Journal: Acta Pharmaceutica Sinica. B

Article Title: ASF1A-dependent P300-mediated histone H3 lysine 18 lactylation promotes atherosclerosis by regulating EndMT

doi: 10.1016/j.apsb.2024.03.008

Figure Lengend Snippet: Lipid peroxidation promotes EndMT by increasing lactate level. (A) Expression profiling by array assessing changes in endothelial cell markers in ox-PAPC-treated human aortic endothelial cells (GSE72633). The results are presented as a heat map, arranged from blue (low values) to red (high values). (B) Lactate levels in untreated and 50 μg/mL ox-LDL-treated HCAECs for 24 h ( n = 6). (C) The morphology of HCAECs after ox-LDL and lactate treatment ( n = 6). (D) EC markers VE-cadherin and CD31, as well as EndMT markers α -SMA, N-cadherin, and Vimentin, were detected in ox-LDL and lactate-treated ECs by Western blot ( n = 6). (E) RT-qPCR analysis of EndMT markers in ox-LDL and lactate-treated HCAECs ( n = 6). Each box color in the heat map corresponds to the average of the data from the corresponding 6 independent experiments. The column graphs with individual values and significance tests for each mRNA in the heat map are shown in Fig. S1A . (F) Real-time ECAR in control and ox-LDL cells with or without si LDHA treatment were measured at 20, 40, 60, 80, and 100 min by using the Seahorse Bioscience Extra Cellular Flux Analyzer ( n = 6). (G) Lactate levels in 2-DG, oxamate, and si LDHA -treated ECs ( n = 6). (H) The morphology of HCAECs after 2 mmol/L 2-DG, 10 mmol/L oxamate, and si LDHA treatment for 24 h ( n = 6). (I)–(K) ECs markers and EndMT markers were detected in 2-DG, oxamate and si LDHA -treated HCAECs by Western blot ( n = 6). (L) RT-qPCR analysis of EndMT markers in si LDHA -treated ECs ( n = 6). Each box color in the heat map corresponds to the average of the data from the corresponding 6 independent experiments. The column graphs with individual values and significance tests for each mRNA in the heat map are shown in Fig. S1D . (M) Cell permeability of HCAECs after 2-DG, oxamate, and si LDHA treatment ( n = 6). (N) Cellular immunofluorescence analysis of ECs permeability after 2-DG treatment ( n = 6). (B) was analyzed by unpaired two-tailed student's t -test. (C)–(M) were analyzed by Ordinary one-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD; ∗∗ P < 0.01, ∗∗∗ P < 0.001. VE-cadherin: vascular endothelial cadherin; eNOS: endothelial nitric oxide synthases; HCAECs: human coronary artery endothelial cells; ox-LDL: oxidized low-density lipoprotein; ECAR: extracellular acidification rate; 2-DG: 2-deoxy- d -glucose; LDHA: lactate dehydrogenase A.

Article Snippet: Human coronary artery endothelial cells (HCAECs) were purchased from ScienCell and cultured in Endothelial Cell Medium (ScienCell) supplemented with 5% fetal bovine serum (FBS; ScienCell), 1% cell growth supplement, and 1% Penicillin/Streptomycin Solution (ScienCell).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Permeability, Immunofluorescence, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: ASF1A-dependent P300-mediated histone H3 lysine 18 lactylation promotes atherosclerosis by regulating EndMT

doi: 10.1016/j.apsb.2024.03.008

Figure Lengend Snippet: Increased histone H3K18 lactylation is involved in endothelial dysfunction induced by lipid peroxidation. (A) Pan Kla was detected in ox-LDL and lactate-treated HCAECs and MAECs by Western blot ( n = 6). (B) Pan Kla, H3K18la, H3K56la, H3K9la, and H3K14la were detected in ox-LDL-treated HCAECs and MAECs by Western blot ( n = 6). (C) H3K18la levels were visualized by immunofluorescence staining ( n = 6). Scale bar, 20 μm. (D) Pan Kla and H3K18la were detected in the aortic tissues from atherosclerotic patients or non-atherosclerotic patients by Western blot ( n = 5 human samples per group). (E) H3K18la levels were observed by enface staining of animal aortic tissues ( n = 6 mice per group). Scale bar, 20 μm. (F)–(H) Pan Kla and H3K18la were detected in 2-DG, oxamate, and si LDHA -treated HCAECs by Western blot ( n = 6). (B)–(D) were analyzed by Unpaired t -test with Welch's correction. (F)–(H) were analyzed by Ordinary one-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; ns, not significant. Pan Kla: pan-lysine lactylation; H3K18la: histone H3 lysine 18 lactylation; MAECs: mouse aortic endothelial cells; AS: atherosclerosis; Apoe: apolipoprotein E; NC: normal chow; HFD: high-fat diet.

Article Snippet: Human coronary artery endothelial cells (HCAECs) were purchased from ScienCell and cultured in Endothelial Cell Medium (ScienCell) supplemented with 5% fetal bovine serum (FBS; ScienCell), 1% cell growth supplement, and 1% Penicillin/Streptomycin Solution (ScienCell).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: ASF1A-dependent P300-mediated histone H3 lysine 18 lactylation promotes atherosclerosis by regulating EndMT

doi: 10.1016/j.apsb.2024.03.008

Figure Lengend Snippet: Lipid peroxidation induces EndMT through H3K18la-enriched SNAI1. (A) SNAI1 was detected in 2-DG, oxamate, and si LDHA -treated HCAECs by Western blot ( n = 6). (B) RT-qPCR analysis of SNAI1 in HCAECs ( n = 6). (C) The morphology of ox-LDL-treated HCAECs after interfering with SNAI1 ( n = 6). (D) EndMT markers were detected in ox-LDL-treated HCAECs after interfering with SNAI1 by Western blot ( n = 6). (E) RT-qPCR analysis of EndMT markers ( n = 6). Each box color in the heat map corresponds to the average of the data from the corresponding 6 independent experiments. The column graphs with individual values and significance tests for each mRNA in the heat map are shown in Fig. S2C . (F) CD31 and α -SMA levels were visualized by immunofluorescence staining ( n = 6). Scale bar, 20 μm. (G) Cell permeability of ox-LDL-treated HCAECs after si SNAI1 treatment ( n = 6). (H) Workflow chart for NRO, bromouridine immunocapture, and RT-qPCR. (I) Transcriptional regulation of SNAI1 was analyzed by NRO assay ( n = 6). (J) ChIP-seq peaks around SNAI1 from GSE171088 and GSE192358. (K) ChIP detection of binding sites of H3K18la at −1 kb from the promoter, the promoter, and +1 kb from the promoter region of SNAI1 . ChIP detection of the degree of binding of control H3 to SNAI1 ( n = 6). (L) Re-ChIP was performed with the first round including H3K27ac or H3K4me1 antibodies and a second round of pull-down with H3K18la antibodies ( n = 6). (A)–(E), (G), (I), (K), and (L) were analyzed by Ordinary one-way ANOVA with Tukey's multiple comparisons test. (C) was analyzed by Brown–Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. Data are shown as mean ± SD; ∗∗ P < 0.01, ∗∗∗ P < 0.001. NRO: nuclear run-on; ChIP: chromatin Immunoprecipitation.

Article Snippet: Human coronary artery endothelial cells (HCAECs) were purchased from ScienCell and cultured in Endothelial Cell Medium (ScienCell) supplemented with 5% fetal bovine serum (FBS; ScienCell), 1% cell growth supplement, and 1% Penicillin/Streptomycin Solution (ScienCell).

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Permeability, ChIP-sequencing, Binding Assay, Control, Chromatin Immunoprecipitation

Journal: Acta Pharmaceutica Sinica. B

Article Title: ASF1A-dependent P300-mediated histone H3 lysine 18 lactylation promotes atherosclerosis by regulating EndMT

doi: 10.1016/j.apsb.2024.03.008

Figure Lengend Snippet: The P300–ASF1A complex constitutes a chromosomal microenvironment to regulate the expression of SNAI1 via H3K18la. (A) P300 and HDAC1–3 were detected in ox-LDL-treated HCAECs by Western blot ( n = 6). (B) Pan Kla, H3K18la, H3K9la, and SNAI1 levels were detected in ox-LDL-treated HCAECs after interfering with P3 00 by Western blot ( n = 6). (C) RT-qPCR analysis of SNAI1 in ox-LDL-treated HCAECs after interfering with P300 ( n = 6). (D) ChIP analysis of the enrichment at the promoter of SNAI1 ( n = 6). (E) H3K18la levels were visualized by immunofluorescence staining ( n = 6). Scale bar, 20 μm. (F) The morphology of ox-LDL-treated HCAECs after interfering with P300 ( n = 6). (G) RT-qPCR analysis of endothelial mesenchymal transition markers in ox-LDL-treated HCAECs after interfering with P300 ( n = 6). Each box color in the heat map corresponds to the average of the data from the corresponding 6 independent experiments. The column graphs with individual values and significance tests for each mRNA in the heat map are shown in Fig. S3B . (H) Endothelial-like cell markers VE-Cadherin and CD31, as well as mesenchymal-like cells α -SMA and Vimentin were detected in ox-LDL-treated HCAECs after interfering with P3 00 by Western blot ( n = 6). (I) Molecular docking diagram ( http://hdock.phys.hust.edu.cn/ ) of ASF1A and P300. (J) Co-immunoprecipitation analysis of ASF1A-P300 binding in ox-LDL-treated HCAECs ( n = 6). (K) RT-qPCR analysis of ASF1A expression in ox-LDL and 2-DG-treated ECs ( n = 6). (L) Pan Kla, H3K18la, H3K9la, and SNAI1 were detected in ox-LDL and siASF1A-treated HCAECs by Western blot ( n = 6). (M) RT-qPCR analysis of endothelial mesenchymal transition markers in ox-LDL-treated HCAECs after interfering with ASF1A ( n = 6). Each box color in the heat map corresponds to the average of the data from the corresponding 6 independent experiments. The column graphs with individual values and significance tests for each mRNA in the heat map are shown in Fig. S3E . (N) In vitro assays to monitor H3K18 lactylation with H3–H4 substrates in the presence of full-length P300 (P300-FL) in the presence or absence of ASF1A ( n = 6). (O) In vitro, assays were performed with P300 in the presence of increasing concentrations of wild-type (WT) ASF1A or ASF1A-V94R mutant (defective in interaction with histones). ASF1A proteins are shown by Coomassie brilliant blue staining. H3K18la was detected by Western blot ( n = 6). (P) The transcription of SNAI1 in HCAECs treated with ox-LDL or si ASF1A was analyzed by NRO-RNAs ( n = 6). (Q) H3K18la and SNAI1 were detected in CTB and si ASF1A -treated HCAECs by Western blot ( n = 6). (R) RT-qPCR analysis of SNAI1 expression in CTB-treated HCAECs after interfering with ASF1A ( n = 6). (S) 3C-qPCR analysis of long–range interactions between the SNAI1 promoter and seven binding sites in ox-LDL-treated HCAECs combined with the deficiency of ASF1A or P300 and CTB-treated HCAECs after interfering with ASF1A ( n = 6). (T) Re-ChIP was performed with the first round with H3K18la antibody and a second round of pull-down with P300 antibodies in HCAECs treated with ox-LDL or si ASF1A ( n = 6). (U) Re-ChIP was performed with the first round with H3K18la antibody and a second round of pull-down with P300 antibodies in HCAECs treated with CTB or si ASF1A ( n = 6). (A) was analyzed by unpaired t -test with Welch's correction. (B)–(D), (G) and (H), (K)–(M), (P)–(R), and (T) were analyzed by Ordinary one-way ANOVA with Tukey's multiple comparisons test. (E), (F) and (U) were analyzed by Brown–Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. (S) was analyzed by Two-way ANOVA followed by a post hoc test. Data are shown as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; ns, not significant. HDAC: histone deacetylase; 3C-qPCR: chromatin conformation capture assay-qPCR; ASF1A: anti-silencing function 1A; CTB: cholera toxin B.

Article Snippet: Human coronary artery endothelial cells (HCAECs) were purchased from ScienCell and cultured in Endothelial Cell Medium (ScienCell) supplemented with 5% fetal bovine serum (FBS; ScienCell), 1% cell growth supplement, and 1% Penicillin/Streptomycin Solution (ScienCell).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Immunoprecipitation, Binding Assay, In Vitro, Mutagenesis, Histone Deacetylase Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: ASF1A-dependent P300-mediated histone H3 lysine 18 lactylation promotes atherosclerosis by regulating EndMT

doi: 10.1016/j.apsb.2024.03.008

Figure Lengend Snippet: PROTAC HK2 Degrader-1 reduces EndMT and atherosclerosis in Apoe KO mice. (A) Chemical Structure and schematic of PROTAC HK2 Degrader-1 mode of action. (B) Western blot analysis of HK2 in HCAECs treated with the indicated concentration of PROTAC HK2 Degrader-1 for 36 h ( n = 6). (C) Western blot analysis of HK2 in HCAECs treated with 0.5 μmol/L PROTAC HK2 Degrader-1 for the indicated incubation time ( n = 6). (D) Viability of HCAECs after treatment with PROTAC HK2 Degrader-1 for 48 h by CCK-8 assay ( n = 6). (E) Western blot analysis of HK2 in HCAECs pretreated with 2 μmol/L MG132 for 2 h before being treated with 0.5 μmol/L PROTAC HK2 Degrader-1 for 36 h ( n = 6). (F) Western blot analysis of HK1 and HK2 protein levels in HCAECs after incubation with 0.5 μmol/L PROTAC HK2 Degrader-1 for 36 h. ( n = 6). (G) Lactate level of HCAECs treated with 0.5 μmol/LPROTAC HK2 Degrader-1 for 36 h ( n = 6). (H) Representative images of aortas stained with Oil Red O from HFD-fed Apoe KO mice given intraperitoneal injections with Vehicle control or PROTAC HK2 Degrader-1 (5 mg/kg, q.o.d. for 12 weeks). The quantitative data of Oil Red O staining of mouse blood vessels ( n = 6 mice per group). (I, J) Atherosclerotic lesion formation detected by H&E, Oil Red O, Sirius red, and Masson's trichrome staining ( n = 6 mice per group). Scale bar, 100 μm. Quantification of lesions area and Oil Red O area. Percentage of necrotic core and collagen area. (K) Western blot analysis of H3K18la and EndMT markers in the MAECs extracted from HFD-fed Apoe KO after PROTAC HK2 Degrader-1 treatment ( n = 6 mice per group). (L) RT-qPCR analysis of EndMT markers in MAECs extracted from HFD-fed Apoe KO after PROTAC HK2 Degrader-1 treatment ( n = 10 mice per group). (B), (C), (D), (E), and (G) were analyzed by Ordinary one-way ANOVA with Tukey's multiple comparisons test. (H)–(J) were analyzed by unpaired two-tailed student's t -test. (K), (F), and (L) were analyzed by unpaired t -test with Welch's correction. Data are shown as mean ± SD; ∗∗ P < 0.01, ∗∗∗ P < 0.001; ns, not significant. PROTAC: proteolysis-targeting chimera; HK2: Hexokinase 2.

Article Snippet: Human coronary artery endothelial cells (HCAECs) were purchased from ScienCell and cultured in Endothelial Cell Medium (ScienCell) supplemented with 5% fetal bovine serum (FBS; ScienCell), 1% cell growth supplement, and 1% Penicillin/Streptomycin Solution (ScienCell).

Techniques: Western Blot, Concentration Assay, Incubation, CCK-8 Assay, Staining, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling reduced TNF-α-induced monocyte-adhesion. Cultured endothelial cells were stimulated with 10 ng/mL recombinant human tumor necrosis factor-α (TNF-α) and supplemented with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) for 18 h. Calcein-labeled THP-1 cells were allowed to adhere to human umbilical vein endothelial cells (HUVECs) (A) or HCAECs (B) for 30 min, then adherent cells were quantified and expressed as a fold change of control ( n = 5 each). In HUVECs, VCAM-1 (C) , and ICAM-1 (D) from whole cell lysates were quantified by Western blotting, normalized to stain-free loading controls and expressed as a fold change of TNF-α ( n = 6 and 4, respectively). Representative Western blots shown. In HCAECs, VCAM-1 (E) , and ICAM-1 (F) from whole cell lysates were quantified by Western blotting, normalized to stain-free loading controls and expressed as a fold change of TNF-α ( n = 3 and 5, respectively). Representative Western blots and stain-free loading controls are shown. *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Recombinant, Control, Labeling, Western Blot, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling restored barrier function in TNF-α-stimulated endothelial cells. Cultured endothelial cells were stimulated with 10 ng/mL recombinant human TNF-α in the presence of either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) for 18 h. Human umbilical vein endothelial cells (HUVECs) (A) or HCAECs (B) were seeded in Transwell inserts and streptavidin-HRP leakage across the endothelial monolayers was quantified. Data are expressed as a fold change of control ( n = 6 and 3, respectively) (C) . In HUVECs, following immunofluorescence for phospho-paxillin (Tyr118), total phospho-paxillin (Tyr118) levels were quantified using a Fiji-based macro, and normalized to cell count ( n = 6). Representative images of HUVECs immunostained (green) for VE-cadherin (D) , ZO-1 (E) or phospho-paxillin (Tyr118) (F) . Nuclei were stained with DAPI (blue). Scale bars represent 10 μm and apply to all panels. *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Recombinant, Control, Immunofluorescence, Cell Counting, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling enhanced platelet binding to TNF-α-stimulated endothelial cells. Cells were treated with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) in the presence or absence of 10 ng/mL recombinant human TNF-α stimulus for 18 h. BCECF-AM-labeled, thrombin-activated platelets were co-cultured with human umbilical vein endothelial cells (HUVECs) (A) and HCAECs (B) for 10 min, bound platelets lysed, and the fluorescent signal quantified. Data are expressed as optical density ( n = 8 and 4, respectively). (C) Representative Western blots for integrins α v and β 3 in whole cell lysates, and vWF and ADAMTS13 in conditioned media, and stain-free controls in HUVECs. (D) Segments of human saphenous vein were co-cultured with BCECF-AM-labeled, thrombin-activated platelets for 10 min, and the number of bound platelets quantified ( n = 4). (E) In HUVECs, integrin α v from whole cell lysates was quantified by Western blotting, normalized to stain-free controls and expressed as a fold change from control ( n = 4). Quantification and representative images of immunofluorescence (green) for vWF in permeabilised (F,G) and non-permeabilised (H,I) HUVECs, to detect intracellular and membrane-tethered vWF, respectively ( n = 5 and 4, respectively). Nuclei were stained with DAPI (blue). Scale bars represent 10 μm and apply to all panels. From cultured HUVECs, ADAMTS13 (J) , and soluble vWF (K) in conditioned culture medium were quantified by Western blotting, normalized to stain-free controls and expressed as a fold change from control ( n = 7 and 3, respectively). *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Binding Assay, Control, Recombinant, Labeling, Cell Culture, Western Blot, Staining, Immunofluorescence, Membrane

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Effects of inhibition of Wnt/β-catenin signaling on wound healing, apoptosis and proliferation in cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) in the presence or absence of 10 ng/mL recombinant human tumor necrosis factor-α (TNF-α) stimulus for 18 h. (A) HUVECs were subjected to scratch wounding and regrowth was quantified; data is expressed in μm ( n = 4 each). (B) HUVECs were subjected to immunofluorescence for cleaved caspase-3, and apoptosis quantified and expressed as the percentage of cleaved caspase-3-positive cells ( n = 3). (C) HUVECs were subjected to fluorescent labeling of incorporated EdU, and proliferation quantified and expressed as the percentage of EdU-positive cells ( n = 4). (D) Representative images of scratch wound assay performed on HUVECs. Dashed line indicates wound edge. Scale bar represents 500 μm. (E) Representative images of HUVECs immunostained (green) for cleaved caspase-3. White arrowhead indicates cleaved caspase-3-positive cell. Nuclei were stained with DAPI (blue). Scale bar represents 10 μm and applies to all panels. *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Control, Recombinant, Immunofluorescence, Labeling, Scratch Wound Assay Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Schematic diagram illustrating effect of inhibition of Wnt/β-catenin signaling on endothelial-platelet interaction in unchallenged and TNF-α-challenged cultured endothelial cells. Endothelial cells were treated with either 0.05% DMSO vehicle control or 25 μM iCRT in the presence or absence of 10 ng/mL recombinant human TNF-α stimulus for 18 h, then co-cultured with thrombin-activated platelets for 10 min. (A) Control: Unchallenged endothelial cells treated with 0.05% DMSO vehicle control. UL-vWF multimers remain stored in the Weibel-Palade bodies. (B) Control + iCRT-14: Unchallenged endothelial cells treated with 25 μM iCRT-14. UL-vWF multimers remain stored in the Weibel-Palade bodies. (C) TNF-α: TNF-α-challenged endothelial cells treated with 0.05% DMSO vehicle control. TNF-α stimulates release of UL-vWF from Weibel–Palade bodies. TNF-α-driven up-regulation in ADAMTS13 results in proteolytic cleavage of membrane-tethered UL-vWF; hence, TNF-α-stimulation does not promote endothelial-platelet interaction. (D) TNF-α + iCRT-14: TNF-α-challenged endothelial cells treated with 25 μM iCRT-14. TNF-α stimulates release of UL-vWF from Weibel–Palade bodies. Treatment with iCRT-14 blocks TNF-a-mediated up-regulation of ADAMTS13 thereby maintaining high levels of membrane-tethered UL-vWF; hence, platelet recruitment is enhanced. Acronyms: ADAMTS13 - a disintegrin-like and metalloprotease with thrombospondin type-1 repeats-13; TNF-α - tumor necrosis factor-a; UL-vWF - ultra-large von Willebrand factor.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Control, Recombinant, Membrane

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Time dependence of the absolute cell numbers of HAAF (A) , HASMC (B) , and HCAEC (C) cells cultured on SilkGraft and on polystyrene. Absolute cell numbers were lower on SilkGraft than on polystyrene because the available surface area was reduced due to the use of the steel ring which kept the silk substrate under water. Total cell growth differences between cells cultured on SilkGraft or on polystyrene are expressed by the areas under the corresponding curves, the statistical levels of significance of which are: for HAAFs, P < 0.001; for HASMCs, P < 0.01; and for HCAECs, P < 0.001.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Living cells density/mm 2 of apparent surface area after 21 days of in vitro culture.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: In Vitro

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Cumulative consumption of glucose and glutamine and release of lactate. Results were normalized per 10 3 cells. (A–C) The cumulative glucose consumption was higher for HAAFs ( P < 0.05) and HCAECs ( P < 0.001) seeded on SilkGraft, whereas it showed only marginal differences between the two substrates for HASMCs ( P > 0.05). (D–F) Glutamine consumption was lower for HAAFs ( P < 0.05) seeded on SilkGraft, similar for HASMCs ( P > 0.05) cultured on the two substrates, and significantly larger for HCAECs ( P < 0.001) grown on the silk substrate. (G,H) The cumulative amount of lactate released by HAAFs and HASMCs was the same whichever the substrate ( P > 0.05). Lactate release could not be assessed for HCAECs because the released lactate was re-uptaken and used for metabolic purposes. The statistical analysis of these data is shown in .

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Comparison of metabolic parameters ^* of the different cell types cultured on SilkGraft and polystyrene.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Comparison, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Relevant cytokines and chemokines secreted by each cell type cultured between 18 and 20 days on SilkGraft and polystyrene: HAFFs (A) , HCAECs (B) , and HASMCs (C) . Results of immunofluorescence intensities were normalized to 10 3 cells. IL-6: Interleukin-6; MCP-1: Monocyte chemoattractant protein-1; TIMP-2: Tissue inhibitor of metal proteinases-2; IP-10: Interferon gamma-induced protein-10; MCP-2: Monocyte chemoattractant protein-2; Eotaxin-1; RANTES: Regulated on activation normal T cell expressed and secreted; MIP-1β: Macrophage inflammatory protein-1β; TNF-β: Tumor necrosis factor-β; GM-CSF: Granulocyte-macrophage colony stimulating factor; IL-1α: Interleukin-1α; IL-1β: Interleukin-1β; ICAM-1: Intercellular adhesion molecule-1. The bars are the mean values of three independent experiments corrected for cell numbers. * P < 0.01; ** P < 0.001. SEMs, not shown, ranged between 5 and 10% of corresponding mean values.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Cell Culture, Immunofluorescence, Activation Assay

Journal: bioRxiv

Article Title: miR-10b Deficiency Affords Atherosclerosis Resistance

doi: 10.1101/248641

Figure Lengend Snippet: A. Nucleotide sequences of miR-10b and those of its target in 3’ UTR in LTBP1. B. Western blotting for LTBP1 and β-tubulin (TUBB) using various human ECs that showed Type-I phenotypes (HUVEC, HAEC, HCAEC, HMVEC and ESdEC[P6]) and those with Type-II phenotypes (iPS(BJ)EC, iPS(HU)EC, ESdEC[P0] and ESdEC[P1]) as reported previously 1 . C . Type-II ECs that were transfected with an empty vector (CMV-vector (+), mock) or an miR-10b expression vector (CMV-vector (+), miR-10b), and Type-II ECs without transfection (CMV-vector (-)) were subjected to Western blotting for using an anti-LTBP1 body or an anti-β-tubulin (TUBB) antibody. D . Western blotting for LTBP1 and β-tubulin (TUBB) proteins using Type-I ECs that were transfected with a control HIV vector (control) or an miR-10b inhibitor-expressing HIV vector (miR-10bi) together with Western blotting using Type-II ECs without transfection were shown as indicated. E. Proliferation indexes of SMCs that were co-cultured with Type-II ECs in the absence or the presence of increasing concentrations of LY2157299, a TGF-β signaling inhibitor, as indicated. N=3. F and G . SMCs were co-cultured with Type-I or Type-II ECs and the percentages of phosphorylated SMAD2/3-positive cells in were calculated by flow cytometry (F) and nuclear localization of SMAD3 in PKH-26 (red)-stained SMCs was estimated by immunostaining studies with an anti-smad3 antibody (green) along with nuclear counterstaining with DAPI (blue) (G). Full-length blots are presented in Supplementary information. Abbreviations: ESdEC[P6], human ES cell-derived ECs at passage 6; ESdEC[P0], human ES cell-derived ECs at passage 0; ESdEC[P1], human ES cell-derived ECs at passage 1.

Article Snippet: Human umbilical vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), human microvascular endothelial cells (HMVEC) and human coronary arterial endothelial cells (HCAEC) were purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka Japan).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Cell Culture, Flow Cytometry, Staining, Immunostaining, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Quinic acid regulated TMA/TMAO-related lipid metabolism and vascular endothelial function through gut microbiota to inhibit atherosclerotic

doi: 10.1186/s12967-024-05120-y

Figure Lengend Snippet: QA improved TMAO-induced inflammatory lesions and endothelial dysfunction in HCAECs. (A) CCK-8 was applied to detect the toxicity of QA on HCAECs. (B) CCK-8 was used to detect HCAECs proliferation. (C) The expression of COX-2, IL-6, E-selectin, ICAM-1, HMGB1 was detected by RT-qPCR. (D) The expression of p-P65, p-MAPK14 protein was detected by western blot. (E) HMGB1 levels were detected by ELISA. (F) The expression of ZO-2, VE-Cadherin and Occludin were detected by western blot. * P < 0.05 vs. Control, # P < 0.05 vs. TMAO

Article Snippet: To investigate the cytotoxicity of QA, human coronary artery endothelial cells (HCAECs, HUM-iCell-c006, iCell) were treated with 1, 2.5, 5, 10 and 20 μM QA.

Techniques: CCK-8 Assay, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Cellular signalling

Article Title: PAI-1 contributes to homocysteine-induced cellular senescence

doi: 10.1016/j.cellsig.2019.109394

Figure Lengend Snippet: Cultures of EA.hy926 endothelial cells (A) and HCAEC (B, C) were pretreated with TM5441 (10 μM) (A, B) or TM5A15 (10 μM) (C) in triplicate followed by Homocysteine (Hcy) treatment for 4–5 days. Whole cell extracts were prepared and equal amount of pooled proteins from three wells were subjected to Western blot analysis for senescence markers and regulators using specific antibodies as indicated (A–C). Bar represents mean ± sem. Quantitative data are shown on the right (A’-C’). The levels of at least 2–3 senescence markers were determined in repeat experiments. D, E. Whole cell extracts (HCAEC) were prepared from two separate experiments and equal amount of pooled proteins from three wells were subjected to Western blot analysis for senescence markers and regulators p53 and pERK1/2 (D), integrin β3 and PAI-1 (E) using specific antibodies. Quantitative data in the lower panel showing the levels of each regulator relative to loading control Actin (D’, E’).

Article Snippet: Endothelial cell culture: treatment with Hcy and small molecule inhibitors of PAI-1 Primary cultures of Human Coronary Artery Endothelial Cells (HCAEC) (Cell Applications; Cat # 300–05a) and EA.hy926 (ATCC cat #CRL-2922) were grown in MesoEndo Cell Growth Media and Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and streptomycin respectively and maintained at 37 °C in a 5% CO 2 incubator.

Techniques: Western Blot, Control