Journal: Chinese Medicine

Article Title: Cinnamic acid alleviates endothelial dysfunction and oxidative stress by targeting PPARδ in obesity and diabetes

doi: 10.1186/s13020-025-01064-7

Figure Lengend Snippet: The impact of cinnamic acid on endothelial nitric oxide synthase (eNOS) and antioxidant pathways in mouse aortas. Representative bands and densitometry of the protein levels of a PPARδ (52 kDa), b Nrf2 (110 kDa), and c HO-1 (33 kDa) relative to GAPDH (36 kDa), d phosphorylated (p)-AMPKα (at Thr172) relative to total AMPKα (62 kDa), e p-Akt (at Ser473) relative to total Akt (60 kDa), and f p-eNOS (at Ser1177) relative to total eNOS (140 kDa). # P < 0.05 vs. control (CON) group. * P < 0.05 vs. diet-induced obese (DIO) group. Data were shown as mean ± SEM (n = 6–8)

Article Snippet: Antibodies against phosphorylated AMP-activated protein kinase α (AMPKα) (Thr172), total AMPKα, phosphorylated eNOS (Ser1177) were obtained from Proteintech Group (USA).

Techniques: Control

Journal: Chinese Medicine

Article Title: Cinnamic acid alleviates endothelial dysfunction and oxidative stress by targeting PPARδ in obesity and diabetes

doi: 10.1186/s13020-025-01064-7

Figure Lengend Snippet: The impact of cinnamic acid on endothelial nitric oxide synthase (eNOS) and antioxidant pathways in rat aortic endothelial cells (RAECs). Representative bands and densitometry of the protein levels of a PPARδ (52 kDa), b Nrf2 (110 kDa), and c HO-1 (33 kDa) relative to GAPDH (36 kDa), d phosphorylated (p)-AMPKα (at Thr172) relative to total AMPKα (62 kDa), e p-Akt (at Ser473) relative to total Akt (60 kDa), and f p-eNOS (at Ser1177) relative to total eNOS (140 kDa) in high glucose (HG, 44.4 mM)-induced RAECs. # P < 0.05 vs. normal glucose (NG) group. * P < 0.05 vs. HG group. Data were shown as mean ± SEM (n = 4–6)

Article Snippet: Antibodies against phosphorylated AMP-activated protein kinase α (AMPKα) (Thr172), total AMPKα, phosphorylated eNOS (Ser1177) were obtained from Proteintech Group (USA).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease

doi: 10.1161/JAHA.118.010816

Figure Lengend Snippet: Representative images of the freshly isolated arterial endothelial cell. Red—von Willebrand Factor; Green—total eNOS ( A ), p‐ eNOS ( B ), VCAM ‐1 ( C ), vascular endothelial‐cadherin ( D ); Blue— DAPI . DAPI indicates 4′,6‐diamidino‐2‐phenylindole stain; eNOS , endogenous nitric oxide synthase; p‐ eNOS , phosphorylated‐ eNOS at Ser1177; VCAM ‐1, vascular cell adhesion molecule‐1; VE ‐cadherin, vascular endothelial cadherin; vWF , von Willebrand Factor.

Article Snippet: The slides were incubated overnight at 4°C with primary antibodies against the following targets: phosphorylated eNOS at ser1177 (p‐eNOS) (1:200 dilution; GeneTex, Inc, Irvine, CA), total eNOS (1:100 dilution; Millipore, MA), vascular cell adhesion molecule‐1 (1:200 dilution; Abcam, Cambridge, MA), vascular endothelial cadherin (1:50; Santa‐Cruz, Dallas, TX).

Techniques: Isolation, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease

doi: 10.1161/JAHA.118.010816

Figure Lengend Snippet: Serum‐stimulation for a positive control. Freshly isolated endothelial cells harvested from a patient was incubated with serum‐free medium as control ( A ) or serum obtained from the patient ( B ) for 30 minutes. The intensity of p‐ eNOS (arbritrary unit) was strongly increased by thrombin in the serum (control: 33±20, serum 125±80, n=18; P <0.001) ( C ). The % increase of serum‐stimulation was 331±267, which was much higher than insulin, VEGF , acetylcholine ( D ). The images of low magnification (control: E ; serum‐stimulation: F ) revealed that the intensity of p‐ eNOS was enhanced in only the vWF positive cells. The white arrows indicate the endothelial cells. DAPI indicates 4′,6‐diamidino‐2‐phenylindole stain; p‐ eNOS , phosphorylated‐endogenous nitric oxide synthase at Ser1177; VEGF , vascular endothelial growth factor; vWF , von Willebrand Factor.

Article Snippet: The slides were incubated overnight at 4°C with primary antibodies against the following targets: phosphorylated eNOS at ser1177 (p‐eNOS) (1:200 dilution; GeneTex, Inc, Irvine, CA), total eNOS (1:100 dilution; Millipore, MA), vascular cell adhesion molecule‐1 (1:200 dilution; Abcam, Cambridge, MA), vascular endothelial cadherin (1:50; Santa‐Cruz, Dallas, TX).

Techniques: Positive Control, Isolation, Incubation, Control, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease

doi: 10.1161/JAHA.118.010816

Figure Lengend Snippet: Correlations of insulin‐induced increase of p‐ eNOS Ser1177 with confounding factors. Freshly isolated arterial endothelial cells were cultured in medium for 30 minutes before fixing with or without insulin (100 nmol/L, 30 minutes). The percentage increase in p‐ eNOS by insulin from the basal p‐ eNOS correlated with Log 10 BNP ( A ), E/E′ ( B ), d‐ ROM s ( C ) and eGFR ( D ), but did not correlate with HbA1C ( E ), fasting glucose level ( F ), fasting insulin level ( G ), HOMA ‐ IR ( H ). d‐ ROM s indicates derivatives of the reactive oxidative metabolites; E/E′, index of LV diastolic dysfunction; eGFR , estimated glomerular filtration rate; HbA1C, hemoglobin A1C; HOMA ‐ IR , homeostatic model assessment of insulin resistance; Log 10 BNP , log‐transformed brain natriuretic peptide (pg/mL); p‐ eNOS , phosphorylated‐endogenous nitric oxide synthase at Ser1177.

Article Snippet: The slides were incubated overnight at 4°C with primary antibodies against the following targets: phosphorylated eNOS at ser1177 (p‐eNOS) (1:200 dilution; GeneTex, Inc, Irvine, CA), total eNOS (1:100 dilution; Millipore, MA), vascular cell adhesion molecule‐1 (1:200 dilution; Abcam, Cambridge, MA), vascular endothelial cadherin (1:50; Santa‐Cruz, Dallas, TX).

Techniques: Isolation, Cell Culture, Filtration, Transformation Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease

doi: 10.1161/JAHA.118.010816

Figure Lengend Snippet: Correlations of VEGF , acetylcholine‐stimulated increase of p‐ eNOS Ser1177 with confounding factors. Freshly acetylcholine arterial endothelial cells were cultured in medium with or without VEGF (20 ng/mL, 30 minutes), or acetylcholine (1 μmol/L, 15 minutes). The percentage increase in p‐ eNOS by VEGF from the basal p‐ eNOS correlated with d‐ ROM s ( A ), AST ( B ) and ALT ( C ). The percentage increase of acetylcholine ‐induced p‐ eNOS correlated with eGFR ( D ). ALT indicates alanine aminotransferase; AST , aspartate aminotransferase; d‐ ROM s, derivatives of the reactive oxidative metabolites; eGFR , estimated glomerular filtration rate; p‐ eNOS , phospho‐endogenous nitric oxide synthase at Ser1177; VEGF , vascular endothelial growth factor.

Article Snippet: The slides were incubated overnight at 4°C with primary antibodies against the following targets: phosphorylated eNOS at ser1177 (p‐eNOS) (1:200 dilution; GeneTex, Inc, Irvine, CA), total eNOS (1:100 dilution; Millipore, MA), vascular cell adhesion molecule‐1 (1:200 dilution; Abcam, Cambridge, MA), vascular endothelial cadherin (1:50; Santa‐Cruz, Dallas, TX).

Techniques: Cell Culture, Filtration

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease

doi: 10.1161/JAHA.118.010816

Figure Lengend Snippet: Images of p‐ eNOS Ser1177 in patients with low and high CAVI scores. A , The images are from a 57‐year‐old non‐ CAD man who had a history of smoking and obesity only (bone mass index: 27.1). CAVI was 7.77. Among laboratory data, BNP (25.4 pg/ mL ) and eGFR (97.4 mL/min) were within normal range. The basal p‐ eNOS intensity was at low level ( CONT ). The intensity was increased by stimulation with insulin (+51%), VEGF (+45%), and acetylcholine (+79%). B , The images from a 75‐year‐old man with severe CAD and low cardiac function (ejection fraction 20%). He had diabetes mellitus, hypertension, hyperlipidemia, and was a current smoker. BNP was 1981.7 pg/ mL (Log 10 BNP : 3.30); however, renal function was within normal range ( eGFR 69.8 mL/min). CAVI score was 10.11. The basal p‐ eNOS was already enhanced ( CONT ), and the responses to stimuli were reduced. These resulted in decreases in the percentage changes induced by insulin (−1.6%) and VEGF (−27%). However, the acetylcholine‐induced p‐ eNOS intensity was not reduced (+11%). Red—von Willebrand Factor; Green—p‐ eNOS ; Blue— DAPI . BMI indicates body mass index; BNP , brain natriuretic peptide (pg/ mL ); CAD , coronary artery disease; CAVI , cardio‐ankle vascular index; CONT , control; DAPI indicates 4′,6‐diamidino‐2‐phenylindole stain; eGFR, estimated glomerular filtration rate; p‐ eNOS , phosphorylated endogenous nitric oxide synthase at Ser1177; VEGF , vascular endothelial growth factor; vWF , von Willebrand Factor.

Article Snippet: The slides were incubated overnight at 4°C with primary antibodies against the following targets: phosphorylated eNOS at ser1177 (p‐eNOS) (1:200 dilution; GeneTex, Inc, Irvine, CA), total eNOS (1:100 dilution; Millipore, MA), vascular cell adhesion molecule‐1 (1:200 dilution; Abcam, Cambridge, MA), vascular endothelial cadherin (1:50; Santa‐Cruz, Dallas, TX).

Techniques: Control, Staining, Filtration

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial Insulin Resistance of Freshly Isolated Arterial Endothelial Cells From Radial Sheaths in Patients With Suspected Coronary Artery Disease

doi: 10.1161/JAHA.118.010816

Figure Lengend Snippet: Correlations between p‐ eNOS Ser1177 and CAVI . CAVI score correlated with percentage change of insulin‐induced p‐ eNOS at Ser1177. CAVI indicates cardio‐ankle vascular index.

Article Snippet: The slides were incubated overnight at 4°C with primary antibodies against the following targets: phosphorylated eNOS at ser1177 (p‐eNOS) (1:200 dilution; GeneTex, Inc, Irvine, CA), total eNOS (1:100 dilution; Millipore, MA), vascular cell adhesion molecule‐1 (1:200 dilution; Abcam, Cambridge, MA), vascular endothelial cadherin (1:50; Santa‐Cruz, Dallas, TX).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Soy Isoflavone Protects Myocardial Ischemia/Reperfusion Injury through Increasing Endothelial Nitric Oxide Synthase and Decreasing Oxidative Stress in Ovariectomized Rats

doi: 10.1155/2016/5057405

Figure Lengend Snippet: SI increased myocardial PI3K/Akt/eNOS pathway during I/R in ovariectomized rats. Expression of p85 α (a), phosphorylation of Akt (Ser473) (b), and eNOS (Ser1177) (c) with western blotting after 24 h perfusion. Plots represent the mean ± SEM, n = 5-6. Statistical significance: ∗∗ P < 0.01, compared with OVS; # P < 0.05, ## P < 0.01 compared with CMC.

Article Snippet: After blocking at room temperature in TBS containing 0.2% Tween 20 (TBS-T) and 5% nonfat dry milk for 2 h, the membrane was incubated with P85 α (rabbit monoclonal, 1 : 1000, Cell Signaling Technology Inc., MA, USA), anti-phosphorylation-Akt (Ser473) (rabbit polyclonal, 1 : 2000, Cell Signaling Technology Inc. MA, USA), anti-Akt (rabbit polyclonal, 1 : 1000, Cell Signaling Technology Inc., MA, USA), anti-phosphorylation -eNOS (Ser1177) (rabbit polyclonal, 1 : 5000, Cell Signaling Technology Inc., MA, USA), anti-eNOS (mouse polyclonal, 1 : 2000, BD Biotechnology Inc., USA), anti-iNOS (rabbit polyclonal, 1 : 5000, Bioworld Technology Inc., MD, USA), anti-I κ B α (rabbit polyclonal, 1 : 5000, Stanza Cruz Biotechnology Inc., CA, USA), β -actin (mouse monoclonal, 1 : 4000, Sigma-Aldrich, MO, USA), or GAPDH (mouse monoclonal, 1 : 6000, Kang Cheng, China) in blocking buffer at 4°C overnight.

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Soy Isoflavone Protects Myocardial Ischemia/Reperfusion Injury through Increasing Endothelial Nitric Oxide Synthase and Decreasing Oxidative Stress in Ovariectomized Rats

doi: 10.1155/2016/5057405

Figure Lengend Snippet: SI improved endothelium-dependent vasorelaxation, decreased oxidative stress and iNOS expression, and increased I κ B α expression and PI3K/Akt/eNOS phosphyration in thoracic aorta in ovariectomized rats. (a and b) Endothelium-dependent vasorelaxation to acetylcholine of precontracted aortic sections was assessed. (c) Dihydroethidium (DHE) fluorescence staining of thoracic aorta. ((d)–(h)) Expression of iNOS (d), I κ B α (e), and p85 α (f), phosphorylation of Akt (Ser473) (g) and eNOS (Ser1177) (h) in thoracic aorta with western blotting after 24 h perfusion. Plots represent the mean ± SEM, n = 5–7. Statistical significance: ∗ P < 0.05, ∗∗ P < 0.01 compared with OVS; # P < 0.05, ## P < 0.01 compared with CMC.

Article Snippet: After blocking at room temperature in TBS containing 0.2% Tween 20 (TBS-T) and 5% nonfat dry milk for 2 h, the membrane was incubated with P85 α (rabbit monoclonal, 1 : 1000, Cell Signaling Technology Inc., MA, USA), anti-phosphorylation-Akt (Ser473) (rabbit polyclonal, 1 : 2000, Cell Signaling Technology Inc. MA, USA), anti-Akt (rabbit polyclonal, 1 : 1000, Cell Signaling Technology Inc., MA, USA), anti-phosphorylation -eNOS (Ser1177) (rabbit polyclonal, 1 : 5000, Cell Signaling Technology Inc., MA, USA), anti-eNOS (mouse polyclonal, 1 : 2000, BD Biotechnology Inc., USA), anti-iNOS (rabbit polyclonal, 1 : 5000, Bioworld Technology Inc., MD, USA), anti-I κ B α (rabbit polyclonal, 1 : 5000, Stanza Cruz Biotechnology Inc., CA, USA), β -actin (mouse monoclonal, 1 : 4000, Sigma-Aldrich, MO, USA), or GAPDH (mouse monoclonal, 1 : 6000, Kang Cheng, China) in blocking buffer at 4°C overnight.

Techniques: Expressing, Fluorescence, Staining, Phospho-proteomics, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cysteine-Rich Whey Protein Isolate (CR-WPI) Ameliorates Erectile Dysfunction by Diminishing Oxidative Stress via DDAH/ADMA/NOS Pathway

doi: 10.1155/2022/8151917

Figure Lengend Snippet: Changes of the DDAH/NOS pathway and apoptosis-related proteins in penile tissues after 100 mg kg −1 of and 300 mg kg −1 CR-WPI supplementation for 60 days. (a) Representative immunofluorescence staining of the eNOS-positive penile specimen (red) in the sham, DM, 100 mg kg −1 CR-WPI, and 300 mg kg −1 CR-WPI groups for 60 days. (b) The effects of CR-WPI on the protein expression of DDAH, NOS, Caspase 3, and Caspase 9 in diabetic rats. β -Actin was used as a loading control. ∗ P < 0.01 and ∗∗ P < 0.01 indicate a significant difference compared with the DM group. # P < 0.05 and ## P < 0.01 indicate a significant difference compared with the sham group.

Article Snippet: The membrane was blocked with 5% skim milk for 1 h and subsequently incubated at 4°C overnight with primary antibodies against β -actin (1 : 1000; sc1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA), α -SMA (1 : 1000; AF1032, Affinity Biosciences), eNOS (1 : 1000; AF0096, Affinity Biosciences), phosphorylated eNOS (Ser 1177) (p-eNOS; 1 : 500; sc81510, Santa Cruz Biotechnology, Santa Cruz, CA, USA), nNOS (1 : 600, SC5302, Santa Cruz Biotechnology, Santa Cruz, CA, USA), DDAH1 (1 : 1000; ab2231, Abcam, Cambridge, UK), DDAH2 (1 : 500; ab184166, Abcam, Cambridge, UK), Caspase 3 (D3R6Y) (1 : 1000; CST14220, Cell Signaling Technology), and Caspase 9 (1 : 2000; ab202068, Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Expressing, Control

Journal: Neurochemical Research

Article Title: Prostaglandin E2 EP4 Receptor Activation Attenuates Neuroinflammation and Early Brain Injury Induced by Subarachnoid Hemorrhage in Rats

doi: 10.1007/s11064-016-2168-6

Figure Lengend Snippet: Effects of EP4 receptor on the expression of p-eNOS. Western blot analysis showed AE1-329 increased the expression of Ser1177 p-eNOS 24 h after SAH and AE3-208 showed opposite effects ( a, b ). Data were expressed as mean ± SEM (n = 8, ### p < 0.001 vs. Sham, *p < 0.05 vs. SAH treated with vehicle, **p < 0.01 vs. SAH treated with vehicle)

Article Snippet: Samples were supplemented with 2% beta-mercaptoethanol and 50 mM DTT and boiled for about 5 min. Proteins were separated on tris-glycine 4–15% acrylamide gels and transferred to PVDF membranes soaked in 5% nonfat milk in PBS-Tween 20 (0.05%) for 2 h. The proteins of total caspase-3, cleaved caspase-3, Ser1177 phosphorylated endothelial nitric oxide synthase (Ser1177 p-eNOS) and β-actin were tested by rabbit monoclonal antibody against caspase-3 (Santa Cruz, 1:500), rabbit polyclonal antibody against cleaved caspase-3 (Biorbyt, 1:500), rabbit polyclonal antibody against Ser1177 p-eNOS (Invitrogen, 1:400) and rabbit polyclonal antibody against β-actin (Santa Cruz, 1:500) respectively.

Techniques: Expressing, Western Blot

Journal: JVS-Vascular Science

Article Title: A mouse model of stenosis distal to an arteriovenous fistula recapitulates human central venous stenosis

doi: 10.1016/j.jvssci.2020.07.003

Figure Lengend Snippet: Expression of mechanosensitive proteins in the mouse inferior vena cava (IVC). A, Representative immunofluorescence photomicrographs of IVC sections at day 21 with anti-von Willebrand factor ( vWF ), anti-Krüppel-like factor 2 ( Klf2 ) (upper panels), and anti-phosphorylated endothelial nitric oxide synthase ( p-eNOS ) (lower panels) antibodies in arteriovenous fistula ( AVF ) vs AVF with stenosis, upstream of stenosis. Scale bar = 20 μm. B, Bar graphs showing quantification of Klf2 (top) and p-eNOS (bottom) positive signal in the endothelium, upstream; Klf2 vWF: P = .6102 ( t -test), p-eNOS vWF: P = .3843 ( t -test); n = 4. C, Representative immunofluorescence photomicrographs of IVC sections at day 21 with anti-vWF, anti-Klf2 (upper panels) and anti-p-eNOS (lower panels) antibodies in AVF vs AVF with stenosis, downstream of stenosis. Scale bar = 20 μm. D, Bar graphs showing quantification of Klf2 (top) and p-eNOS (bottom) positive signal in the endothelium, downstream; vWF Klf2: P = .2243 ( t -test), vWF p-eNOS: P = .0905 ( t -test); n = 4. L , Lumen.

Article Snippet: Primary antibodies used were anti-vWF (1:400, ab11713; Abcam, Cambridge, Mass), anti-Krüppel-like factor 2 (Klf2, 1:100, LS-B5627, LifeSpan Biosciences, Seattle, Wash), anti-phosphorylated endothelial nitric oxide synthase (p-eNOS, 1:50, 9570; Cell Signaling Technology, Danvers, Mass), anti-α-smooth muscle actin (1:500, 14-9760-82; eBioscience, San Diego, Calif), anti-CD68 (1:100, MCA1957, Bio-Rad, Hercules, Calif), and anti-CD3 (1:20, MAB4841, R&D, Minneapolis, Minn).

Techniques: Expressing, Immunofluorescence