Journal: Cells

Article Title: HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells

doi: 10.3390/cells12182231

Figure Lengend Snippet: VEGF induces rapid NICD expression through ERK1/2. ( A ) NICD expression was assessed 30 min after VEGF treatment in HPMEC-Im, as shown by immunoblotting, with densitometry shown graphically; ( B ) n = 3 independent experiments, * p < 0.05, VEGF vs. Ctrl; ( C ) mRNA expression of DLL4 , HES1 , HEY1/2 , and NRARP was quantified by qRT-PCR after transfection of empty or NICD-contained plasmid in HPMEC-Im, n = 10 independent experiments; * p < 0.05, empty plasmid vs. NICD overexpression (OE). ( D ) HPMEC-Im were treated with 50 μg/mL VEGF for 30 min with or without U0126 (ERK inhibitor) pretreatment, and DLL4, NICD, ERK, and p-ERK expression were quantified by immunoblotting, with densitometry analysis presented graphically ( E ), n = 5 independent experiments, * p < 0.05, VEGF vs. Ctrl. ( F ) DLL4 and HES1 mRNA expression was assessed by qRT-PCR 5 h after 50 μg/mL VEGF treatment. n = 9 independent experiments, * p < 0.05.

Article Snippet: Cell culture and reagents: Embryonic human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell (Carlsbad, CA, USA) and were immortalized (HPMEC-Im) with SV40 large T antigen transformation as described before by our lab [ ].

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression

Journal: Cells

Article Title: HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells

doi: 10.3390/cells12182231

Figure Lengend Snippet: VEGF induces rapid NICD expression through DLL4, ADAM17, and ɣ-secretase. NICD expression was quantified by immunoblotting 30 min after VEGF treatment in HPMEC-Im ( A ), with densitometry analysis shown graphically ( B ), n = 4 independent experiments, * p < 0.05, ns means no significance. HPMEC-Im transduced with lentivirus containing sc shRNA or DLL4 shRNA were treated with VEGF, and DLL4 , HES1 , and HEY1 mRNA expression was assessed by qRT-PCR ( C ), n = 6 independent experiments, * p < 0.05. NICD protein expression was assessed by immunoblotting after VEGF treatment with or without KP457 pretreatment ( D ). Densitometry analysis is presented graphically ( E ), n = 5 independent experiments, * p < 0.05. HPMEC-Im were treated with VEGF with or without KP457 pretreatment, and HES1 and HEY1 mRNA expression was assessed by qRT-PCR ( F ), n = 9 independent experiments, * p < 0.05. NICD protein expression was assessed by immunoblotting after VEGF treatment with or without DBZ pretreatment ( G ). Densitometry analysis is presented graphically ( H ), n = 3 independent experiments, * p < 0.05.

Article Snippet: Cell culture and reagents: Embryonic human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell (Carlsbad, CA, USA) and were immortalized (HPMEC-Im) with SV40 large T antigen transformation as described before by our lab [ ].

Techniques: Expressing, Western Blot, Transduction, shRNA, Quantitative RT-PCR

Journal: Cells

Article Title: HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells

doi: 10.3390/cells12182231

Figure Lengend Snippet: Inhibition of HDAC6 leads to increased expression of NICD but suppresses the Notch signaling pathway. HPMEC-Im transduced with lentivirus containing sc shRNA or HDAC6 shRNA. ( A ) DLL4 , HES1 , HEY1, and JAG1 mRNA expression was assessed by qRT-PCR sc shRNA vs. HDAC6 shRNA, n = 6 independent experiments, * p < 0.05. ( B ) NICD protein expression was assessed by immunoblotting, with densitometry shown graphically ( C ), n = 4 independent experiments, and * p < 0.05. ( D ) Acetylated tubulin expression and ERK phosphorylation were assessed by immunoblotting, with densitometry shown graphically ( E ), n = 3 independent experiments, and * p < 0.05. ( F ) TP1-Luc2 luciferase assay was applied to assess Notch signaling activity in sc shRNA or HDAC6 shRNA-expressed HEK293 cells, n = 5 independent experiments and * p < 0.05.

Article Snippet: Cell culture and reagents: Embryonic human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell (Carlsbad, CA, USA) and were immortalized (HPMEC-Im) with SV40 large T antigen transformation as described before by our lab [ ].

Techniques: Inhibition, Expressing, Transduction, shRNA, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Luciferase, Activity Assay

Journal: Cells

Article Title: HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells

doi: 10.3390/cells12182231

Figure Lengend Snippet: Regulation of NICD and SNW1 binding via HDAC6-mediated deacetylation. HPMEC-Im transduced with lentivirus containing sc shRNA or HDAC6 shRNA were treated with 50 ng/mL VEGF for 45 min. Immunoprecipitation (IP) with NICD antibody was performed with subsequent Western blot analysis carried out with an acetylated antibody and HDAC6 antibody on immunoprecipitated protein to quantify changes in NICD acetylation and HDAC6-NICD binding ( A ). Densitometric quantification of Western blot images in A is shown graphically ( B ), n = 5 independent experiments; * p < 0.05. Similarly, NICD acetylation and NICD-SNW1 binding were assessed after IP with NICD antibody ( C ), with the results presented graphically ( D ), n ≥ 4 independent experiments, * p < 0.05. SNW1 acetylation and NICD-SNW1 binding were measured through immunoblotting after IP with SNW1 antibody ( E ), and densitometry analysis shown graphically ( F ), n = 5 independent experiments and * p < 0.05. ( G ) SNW1 binding to RBPJ binding sites on the promoters of HES1 and HEY2 was quantified using Chromatin IP (ChIP) with SNW1 antibody. n = 4 independent experiments, * p < 0.05.

Article Snippet: Cell culture and reagents: Embryonic human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell (Carlsbad, CA, USA) and were immortalized (HPMEC-Im) with SV40 large T antigen transformation as described before by our lab [ ].

Techniques: Binding Assay, Transduction, shRNA, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

Journal: Cells

Article Title: HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells

doi: 10.3390/cells12182231

Figure Lengend Snippet: HDAC6 regulates pulmonary EC angiogenesis ( A – D ). - 3D angiogenesis was performed using a validated bead assay as described in the methods section in HPMEC-Im and treated with sc shRNA and HDAC6 shRNA ( A ). Magnified image of a representative sample from HPMEC-Im treated with sc shRNA showing an angiogenic tube with lumen ( B ). Quantification of roots, branches ( C ), and length of roots ( D ) is shown; 1-3 representative beads/well from 8 (sc shRNA) and 13 (HDAC6 shRNA) wells were evaluated for 3D angiogenesis. Data is derived from two independent experiments. (* p < 0.05). ( E – I ) Mouse lungs were inflation-fixed on P14 after three doses of HDAC6-I ip injection. CD31 immunohistochemical staining was performed ( E ) (scale bar: 50 µm) and H&E staining was performed ( G ) (scale bar: 200 µm) with mouse lung sections. Arrows in figure E show single vs. double layer capillaries in and around alveolar septum. Radial alveolar counts (RAC) ( G ) and mean linear intercepts (MLI) ( H ) graphically presented. The results were obtained from at least four mice per group, indicating statistical significance (* p < 0.05). ( I ) Western blot depicting acetylated tubulin from clarified mouse whole lung lysates.

Article Snippet: Cell culture and reagents: Embryonic human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell (Carlsbad, CA, USA) and were immortalized (HPMEC-Im) with SV40 large T antigen transformation as described before by our lab [ ].

Techniques: shRNA, Derivative Assay, Injection, Immunohistochemical staining, Staining, Western Blot

Journal: Molecules

Article Title: Iloprost Attenuates Oxidative Stress-Dependent Activation of Collagen Synthesis Induced by Sera from Scleroderma Patients in Human Pulmonary Microvascular Endothelial Cells

doi: 10.3390/molecules26164729

Figure Lengend Snippet: Figure 1. (A,B) Effect of SSc and SSc iloprost-treated sera on human pulmonary microvascular endothelial cells (HPMECs) intracellular ROS levels. Before sera treatment, subconfluent HPMECs were loaded with 10 µM of H2-DCFDA, and then cultured in basal medium containing 10% (v/v) of sera from SSc patients (SSc), sera from iloprost-treated SSc patients (SSc + I),

Article Snippet: In this study, human pulmonary microvascular endothelial cells (HPMECs) isolated from the human lung of healthy donors were used (Innoprot, Bizkaia, Spain).

Techniques: Cell Culture

Journal: Respiratory Research

Article Title: The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS

doi: 10.1186/s12931-026-03928-4

Figure Lengend Snippet: A A schematic setup is shown for stimulation with the Tie2 agonist PMC-403 and its effects on HUVECs and HPMECs, accordingly. Created in BioRender. Stahl, K. (2026) https://BioRender.com/yc7s3fj . B HUVECs were stimulated with different concentrations of the Tie2 agonist PMC-403 or 500 ng/ml Angpt-1 for 1 hour. A western blot was performed and pAKT and AKT were analyzed. C HPMECs were incubated with 1 µg/ml PMC-403 for 1 hour and then stimulated with LPS. TER was measured by ECIS over time and group differences over time were analyzed by a linear mixed-effects model accordingly. D Group differences are shown 6.5 hours after LPS stimulation. Results are shown as dot plots with mean ± SD and each group consisted of n =4. P -values < 0.05 are considered significant. Abbreviations: Angpt-1 - Angiopoietin-1, ECIS - Electric cell-substrate impedance sensing, HPMECs - human pulmonary microvascular endothelial cells, HUVECs – human umbilical vein endothelial cells, LPS - lipopolysaccharide, SD – standard deviation, TER – transendothelial electrical resistance

Article Snippet: As an additional endothelial cell type, primary human pulmonary microvascular endothelial cells (HPMECs; PromoCell, Heidelberg, Germany; Cat. No. C-12281) were used and cultured in endothelial cell growth medium MV2 (PromoCell; Cat. No. C-22022).

Techniques: Western Blot, Incubation, Electric Cell-substrate Impedance Sensing, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Lycium barbarum polysaccharide reduces hyperoxic acute lung injury in mice through Nrf2 pathway.

doi: 10.1016/j.biopha.2018.12.073

Figure Lengend Snippet: Fig. 4. Effects of Lycium barbarum polysaccharide (LBP) on cell viability (A) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels in pulmonary microvascular endothelial cells. Con, control; VEH, vehicle; siRNA, AMPKα1-specific siRNA. Values are shown as means ± s.e.m. *P < 0.05.

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were isolated from the lung of WT BALB/c mice using an anti–CD31 antibody (Boster, Wuhan, China) [26].

Techniques: Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Differential sex-specific expression on miR-30a in vitro. A and B: miR-30a-3p and miR-30a-5p expression in human pulmonary microvascular endothelial cells (HPMECs) exposed to room air (RA) (RA-5% CO2) and 72 h of hyperoxia (95% O2-5% CO2) (n = 3/group). Values are means ± SE. Significant differences between RA and hyperoxia within each sex are indicated by ***P < 0.001. Significant differences between male and female mice in normoxia or hyperoxia are indicated by #P < 0.05 and ##P < 0.01.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Expressing, In Vitro

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Delta-like ligand 4 (Dll4) expression in male and female human neonatal pulmonary microvascular endothelial cells upon exposure to hyperoxia in vitro: DLL4 mRNA (A) and protein (B) expression in male and female neonatal human pulmonary microvascular endothelial cells (HPMECs) exposed to room air (RA) (RA-5% CO2) and 72 h of hyperoxia (95% O2-5% CO2) (n = 3/group). Values are means ± SE. Significant differences between RA and hyperoxia within each sex are indicated by *P < 0.05 and **P < 0.01. Significant differences between male and female mice in normoxia or hyperoxia are indicated by ##P < 0.01. NS, not significant.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Expressing, In Vitro

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Delta-like ligand 4 (Dll4) is an miR-30a-5p target in neonatal human pulmonary microvascular endothelial cells. TargetScan reported miR-30a binding sites 59–66bp in the 3′UTR of DLL4 (A). Increased mir30a-5p expression after mir30a-5p mimic transfection in HPMECs (B). DLL4 mRNA (C) and protein (D) expression after mir30a-5p mimic transfection in HPMECs. DLL4 mRNA (E) and protein (F) expression after mir30a-5p inhibitor transfection in HPMECs. DLL4 protein expression (G) after mir30a-5p mimic transfection with and without target protector in HPMECs. Values are means ± SE from 3 independent experiments (n = 3/group). Significant differences between indicated groups are indicated by *P < 0.05 and **P < 0.01. HPMEC, human pulmonary microvascular endothelial cell; UTR, untranslated region; NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Binding Assay, Expressing, Transfection, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Effect of miR30a-3p overexpression and inhibition on DLL4 expression in HPMECs. Increased mir30a-3p expression after mir30a-3p mimic transfection in HPMECs (A). DLL4 mRNA (B) and protein (C) expression after mir30a-3p mimic transfection in HPMECs. DLL4 mRNA (D) and protein (E) expression after mir30a-3p inhibitor transfection in HPMECs. Values are means ± SE from three independent experiments (n = 3/group). Significant differences between indicated groups are indicated by *P < 0.05 and **P < 0.01. DLL4, delta-like ligand 4; HPMEC, human pulmonary microvascular endothelial cell. NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Over Expression, Inhibition, Expressing, Transfection, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: miR30a-5p inhibition under hyperoxic conditions decreases increases Dll4 expression in female HPMECs. DLL4 mRNA (A) and protein (B) expression after inhibition of mir30a-5p in HPMECs upon exposure to hyperoxia. Values are means ± SE from three independent experiments (n = 3/group). Significant differences between indicated groups are indicated by **P < 0.01. DLL4, delta-like ligand 4; HPMEC, human pulmonary microvascular endothelial cell. NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Inhibition, Expressing, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: miR30a-5p mimic increases sprouting angiogenesis in female HPMECs. Angiogenic potential was quantified based on the maximum length of sprouts protruding from HPMEC-coated cytodex-3 microcarrier beads suspended in a fibrin gel (n = 3/group). A: representative images from male and female HPMECs subjected to sprouting angiogenesis assay treated with miR30a-5p mimic or negative control. Scale bar represents 200 μm. B: maximum sprouting distance in male and female HPMECs. Values are means ± SE. Significant differences between treatment and control groups is indicated by *P < 0.05. HPMEC, human pulmonary microvascular endothelial cell.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Angiogenesis Assay, Negative Control, Control

Journal: International Journal of Molecular Sciences

Article Title: Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation

doi: 10.3390/ijms24044087

Figure Lengend Snippet: Effects of Ca V channel inhibition with Nifedipine and the selective BK channel opener NS1619 on inflammatory cytokine secretion: ( A , B ). The inhibition of L-type Ca V channels with Nifedipine (10 µM; 24 h) decreases the baseline and TNF-α-induced (5 ng/mL; 24 h) CCL-2 and IL-6 secretion from HULEC5a cells ( n = 5–20, * p ≤ 0.05), shown by ELISA. ( C , D ) Fluorescence intensity (IF; A.U. = arbitrary units) measured by fluorometric FLIPR assays depicts Em changes in HULEC5a cells: ( C ) A representative recording, showing NS1619-induced (30 µM; 5 min) Em hyperpolarization (decrease in IF), which persists even after TNF-α treatment (5 ng/mL; 5 min). TNF-α itself did not alter the Em. As an internal control, we induced Em depolarization with 45 mM K 2 SO 4 . ( D ) Summary of n = 6 individual FLIPR experiments; * p ≤ 0.05. ( E , F ) Em hyperpolarization with NS1619 (30 µM; 24 h) decreases CCL-2 secretion from human pulmonary microvascular endothelial (HULEC5a) and primary human pulmonary artery endothelial (HPAEC) cells when stimulated with 5 ng/mL or 25 ng/mL of TNF-α, respectively, shown by ELISA. ( G ) Em hyperpolarization with NS1619 (30 µM; 24 h) does not change TNF-α-induced (5 ng/mL; 24 h) IL-6 secretion from human pulmonary endothelial cells (HULEC5a), shown by ELISA. Baseline CCL-2 and IL-6 secretion are not affected by NS1619 treatment ( n = 4–11, * p ≤ 0.05). n = number of separate cell passages, which are considered biological replicates.

Article Snippet: Human pulmonary microvascular endothelial cells (HULEC5a; ATCC-CRL-3244) were purchased from ATCC (Manassas, VA, USA) and cultured in an MCDB131 medium (Calsson Labs, Smithfield, UT, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA), 1% Penicillin/Streptomycin (Gibco, Waltham, MA, USA), 10 mM L-Glutamine (Gibco, Waltham, MA, USA), 10 ng/mL Epidermal Growth Factor (GenScript, Piscataway, NJ, USA), and 1 μg/mL hydrocortisone (Alfa Aesar, Waltham, MA, USA).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Fluorescence, Control