primary haoecs  (Cell Applications Inc)

Journal: Cardiovascular research

Article Title: Disturbed flow induces reprogramming of endothelial cells to immune-like and foam cells under hypercholesterolaemia during atherogenesis

doi: 10.1093/cvr/cvaf233

Figure Lengend Snippet: scRNA-seq data clustering analysis of mouse carotid artery 1 cells exposed to d-flow and/or hypercholesterolaemia during atherogenesis. ( A ) C57BL/6 mice were treated with or without AAV-PCSK9 injection and Western diet for 2 or 4 weeks, with or without PCL surgery. Representative macroscopic images of mouse carotid arteries and aortic arch are shown for 4 weeks post-PCL time points. Atherosclerotic plaque development occurred only in the LCAs of the d-flow and hypercholesterolaemia group (white arrow). Scale bar: 1 mm. (B ) UMAP plot of 98 553 cells from the scRNA-seq data of Ctrl (s-flow, normal cholesterol), D-flow (d-flow, normal cholesterol), HighChol (s-flow, hypercholesterolaemia), and D-flow_HighChol (d-flow, hypercholesterolaemia) groups at 2 and 4 weeks post-PCL mice reveals 25 unique cell clusters. Major cell populations include endothelial cells (ECs), vascular smooth muscle cells (SMCs), fibroblasts (FBs), macrophages (MΦs), dendritic cells (DCs), neutrophils (NTs), B cells (BCs), T cells (TCs), and natural killer cells (NKs). Leukocytes include MΦs, DCs, NTs, BCs, TCs, and NKs. ( C ) Stacked violin plot shows the expression levels of canonical marker genes used to annotate each cell cluster. ( D ) UMAP plot of each experimental condition is shown across time (2 days, 2 weeks, and 4 weeks). S-flow (top): Ctrl (left, boxed in green) and HighChol (right, blue) and D-flow (bottom): D-flow (left, red) and D-flow_HighChol (right, purple) are shown. N = 5–20 mice for each condition. Note that Ctrl_2d, D-flow_2d, and D-flow_2wk conditions contain only luminally collected cells from the previous work.

Article Snippet: Primary human aortic endothelial cells (HAECs; Cell Applications #304–05a) were cultured in complete medium composed of MCDB 131 (Corning, Corning, NY, #15–100-CV) supplemented with 10% FBS (R&D Systems, Minneapolis, MN, #S11550), 1% L-glutamine (Gibco, Billings, MT, #25030–081), 1% penicillin-streptomycin (Gibco, Billings, MT, #15140–122), 1% endothelial cell growth supplement (ECGS; bovine brain extract), 50 μg/mL L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, #A5960), 1 μg/mL hydrocortisone (Sigma–Aldrich, St. Louis, MO, #H088), 10 ng/mL EGF (STEMCELL Technologies, Vancouver, Canada, #78006), 2 ng/mL FGF (ProSpec, Mount Vernon, NY, #CYT-218-b), 2 ng/mL IGF-1 (R&D Systems, Minneapolis, MN, #291-G1), and 1 ng/mL VEGF (BioLegend, San Diego, CA, #583706).

Techniques: Injection, Western Blot, Expressing, Marker

Journal: Cardiovascular research

Article Title: Disturbed flow induces reprogramming of endothelial cells to immune-like and foam cells under hypercholesterolaemia during atherogenesis

doi: 10.1093/cvr/cvaf233

Figure Lengend Snippet: Lineage tracing study on EC-Confetti mice validates FIRE (endothelial inflammation, EndMT, EndIT, and EndFT) under d-flow and hypercholesterolaemia at 4 weeks post-PCL. EC-Confetti mice treated with d-flow and hypercholesterolaemia at 4 weeks post-PCL ( N = 6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B–R ), and quantified ( S – V ). ( A ) shows a representative gross image of LCA, RCA, and aortic arch. ( B – R ). LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B–D ); EndMT (Acta2, Snai1, and Cnn1, E – H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I – M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N – R ). ( B ), ( E ), ( I ), and ( N ) show merged images of confetti and FIRE markers at low magnification (10×), while the rest show 40× images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. ( S – V ) Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined Matlab and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot (male is black, female is red) represents % of confetti + ECs co-expressing FIRE markers in each longitudinal section used for quantification ( N = 10–13 longitudinal sections for RCA; N = 11–24 longitudinal sections for LCA). P values were calculated by two-tailed unpaired Student’s t -test with or without Welch’s correction for normal data and two-tailed unpaired Mann–Whitney U test for non-normal data.

Article Snippet: Primary human aortic endothelial cells (HAECs; Cell Applications #304–05a) were cultured in complete medium composed of MCDB 131 (Corning, Corning, NY, #15–100-CV) supplemented with 10% FBS (R&D Systems, Minneapolis, MN, #S11550), 1% L-glutamine (Gibco, Billings, MT, #25030–081), 1% penicillin-streptomycin (Gibco, Billings, MT, #15140–122), 1% endothelial cell growth supplement (ECGS; bovine brain extract), 50 μg/mL L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, #A5960), 1 μg/mL hydrocortisone (Sigma–Aldrich, St. Louis, MO, #H088), 10 ng/mL EGF (STEMCELL Technologies, Vancouver, Canada, #78006), 2 ng/mL FGF (ProSpec, Mount Vernon, NY, #CYT-218-b), 2 ng/mL IGF-1 (R&D Systems, Minneapolis, MN, #291-G1), and 1 ng/mL VEGF (BioLegend, San Diego, CA, #583706).

Techniques: Staining, Fluorescence, Microscopy, Expressing, Marker, Two Tailed Test, MANN-WHITNEY

Journal: Cardiovascular research

Article Title: Disturbed flow induces reprogramming of endothelial cells to immune-like and foam cells under hypercholesterolaemia during atherogenesis

doi: 10.1093/cvr/cvaf233

Figure Lengend Snippet: Summary and two-hit hypothesis of d-flow and hypercholesterolaemia in atherogenesis. D-flow is the initial instigator of partial FIRE, including endothelial inflammation, EndMT, and partial EndIT. D-flow under hypercholesterolaemic conditions triggers a robust FIRE, involving endothelial inflammation, EndMT, full EndIT, and EndFT, leading to atherosclerotic plaque development.

Article Snippet: Primary human aortic endothelial cells (HAECs; Cell Applications #304–05a) were cultured in complete medium composed of MCDB 131 (Corning, Corning, NY, #15–100-CV) supplemented with 10% FBS (R&D Systems, Minneapolis, MN, #S11550), 1% L-glutamine (Gibco, Billings, MT, #25030–081), 1% penicillin-streptomycin (Gibco, Billings, MT, #15140–122), 1% endothelial cell growth supplement (ECGS; bovine brain extract), 50 μg/mL L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, #A5960), 1 μg/mL hydrocortisone (Sigma–Aldrich, St. Louis, MO, #H088), 10 ng/mL EGF (STEMCELL Technologies, Vancouver, Canada, #78006), 2 ng/mL FGF (ProSpec, Mount Vernon, NY, #CYT-218-b), 2 ng/mL IGF-1 (R&D Systems, Minneapolis, MN, #291-G1), and 1 ng/mL VEGF (BioLegend, San Diego, CA, #583706).

Techniques: