oxygen species (ROS), including superoxide (O 2 •– ). Cultured cells were treated with 0, 50, 200, or 500 μM TBHP for 1 h and recovered in new culture medium without TBHP for 1 or 3 days. The reaction between O 2 •– and non-fluorescent hydroethidine generates highly specific red fluorescent products, ethidium and 2-hydroxyethidium were used to determine (B) concentration-dependent effect of TBHP on intracellular O 2 •– production. (C–F) Cells had been incubated with 500 μM TBHP for 1 h and were incubated in fresh culture medium without TBHP in an absence or presence of the PKCε activator bryostatin (bry, 25 nM) or DCPLA-ME (DCP, 100 nM) for 3 days. (C) Effects of bryostatin and DCPLA-ME on O 2 •– production, determined by ethidium and 2-hydroxyethidium as in the panel (A) . Quantitative PCR (qPCR) was used to determine (D) PKCε, (E) MnSOD, and (F) VEGF mRNA expression. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 110–465 random cells from 3 to 4 cultures/group) or Student’s t -test for double measurement qPCR ( n = 4–5 cultures/group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Expressing, Cell Culture, Concentration Assay, Incubation, Real-time Polymerase Chain Reaction, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: PKCε activation increases PKCε, MnSOD, and VEGF protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were treated with 500 μM TBHP for 1 h and incubated in new culture medium with or without the PKCε activators bryostatin (bry, 25 nM) and DCPLA-ME (DCP, 100 nM) for 3 days. (A,B,E,F,I,J) Immunohistochemistry imaged with confocal microscopy and (C,D,G,H,K,L) western blot analysis of (A–D) PKCε, (E–H) MnSOD, and (I–L) VEGF. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t-test (n = 3 cultures/western blot group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Activation Assay, Expressing, Cell Culture, Incubation, Immunohistochemistry, Confocal Microscopy, Western Blot, Molecular Weight, Marker, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: Reactive oxygen species (ROS) affects MnSOD mRNA and protein expression in cultured human brain microvascular endothelial cells (HBMEC) treated with tert-butyl hydroperoxide (TBHP). Cultured cells were incubated with the ROS scavenger N -acetylcysteine (Nac, 5 mM for 15 h) or the cell-permeable SOD mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 25 μM for 45 min) and were then treated with TBHP at 500 μM for 1 h and recovered without TBHP, Nac, and MnTMPyP for 3 days. (A,B) Immunohistochemistry and (C,D) western blot analysis were used to study MnSOD protein expression. (E) Quantitative PCR (qPCR) was used to determine MnSOD mRNA expression. Data are reported as mean ± SEM. Asterisks over the bars (* p < 0.05; ** p < 0.01; *** p < 0.001) compared with their according controls, set as 100%. Student’s t -test for mRNA expression and western blot analysis ( n = 4–5 cultures/group) or one-way ANOVA with post hoc Tukey’s multiple comparison test for immunohistochemistry ( n = 42–160 random cells from 3 to 4 cultures per group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Expressing, Cell Culture, Incubation, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: The mRNA-stabilizing protein HuR involved in PKCε-activated MnSOD and VEGF expression in human brain microvascular endothelial cells (HBMEC). HBMEC cells were treated with the HuR inhibitor CMLD-2 (35 μM) or dihydrotanshinone-I (DHTS, 10 μM) for 30 min before and during the 3-day incubation in the presence of the PKCε activator bryostatin (25 nM) or DCPLA-ME (100 nM). (A) Immunohistochemistry of HuR was used to study nuclear export of the HuR protein. (B) Immunohistochemistry and (C,D) western blot analysis of MnSOD protein expression. (E) Quantitative PCR (qPCR) of MnSOD mRNA expression. (F) Immunohistochemistry and (G,H) western blot analysis of VEGF protein expression. M, molecular weight marker. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 59–134 MnSOD-immunostained cells or 451–907 PKCε or VEGF-immunostained cells from 3 to 4 cultures/group or t -test ( n = 3 cultures/western blot group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Expressing, Incubation, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Molecular Weight, Marker, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: The PKCε activator prevents a decrease in vascular VEGF and MV loss in the CA1 hippocampal stratum radiatum from age-related memory impairment rats. Tissue sections from rats in were used to stained with cytochemistry of the vascular endothelial cell marker IB4. (A) Colocalization of histochemistry of the vascular endothelium marker IB4 and (B) immunohistochemistry VEGF levels. (C) Low magnification of confocal microscope of the vascular endothelium marker IB4 was used to determine (D) . MV density in random hippocampal CA1 areas. Data are presented as mean ± SEM; ** p < 0.01, *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test ( n = 63–95 random MV cells or 32–119 random areas from 3 to 5 rats/group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Staining, Marker, Immunohistochemistry, Microscopy, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: Reduction of VEGF protein, but not mRNA, expression and microvascular loss in the CA1 stratum radiatum of autopsy-confirmed AD human hippocampus. (A) Quantitative PCR (qPCR) was used to detect VEGF mRNA at the whole hippocampus level. (B,C) Immunohistochemistry and confocal microscopy were used to determine VEGF protein expression in MV wall cells in hippocampal CA1 area (N, the nucleus of MV wall cell). (D,E) Immunofluorescence of the vascular endothelial cell marker CD31/PECAM was used to determine MV density. AC, age-matched control; AD, autopsy-confirmed Alzheimer’s disease. Although VEGF mRNA was not different among the experiment groups, VEGF and MV density was decreased in AD hippocampi at the early Braak stages II–III, but not AD at the late Braak stages IV–VI, compared to AC group. Data are reported as mean ± SEM, * p < 0.05; ** p < 0.01; two-tailed t -test compared with their according controls ( n = 11 hippocampi per group for qPCR or n = 182–365 random MV cells from 11 hippocampi per group, or 87–105 random CA1 areas from 5 AD Braak II–III, 14 AD Braak IV–VI and 19 AC).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Confocal Microscopy, Immunofluorescence, Marker, Control, Two Tailed Test

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: The PKCε activator protects a reduction of PKCε and MnSOD in the hippocampal CA1 area from Tg2576 transgenic AD mice. Mice at 2 months of age were injected (i.p., twice a week) with normal saline in the presence or absence of bryostatin (30 μg/kg body weight) for a 3-month period. Mice were then studied at the age of 5–6 months old when an increase in soluble amyloid-beta (Aβ) and memory defect were seen in the hippocampus of Tg2576 mice . Bryostatin was withdrawn for 2 weeks to avoid the acute effect of bryostatin. Double immunohistochemistry and confocal microscopy of (A,B) PKCε, (A,C) PKCα, and (D,E) MnSOD in vascular endothelial cells that were marked with PECAM/CD31. Bryostatin (bry) prevented the loss of PKCε and MnSOD and promoted PKCα in Tg2576 (Tg) mice. Data are represented as mean ± SEM, * p < 0.05; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells from 3 to 5 mice/group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Transgenic Assay, Injection, Saline, Immunohistochemistry, Confocal Microscopy, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer’s Disease Hippocampus

doi: 10.3389/fnagi.2022.836634

Figure Lengend Snippet: The PKCε activator protects a reduction of VEGF in MV endothelial cells and MV density in the hippocampal CA1 area from Tg2576 transgenic AD mice. Tissue sections from animals in were used. (A) Double immunohistochemistry and confocal microscopy of (B) VEGF in vascular endothelial cells that were marked with PECAM/CD31. (C) Cytochemistry of the vascular endothelial cells marker IB4, imaged with a confocal microscope, was used to determine (D) MV density in random CA1 areas. Bryostatin (bry) prevented the loss of VEGF and MV density in Tg2576 (Tg) mice. Data are represented as mean ± SEM, ** p < 0.01; one-way ANOVA and post hoc Tukey’s multiple comparison test. ( n = 35–74 random MV cells or 19–28 random areas from 3 to 5 mice/group).

Article Snippet: Human brain MV endothelial cells (Neuromics, Edina, MN, United States, cat # HEC02 or ScienCell Research Laboratories, Carlsbad, CA, United States, cat # 1000) at subculture passage 2–5 were grown on fibronectin-coated cover glasses (0.8 mm diameter) in 12-well plates or fibronectin-coated 6-well plates.

Techniques: Transgenic Assay, Immunohistochemistry, Confocal Microscopy, Marker, Microscopy, Comparison

Journal: PLoS Biology

Article Title: Blood–brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans

doi: 10.1371/journal.pbio.3000946

Figure Lengend Snippet: (A) Primary BMECs were isolated from 12-week-old C57Bl/6 mice, and Dhh , Ihh , Shh , Pecam1 , Sma , Ng2 , Cd45 , and Gfap expressions were quantified by qRT-PCR (cycle threshold mean values). β-actin is used as a reference. (B) Human cortical sections from healthy donors were obtain from the NeuroCEB biobank and immunostained with anti-CDH5 (in red), anti-PECAM1 (in red), and anti-DHH (in green) antibodies. Nuclei were stained with DAPI (in blue). (C–E) HBMECs were cultured until confluency and starved for 24 h. HBMECs were then treated with PBS (control condition) or IL-1β 10 ng/mL for 24 h and (C) DHH , (D) ICAM1 , and (E) VCAM1 expression were quantified by qRT-PCR. (F–H) Twelve-week-old C57Bl/6 females (6 animals per group) were induced with MOG 35-55 EAE versus placebo. At day 13 post induction, mice were humanely killed, and spinal cord microvascular endothelial cells were isolated. (F) Dhh , (G) Icam1 , (H) Vcam1 , (I) Cldn5 , and (J) Zo1 expression were measured via qRT-PCR in both groups (MOG 35-55 versus placebo). ** P ≤ 0.01, **** P ≤ 0.0001 Mann – Whitney U test. The underlying data for Fig 1 can be found in S1 Data ( https://doi.org/10.6084/m9.figshare.12625034.v6 ). *It is important to note that CNS endothelial cells are from a pooled source including both brain and spinal cord tissues. Therefore, the resulting cell cultures/lysates may be heterogeneous in their use of DHH. This remark applies to Figs – . BMECs, brain microvascular endothelial cells; CD45, cluster of differentiation 45; CDH5, cadherin5; Cldn5, claudin5; CNS, central nervous system; Ctrl, control; DHH desert hedgehog; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic protein; HBMECs, human brain microvascular endothelial cells; IHH, Indian hedgehog; cam1, intercellular adhesion molecule 1; IL-1β, interleukin 1 beta; MEC, microvascular endothelial cell; MOG 35-55 , myelin oligodendrocyte glycoprotein-35-55; NS, non-significant; NG2, neural/glia antigen 2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PECAM1, platelet/endothelial cell adhesion molecule 1; SMA, smooth muscle actin; SHH, Sonic hedgehog; Vcam1, vascular cell adhesion molecule 1; ZO1, zonula occludens 1.

Article Snippet: HBMECs (Alphabioregen–CliniSciences, Nanterre, France) were cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 BulletKits (Lonza).

Techniques: Isolation, Quantitative RT-PCR, Staining, Cell Culture, Control, Expressing, MANN-WHITNEY, Reverse Transcription, Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Blood–brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans

doi: 10.1371/journal.pbio.3000946

Figure Lengend Snippet: (A–K) NHA were cultured until confluency and starved for 24 h. NHA were then treated for 24 h with HBMEC medium from untreated cells (control condition), conditioned media from HBMECs treated with VEGFA, conditioned media from HBMECs treated with Mannitol, or HBMEC medium with 20% plasma from healthy donors (Mannitol and VEGFA were washed out of the HBMEC cultures before the medium was used to treat the NHA cultures). (A) Gfap , (B) Aldh1l1 , (C) Vim , and (D) Cldn4 mRNA expression was quantified by qRT-PCR. (Post-VEGFA n = 5, Mannitol n = 8 to 9, Plasma n = 7 to 8, Vehicle control n = 14 to 15). (E–H) GFAP (in green) and (E) CLDN4 (in red) localizations were evaluated by immunofluorescent staining of a confluent NHA monolayer. Nuclei were stained with DAPI (in blue). The experiment was repeated 3 times. (I, J) GFAP and CLDN4 positive areas were then quantified. (Post-VEGFA n = 7 to 8, Mannitol n = 7 to 8, Plasma n = 8, Vehicle control n = 7 to 8). (K–O) Cerebral cortices of 10-week-old C57BL/6 mice were harvested 24 h following stereotactic microinjection of murine VEGFA (60 ng in 3 μL PBS), healthy C57BL/6 mouse plasma (3 μL), or vehicle control (3 μL PBS). (K–M) Cortical lesions were immunostained with anti-GFAP and anti-CLDN4 antibodies. (N) GFAP positive areas and (O) CLDN4 positive areas were quantified (VEGFA n = 4, healthy C57BL/6 mouse plasma n = 4, Vehicle control n = 4). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 Kruskal–Wallis test. The underlying data for Fig 5 can be found in S1 Data ( https://doi.org/10.6084/m9.figshare.12625034.v6 ). Aldh1l1, aldehyde dehydrogenase 1 family, member l1; AU, arbitrary units; CLDN4, Claudin4; Ctrl, control; GFAP, glial fibrillary acidic protein; HBMEC, human brain microvascular endothelial cells; NHA, normal human astrocytes; NS, non-significant; qRT-PCR, quantitative reverse transcription polymerase chain reaction; VEGFA, vascular endothelial growth factor A; Vim, vimentin.

Article Snippet: HBMECs (Alphabioregen–CliniSciences, Nanterre, France) were cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 BulletKits (Lonza).

Techniques: Cell Culture, Control, Clinical Proteomics, Expressing, Quantitative RT-PCR, Staining, Microinjection, Reverse Transcription, Polymerase Chain Reaction