primary human aortic endothelial cells  (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:

Journal: bioRxiv

Article Title: Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

doi: 10.64898/2025.12.17.695030

Figure Lengend Snippet: A . Recycled VEGFR2 stained with Alexa674 (Cyan) and nuclei stained with Hoechst (Red) in human aortic endothelial cells (HAEC) RUBCN KO and RUBCN WT exposed to static or 5 dynes of laminar flow during 24h. Scale bars, 1mm. Images are representative of three independently performed experiments. B. Volume of recycled VEGFR2 per cell with each point representing the average volume for at least 3 cells per field view from HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h. IgG controls as a control for VEGFR2 antibody. Values are expressed as mean±SEM. Significance was calculated for the Volume of recycled VEGFR2 with a 2-way ANOVA followed by tukey’s multiple comparison test (Flow exposure effect: *P<0.05 ; Genotype effect: ****P<0.0001; Flow exposure Genotype effect: *P<0.05 ). C. Volume of recycled VEGFR2 per cell with each point representing the average volume for at least 3 cells per field view from murine PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3 then exposed to static or 5 dynes of laminar flow during 24h. Values are expressed as mean±SEM. Significance was calculated for the volume of VEGFR2 recycled per cell with an unpaired Student t test . *P<0.05 D. Representative immunoblots of VEGFR2 and actin in isolated surface, whole-cell lysate (WCL) and biotin unbound fractions across murine PMVEC isolated from 2-month-old Atg16l1 wt and Atg16l1 DWD ki mice then exposed to static or 5 dynes of laminar flow during 24h. E. Immunoblots of total protein S-nitrosylation in whole cell lysate from PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3, then exposed to static or 5 dynes of laminar flow during 24h. F. Total protein S-nitrosylation densitometry measured by western blot in PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3 then exposed to static or 5 dynes of laminar flow during 24h. Values are expressed as mean±SEM. Significance was calculated for the densitometry with a 2-way ANOVA followed by tukey’s multiple comparison test. G. Nitrites measured in cell culture medium in HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h.Values are expressed as mean±SEM. Significance was calculated for the nitrites with a 2-way ANOVA followed by tukey’s multiple comparison test (Flow exposure effect: **P<0.01 ; Genotype effect: *P<0.05 ). H. Immunoblots of total VEGFR2, Y1175-phosporylated-VEGFR2, Ser1177-phosphorylated-eNOS, total eNOS and b-actin proteins in whole cells lysates of HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h.

Article Snippet: Human aortic endothelial cells (HAEC) were purchased at ATCC (Cat#PCS-100-011), plated on fibronectin coated plates (1μg/cm , Corning; Cat# 354008) and maintained in EBM-2 medium (Lonza, Cat# CC-3156) completed with Endothelial Cell growth medium-2 bullet kit (Lonza, Cat# CC-3162) in a humidified 5% CO chamber at 37°C.

Techniques: Staining, Control, Comparison, Isolation, Western Blot, Cell Culture