Journal: Annals of biomedical engineering

Article Title: Development of a stereolithography 3D printing-based micropatterning method to study endothelial-to-mesenchymal transition mechanobiology

doi: 10.1007/s10439-025-03921-w

Figure Lengend Snippet: (a) Human aortic endothelial cells (HAECs) treated with TNF-α (50 ng/ml) for 96 hours exhibit morphological changes, transitioning from a circular, cobblestone pattern (left) to an elongated, spindle-like shape (right). Scale bar, 100 μm (b) RT-qPCR analysis of HAECs demonstrates that TNF-α treatment (50 ng/ml; 96 hours) results in decreased mRNA expression of the endothelial marker, ENOS3, and increased expression of the mesenchymal marker, CDH2 . n=3 for each experiment. Statistical analysis by unpaired t-test. Data presented are representative of 3 separate experiments (c) Western blot analysis of HAECs reveals that TNF-α treatment (50 ng/ml) for 24 hours results in significantly reduced expression of the endothelial marker ENOS3 (n=9), and increased expression of the mesenchymal marker, N-cadherin (n=6), the protein encoded for by CDH2 . Quantification of bands shown on the right. (d) Western blot analysis after 96 hours of treatment results in decreased ENOS3 expression (n=3), and increased expression of the mesenchymal markers, transgelin (TAGLN; n=3) and N-cadherin (n=9). Immunoblots shown are representative images. Quantification of bands shown on the right. Statistical analyses by unpaired two-tailed t-test for (b), Mann-Whitney test for ENOS3 in (c), and paired two-tailed t-tests for N-cadherin in (c) and all comparisons in (d).

Article Snippet: Human aortic endothelial cells (HAECs; Cell Applications) were thawed and seeded in 100mm Petri dishes coated in EmbryoMax 0.1% Gelatin Solution (ES-006B; EMD Millipore), starting at passage 5.

Techniques: Quantitative RT-PCR, Expressing, Marker, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Annals of biomedical engineering

Article Title: Development of a stereolithography 3D printing-based micropatterning method to study endothelial-to-mesenchymal transition mechanobiology

doi: 10.1007/s10439-025-03921-w

Figure Lengend Snippet: HAECs were seeded and incubated without (Control) or with TNF-α for 24 hours. Rose plots of cell orientations for (a) Control groups and (b) TNF-α treated groups. The average orientations are denoted by the dashed red line. Comparison of the two groups revealed a significant difference in the mean cellular orientations, with p <0.00025. Statistical analysis by Watson-Williams test. Cells from passages 11 through 13 were used in the samples constructing this analysis.

Article Snippet: Human aortic endothelial cells (HAECs; Cell Applications) were thawed and seeded in 100mm Petri dishes coated in EmbryoMax 0.1% Gelatin Solution (ES-006B; EMD Millipore), starting at passage 5.

Techniques: Incubation, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: EGCG stimulates autophagy. BAEC were treated with EGCG for the indicated times. Cell lysates were analyzed by Western blot using antibodies against the indicated proteins. A and B, time course of EGCG (10 μm) effect on LC3-II formation. Data are mean ± S.E. *, p < 0.05; **, p < 0.01 versus control (0 time point). C and D, dose-dependent response to EGCG on LC-3-II formation. Data are mean ± S.E. **, p < 0.01; ***, p < 0.001 versus control (no treatment). E and F, BAEC were transfected with control scrambled or ATG5 siRNA and incubated for 48 h. The cells were serum-starved for 2 h and then treated without or with EGCG (10 μm) for 4 h. LC3-II bands from three independent experiments were quantified and normalized for β-actin. Data are mean ± S.E. (**, p < 0.05, versus scrambled siRNA treated).

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Western Blot, Control, Transfection, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: Electron microscopic images of BAEC treated without or with EGCG. A and B, BAEC were treated without (upper panels) or with (lower panels) 10 μm EGCG for 2 h. Autophagosomes (thin solid arrows), autophagolysosomes (white triangle), and phagophore (black arrow) are indicated. C and D, areas of autophagosome and autophagolysosome were calculated as described under “Experimental Procedures.” Data are mean ± S.E. (***, p < 0.001, versus nontreated (NT) cells).

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: EGCG enhances autophagic flux. A and B, BAEC were treated without or with EGCG (10 μm, 4 h). The lysosomal inhibitor (NH4Cl (20 mm), Leu (200 μm)) was treated 1 h prior to cell harvest, and cell lysate was collected and analyzed by immunoblotting for LC3-II formation. Three independent experiments were performed, and the density of LC3-II/LC3-I was quantified. Data are mean ± S.E. (***, p < 0.001). C, differences between the absence and presence of NH4Cl/Leu were calculated for the indication of autophagic flux. Three independent experiments were quantified and calculated. Data are mean ± S.E. (*, p < 0.05). D and E, BAEC were treated without or with EGCG (10 μm) for the indicated time points. Cell lysate was harvested and analyzed by immunoblot with anti-SQSTM1(p62) antibody. SQSTM1 was significantly degraded by treatment with EGCG, which indicates autophagic degradation was enhanced. Three independent experiments were performed, and data are mean ± S.E. (***, p < 0.001). NT, not treated.

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: EGCG-stimulated autophagy is through AMPK and CaMKKβ but not through reactive oxygen species production. A, BAEC were treated with EGCG (10 μm) for the indicated times, and then cell extracts were analyzed by immunoblotting with the indicated antibodies. Three independent experiments were performed and quantified. B and C, BAEC were pretreated with inhibitors for 30 min; 3-methyladenine (3-MA, 5 mm), PD98059 (PD, 25 μm), SB203508 (SB, 10 μm), compound C (CC, 10 μm), or STO-609 (STO, 10 μm), and then treated with EGCG (10 μm, 4 h). Cell extracts were analyzed by Western blot by using the indicated antibodies. Data are mean ± S.E. (*, p < 0.05; **, p < 0.01 versus EGCG alone). D–G, BAEC were pretreated with the indicated dose of N-acetylcysteine and then treated with EGCG (10 μm, 4 h, D and E, or 3 h, F and G). Cell extracts were analyzed by Western blot using the indicated antibodies. Experiments were repeated three times and quantified by densitometry. Data are mean ± S.E., ***, p < 0.001.

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: CaMKKβ is involved in EGCG-stimulated AMPK, ULK1, and autophagy but not mTOR. A–C, BAEC were pretreated with STO-609 (10 μm) for 30 min and then treated with EGCG (10 μm) for 2 min. Cell lysates were analyzed by immunoblotting with the indicated antibody. The increased fold was calculated by LC3-II/β-actin. Experiments were repeated three times and quantified by densitometry. Data are mean ± S.E.; ns > 0.05, **, p < 0.01, and ***, p < 0.001. D, BAEC were transfected with control scrambled or CaMKKβ siRNA and incubated for 48 h. The cells were serum-starved for 2 h and then treated without or with EGCG (10 μm) for 4 h. LC3-II bands from three independent experiments were quantified and normalized for β-actin. Reduction of CaMKKβ was examined by RT-PCR. Data are mean ± S.E. (**, p < 0.01, and ***, p < 0.001).

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Western Blot, Transfection, Control, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: Elevation of intracellular calcium is required for EGCG-stimulated autophagy. A, serum-starved BAEC were pre-loaded with fluo-3 (10 μg/ml) and then stimulated with EGCG (10 μm). Cytosolic fluorescence was observed and quantified. The pseudocolor scale is a linear representation of the fluorescence intensity ranging from 200 to 1200 intensity units. Oscillation of calcium signaling was observed in EGCG-treated cells. B, BAEC were pretreated with CPA (20 μm) 30 min prior to EGCG treatment. C–E, pretreatment of BAEC with CPA reduced calcium signaling. C, number of events; D, peak of signaling; and E, cumulative fluorescence were quantified as described under “Experimental Procedures”; F, bovine aortic endothelial cells were pretreated with Ca2+ chelators for 30 min, EGTA (1 mm), or BAPTA-AM (10 μm), and then treated with EGCG (10 μm, 4 h). **, p < 0.01. Cell lysates were harvested and analyzed by immunoblotting. LC3-II bands from three independent experiments were quantified and normalized for β-actin. Data are mean ± S.E. (*, p < 0.05).

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: Palmitate-induced inhibition of autophagic flux, which was opposed by co-treatment with EGCG. BAEC were treated with BSA (0.1%) or palmitate (200 μm)/BSA conjugate in the presence or absence of EGCG (10 μm) for 4 h. Lysosomal inhibitor (20 mm NH4Cl, 200 μm leupeptin) was treated 1 h prior to cell harvest. Cell lysate was analyzed by immunoblotting for LC3-II formation. Three independent experiments were performed, and density of LC3-II/LC3-I was quantified. Data are mean ± S.E. (*, p < 0.05; ***, p < 0.001). ns, not significant.

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Inhibition, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

doi: 10.1074/jbc.M113.477505

Figure Lengend Snippet: EGCG enhances lipophagy. BAEC were seeded on glass coverslips. Cells were treated with BSA (0.1%) (the top two rows in A–D) or palmitate (200 μm)/BSA (0.1%)) conjugate (the bottom two rows in A–D) in the absence or presence of EGCG (10 μm) for 4 h without (A and C) or with (B and D) lysosomal inhibitors (20 mm NH4Cl, 200 μm leupeptin). The accumulation of lipid droplets was stained with BODIPY 493/503 (green). The co-localization of lipid droplets with LC3 (A and B, red) or LAMP-1 (C and D, red) was by immunocytochemistry using anti-LC3 and anti-LAMP-1 antibodies as described under “Experimental Procedures.” Insets show the higher magnification of the areas in the squares. Arrows indicate co-localization events. Nucleus was stained with Hoechst 33342 (blue). E, number of lipid droplets (LDs) was counted, and the average number of lipid droplets per cell was calculated. Data are mean ± S.E. (*, p < 0.05; **, p < 0.01, and ***, p < 0.001, n = 12). The percentage of lipid droplet co-localization with LC3 (F) or LAMP-1 (G) in cells treated with palmitate in the absence or presence of EGCG without or with NH4Cl/leupeptin was quantified. Data are mean ± S.E. (*, p < 0.05, n = 6). H and I, BAEC were incubated with palmitate (200 μm)/BSA (0.1%) conjugate without or with EGCG (10 μm) for 4 h (left column) or preincubated with palmitate for 4 h and then treated without or with EGCG (10 μm) for another 4 h (right column). Cells were fixed with paraformaldehyde and then stained with BODIPY 493/503 (green). Nuclei were stained with Hoechst 33342 (blue). The number of lipid droplets (LDs) was counted, and the average number of lipid droplets per cell was calculated. Data are mean ± S.E. (***, p < 0.001, n = 10).

Article Snippet: Cell Culture and Transfection Bovine aortic endothelial cells (BAEC) in primary culture were obtained from Cell Applications (San Diego) and maintained in F-12K media containing 5% fetal bovine serum (FBS), endothelial cell growth supplement (15 μmol/ml, CB40006B, BD Biosciences), heparin sulfate (50 μg/ml, Sigma, H3393), penicillin (100 units/ml), and streptomycin (100 μg/ml).

Techniques: Staining, Immunocytochemistry, Incubation

Journal: PLoS ONE

Article Title: Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein

doi: 10.1371/journal.pone.0004627

Figure Lengend Snippet: A–D: F-Aβ40 uptake into the acidic compartments of bovine brain microvascular endothelial (BBME) cells labeled by Lysotracker Red® (60×). (A) Uptake of F-Aβ40 (B) Uptake of Lysotracker Red®; (C) Superimposition of images A and B; (D) A magnified portion of image C (enclosed in the white rectangle) to show co-localization of both fluorophores. I–II: Histograms of fluorescence intensity in BBME cells treated with (I) F-Aβ40: (A) Untreated control, (B) at 4°C, (C) at 37°C; and (II) AF633-Trf: (A) Untreated control, (B) at 4°C, (C) at 37°C.

Article Snippet: Bovine brain microvascular endothelial (BBME) cells were obtained frozen from Cell Applications Inc. (San Diego, CA) and were grown in 75 cm 2 cell culture flasks coated with collagen.

Techniques: Labeling, Fluorescence, Control