haoecs  (PromoCell)

Journal: FASEB BioAdvances

Article Title: Age and Sex Impact the Role of Thrombospondin‐2 and Thrombospondin‐5 in Response to Hindlimb Ischemia

doi: 10.1096/fba.2025-00258

Figure Lengend Snippet: TSP‐2 inhibits angiogenesis in early passage ECs, and TSP‐5 may be protective against tubule disruption. (A, B) Angiogenesis was assessed by plating ECs overnight on a Matrigel‐based endothelial tubule formation assay and pretreating with or without TSP‐2 (5 μg/mL) or TSP‐5 (5 μg/mL). TSP‐2 inhibited tubule formation in early passage ECs, but TSP‐5 inhibited tubule formation in late passage ECs. (C, D) To assess tubule disruption, EC tubules were allowed to form overnight and then treated for 24 h with TSP‐2 (5 μg/mL) or TSP‐5 (5 μg/mL). TSP‐2 inhibited tubule formation in late passage ECs (D), TSP‐5 increased tubule formation in early passage ECs (C). In the postformation treatment groups, late passage ECs had more tubule disruption than early passage cells. Values are means ± SE. Student's t ‐test performed for analysis, * p < 0.05 compared to nontreatment.

Article Snippet: Human aortic endothelial cells (Cell Applications Inc., San Diego, CA, USA) were used in early passage (P3–P8) to represent young cells or late passage (P15–P18) to represent aged cells.

Techniques: Disruption, Tube Formation Assay

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: