Journal: International Journal of Molecular Sciences

Article Title: High-Content Imaging and Machine Learning Classify Phenotypical Change in Coronary Artery Endothelial Cells Caused by BPS

doi: 10.3390/ijms27073259

Figure Lengend Snippet: Representative high-content microscopy images of human coronary artery endothelial cells (HCAEC) exposed to vehicle control (CTRL) or 0.1 µM Bisphenol S (BPS) for 96 h and stained using the PhenoVue Cell Painting assay. For each condition, a representative field acquired at 40× magnification and a higher-magnification inset are shown. Rows correspond to the individual fluorescence channels: Hoechst 33342 (nuclei), PhenoVue Fluor 488 Concanavalin A (endoplasmic reticulum and intracellular membranes), PhenoVue 512 nucleic acid stain (RNA/nucleoli), PhenoVue Fluor 555 wheat germ agglutinin (plasma membrane), PhenoVue 641 mitochondrial stain (mitochondria), and the merged image. White boxes represent the part of the image used for the related inset. Scale bar: 50 µm, 40× objective.

Article Snippet: Primary human coronary artery endothelial cells (HCAEC; ATCC ® PCS-100-020TM, Innovation, VA, USA) were cultured according to the supplier’s recommendations.

Techniques: Microscopy, Control, Staining, Fluorescence, Clinical Proteomics, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice

doi: 10.1161/atvbaha.117.310082

Figure Lengend Snippet: Figure 1. PAR2 (protease-activated receptor 2) mRNA and protein are increased in the atherosclerotic prone regions of mice and humans. Normal mouse aortic arch (low-density lipoprotein receptor–deficient [Ldlr−/−] mice fed a chow diet for 24 wk) or atherosclerosis-containing aortic arches (Ldlr−/− mice fed a Western diet for 24 wk) were examined for (A) mRNA extrapolated to normal and (B) protein expression (n=5 individual samples per group). Normal or diseased human carotid arteries were isolated and examined for (C) mRNA extrapolated to normal and (D) protein expression (n=5 individual samples per group). Immunologic detection of PAR2 expression was also performed on mouse atherosclerotic aortic sinus (E, F) and human carotid atherosclerotic lesions (H, I; n=4–5 individual samples per species; ×4 magnification E and H; ×10 magnification F and I). G, J, Quantification of PAR2 staining in the lesion or media of atherosclerotic lesions. Solid white box is zoomed in area. Dashed white line is the area of the lesion. Histobars represent mean±SEM. *P<0.05 for comparisons to normal aorta/artery or lesion vs tunica media (2-tailed Student t test).

Article Snippet: Immunostaining was performed on frozen serial sections as described previously.26 Human carotid atherosclerotic sections were obtained from Origene Technologies, Inc.

Techniques: Western Blot, Expressing, Isolation, Staining

Journal: Advanced Healthcare Materials

Article Title: Nitric Oxide‐Releasing Catheters with Phenol‐Amine Catalytic Coatings for Improved Anti‐Inflammatory Performance

doi: 10.1002/adhm.202500457

Figure Lengend Snippet: a) HCASMCs viability, b) number of cells, and c) endogenous NO generation after incubation with uncoated and coated catheter segments compared to the blank group, measured using the Live/Dead assay, Hoechst staining, and DAF‐FM diacetate, respectively, at i) 48 h and ii) 72 h. Statistical significance relative to control tests was calculated using one‐way ANOVA, ns = not significant, * p < 0.1, ** p < 0.01, **** p < 0.0001. n = 6; error bars represent standard deviation.

Article Snippet: Human coronary artery smooth muscle cells (HCASMCs) and smooth muscle cell growth medium kit were purchased from Cell Applications.

Techniques: Incubation, Live Dead Assay, Staining, Control, Standard Deviation

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMP9 and BMP10 selectively activate SMAD1/5/8 signaling and induce proliferation in pulmonary artery endothelial cells but not pulmonary artery smooth muscle cells. ( A ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PAECs treated with the indicated TGF-β superfamily ligands (0.8 nM) or untreated control (UT); β-actin serves as a loading control. ( B ) PAEC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. ( C ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PASMCs treated with the indicated ligands (0.8 nM); β-actin serves as a loading control. ( D ) PASMC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. Data are shown as mean ± SD ( n = 3 replicate wells). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, BrdU Incorporation Assay

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMPR2 dosage-dependent model for BMP9/10 signaling output in pulmonary artery endothelial cells. Schematic illustrating how BMPR2 abundance constrains BMP9/10 (ALK1-dependent) canonical signaling output and downstream cellular programs in PAECs. ( A ) BMPR2-sufficient (~100%) state: BMP9/10 predominantly signal through ALK1–BMPR2 complexes, generating pSMAD1/5/8 output consistent with a threshold-like requirement for proliferation; bimagrumab (BiMab) produces no effect detected under BMPR2-replete conditions. ( B ) BMPR2-limiting (~50%) state: Reduced BMPR2 attenuates BMP9/10-induced canonical output and is associated with reduced proliferation and increased caspase-3/7 activity consistent with stress/injury. Under BMPR2-limiting conditions, residual canonical output becomes bimagrumab-sensitive, consistent with context-dependent contribution of Activin type II receptors (predominantly ACVR2A in PAECs; see for BMP10 affinity comparisons) to the remaining pSMAD1/5/8 signal. A putative non-canonical stress-signaling arm is shown as a proposed intermediate. Solid arrows denote observed relationships; dashed arrows and dashed-outline boxes denote proposed steps. Node shading and output gauges depict relative canonical signaling output.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice

doi: 10.1161/atvbaha.117.310082

Figure Lengend Snippet: Figure 1. PAR2 (protease-activated receptor 2) mRNA and protein are increased in the atherosclerotic prone regions of mice and humans. Normal mouse aortic arch (low-density lipoprotein receptor–deficient [Ldlr−/−] mice fed a chow diet for 24 wk) or atherosclerosis-containing aortic arches (Ldlr−/− mice fed a Western diet for 24 wk) were examined for (A) mRNA extrapolated to normal and (B) protein expression (n=5 individual samples per group). Normal or diseased human carotid arteries were isolated and examined for (C) mRNA extrapolated to normal and (D) protein expression (n=5 individual samples per group). Immunologic detection of PAR2 expression was also performed on mouse atherosclerotic aortic sinus (E, F) and human carotid atherosclerotic lesions (H, I; n=4–5 individual samples per species; ×4 magnification E and H; ×10 magnification F and I). G, J, Quantification of PAR2 staining in the lesion or media of atherosclerotic lesions. Solid white box is zoomed in area. Dashed white line is the area of the lesion. Histobars represent mean±SEM. *P<0.05 for comparisons to normal aorta/artery or lesion vs tunica media (2-tailed Student t test).

Article Snippet: Human carotid artery atherosclerotic samples were obtained from Origene Technologies, Inc.

Techniques: Western Blot, Expressing, Isolation, Staining

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Glutamate Exacerbates Traumatic Brain Injury‐Induced Acute Lung Injury Through NMDAR / ROS /Ca 2+ Signaling Pathway in Pulmonary Endothelial Cells

doi: 10.1002/kjm2.70087

Figure Lengend Snippet: Changes of serious lung injury after TBI‐ALI. (A) The TTC staining was performed to evaluate the brain injury volume. (B) The level of cytokines (TNF‐α, IL‐1β, and IL‐6) in peripheral blood. (C) The level of cytokines (TNF‐α, IL‐1β, and IL‐6) in BALF. (D, E) The wet/dry weight ratio and the protein concentration are detected. (F, G) Corresponding lung H&E staining and acute lung injury scores. (H) The expression of GluN1 in HPMVECs. Scale bar, 200 μm. Results represent the mean ± SEM of independent experiments of animals ( n = 8). * p < 0.05 versus Sham group; # p < 0.05 versus TBI group.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMVECs) were purchased from Cell Applications Inc. (San Diego, CA, USA; Catalog No. 300K‐05a) and cultured in RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS, Gbico, Grand Island, NY, USA) at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Staining, Protein Concentration, Expressing

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Glutamate Exacerbates Traumatic Brain Injury‐Induced Acute Lung Injury Through NMDAR / ROS /Ca 2+ Signaling Pathway in Pulmonary Endothelial Cells

doi: 10.1002/kjm2.70087

Figure Lengend Snippet: Glutamate alters NMDAR/ROS/Ca 2+ pathway. (A) Cell viability (percentage of untreated control) of HPMVECs after the treatment of glutamate. (B) Immunofluorescence images showing ROS production in HPMVECs. (C) Comparison of Ca 2+ concentration in each group. (D) The levels of p‐NFAT and p‐p65 in cytoplasm and nucleus were tested by western blot. (E) Immunofluorescence stain of p‐NFAT and p‐p65 in nucleus. Results represent the mean ± SEM of independent experiments of cells ( n = 3). * p < 0.05 versus Sham group; # p < 0.05 versus Glu group.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMVECs) were purchased from Cell Applications Inc. (San Diego, CA, USA; Catalog No. 300K‐05a) and cultured in RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS, Gbico, Grand Island, NY, USA) at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Control, Immunofluorescence, Comparison, Concentration Assay, Western Blot, Staining

Journal: Nature communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis.

doi: 10.1038/ncomms9995

Figure Lengend Snippet: Figure 1 | Increased MAP4K4 expression in atherosclerosis. (a) Eight-to-ten-week-old mice were fed chow or 60% HFD for 16 weeks, messenger RNA (mRNA) was extracted from the indicated tissues, and quantitative RT–PCR was performed for Map4k4 and normalized to 36b4. The data represent the mean±s.e.m. (*Po0.05, **Po0.005, N ¼ 3–7). (b–d) Aortas were extracted from age-matched chow-fed wild-type or Apoe / mice or WD-fed Apoe / mice. (b) Immune-complex kinase assays were performed in Map4k4 immunoprecipitates using MBP as an exogenous substrate. Lysates were immunoblotted for tubulin as a loading control. (c) Densitometric quantification of 32P MBP as normalized to tubulin. (d) Densitometric quantification of immunoprecipitated Map4k4 as normalized to tubulin (analysis of variance þP ¼ 0.05, *Po0.05, N ¼ 4–5). (e) mRNA was isolated from normal human arteries or atherosclerotic plaques, and quantitative RT–PCR was performed for MAP4K4 or GAPDH (*Po0.05, N ¼ 3–5).

Article Snippet: One additional human atherosclerotic plaque RNA sample was purchased from Origene (CR562657).

Techniques: Expressing, Quantitative RT-PCR, Immune Complex Kinase Assay, Control, Immunoprecipitation, Isolation

Journal: Nature communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis.

doi: 10.1038/ncomms9995

Figure Lengend Snippet: Figure 4 | Less macrophages in plaques from M4K4 ECKO and KD mice. (a,b) Messenger RNA was prepared from whole aortas, and qPCR was performed. (a) Macrophage markers F4/80 and Cd68. (b) Chemokines Ccl2, Cxcl1, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl9 and Cxcl10. Data represent the mean±s.e.m. as normalized to 36b4 (*Po0.05, **Po0.005, N ¼ 9–10). (c,d) Homing of GFP leukocytes into atherosclerotic lesions 48 h after intravenous injection into control or MAP4K4 KD mice that were fed WD for 16 weeks. (c) Fluorescence micrograph of atherosclerotic plaque demonstrating four GFP leukocytes within the aortic arch. The dashed line indicates the plaque border. Inset, magnification of three GFP leukocytes. Left, 4,6-diamidino-2- phenylindole; middle, GFP; right, merge. Scale bars, 100 mm. (d) Quantification of GFP leukocytes per square millimetre of plaque. Data represent the mean±s.e.m. (**Po0.005, N ¼ 4,7).

Article Snippet: One additional human atherosclerotic plaque RNA sample was purchased from Origene (CR562657).

Techniques: Injection, Control, Fluorescence