Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 1. Characterization of rabbit EPCs and comparison with HUVECs. A, B, Cobblestone morphology of EPCs and HUVEC 10 days after plating (40); C, D, immunohistochemical detec- tion of CD31; immunofluorescence detection of (E, F), VE-cadherin; (G, H), eNOS; (I, J), vWF. Preparations were viewed at 400 unless otherwise indicated. Preparations in G, H, I, and J were counterstained with Hoechst 33342.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Comparison, Immunohistochemical staining

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 2. Ex vivo retroviral transduction and transgene expression in EPCs. A, Confluent monolayer of EPCs trans- duced with MSCV retrovirus expressing GFP viewed under normal (white) light (100); B, view of same field under green fluorescent light; C, D, human eNOS transgene mRNA and protein expression in untransduced and trans- duced EPCs, respectively; E, F, HO-1 mRNA and protein expression in untransduced and transduced EPCs, respectively.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Ex Vivo, Retroviral, Transduction, Expressing

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 3. In vivo transgene expression after transplantation of EPCs onto injured carotid arteries. A, Immunohistochemical detection of eNOS (arrows) in frozen sections from denuded vessel trans- planted with eNOS-EPCs (250). B, eNOS immunoreactivity in sections from control vessel transplanted with GFP- EPCs (250); C, immunohistochemical detection of human HO-1 (arrows) in fro- zen sections from vessel transplanted with HO-1-EPCs (250); D, control ves- sels transplanted with GFP-EPCs (250); E, detection of human eNOS transgene mRNA expression by RT-PCR; F, human eNOS protein expression in homoge- nates from eNOS-EPC–transplanted ves- sels; G, human HO-1 transgene mRNA expression; H, HO-1 protein expression in homogenates from HO-1-EPC–trans- planted vessels. GFP-EPC–transplanted vessels served as controls.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: In Vivo, Expressing, Transplantation Assay, Immunohistochemical staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 4. Reendothelialization of injured arteries after transplan- tation of EPCs expressing GFP, eNOS, or HO-1 genes. Low- power (100) magnification of vessel profiles stained with anti- CD31 antibody in A, unseeded; B, GFP-EPCs; C, eNOS-EPCs; and D, HO-1-EPCs. Red CD31-positive staining indicates pres- ence of an endothelial monolayer. E, Quantification of endotheli- alization by planar morphometry. Endothelialization was enhanced in EPC-transplanted vessels compared with unseeded saline-treated controls (*P0.05, n4/group).

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Expressing, Staining, Saline

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 5. Inhibition of neointimal proliferation by EPCs in injured carotid arteries. All sections were stained with Accustain elastic stain. Low (40, left) and high (200, right) magnification of vessel profiles. A, B, Saline-injured artery transplanted with eNOS-EPCs (n6); C, D, balloon-injured artery transplanted with EPC expressing GFP (n6); E, F, balloon-injured artery transplanted with EPC expressing eNOS (n6); G, H, balloon- injured artery transplanted with HO-1-EPCs (n6); I, neointima/ media ratios in unseeded and EPC-transplanted injured vessels. Arrows indicate neointima. *Saline vs GFP-EPCs, eNOS-EPCs, HO-1-EPCs; †eNOS-EPCs vs GFP-EPCs, P0.05.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Inhibition, Staining, Saline, Expressing

Journal: Nutrients

Article Title: Analysis of the Combined Effects of a Novel Combination of Hypersmin, Pumpkin Seed and Amaranthus Extracts in an In Vitro Model of Chronic Venous Insufficiency.

doi: 10.3390/nu17111807

Figure Lengend Snippet: Figure 3. Vascular tone analysis on in vitro model of vein after intestinal passage. In (A) NO production; in (B) eNOS levels; in (C) endothelin-1 levels. From (A) to (C) all the data are obtained with specific ELISA kits; * p < 0.05 vs. Control; α p < 0.05 vs. single agents; β p < 0.05 vs. Commercial product; γ p < 0.05 vs. KCl.

Article Snippet: The eNOS concentration was measured following the manufacturer’s instructions for the DuoSet ELISA kit for Human eNOS (R&D Systems, Minneapolis, MN, USA) in HUVEC cells [39].

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of the American Heart Association

Article Title: Interleukin‐1β Disruption Protects Male Mice From Heart Failure With Preserved Ejection Fraction Pathogenesis

doi: 10.1161/jaha.122.029668

Figure Lengend Snippet: Figure 3. Western blot analysis and nitrate/nitrite levels. Western blot analysis and cumulative data for phosphorylated endothelial nitric-oxide synthase (P-eNOS) in mesenteric resistance arteries (MRAs) isolated from C57/BL6J (C57), C57/BL6J+high-fat diet (HFD)+N[w]-nitro-l-arginine methyl ester (L-NAME), interleukin- 1β−/− (IL-1β−/−), and IL-1β−/−+HFD+L-NAME male mice (n=5) (A) and nitrate/nitrite level in the urine (B) from all groups of mice. One-way ANOVA followed by Tukey’s multiple comparisons post hoc test was applied for (A and B). *P<0.05, ****P<0.0001 for C57BL/6J vs C57/ Bl6+HFD+L-NAME; (n=5). ns indicates not significant; and T-eNOS, total nitric oxide synthase.

Article Snippet: We used specific antibodies against endothelial nitric oxide synthase (eNOS; Cell Signaling, D9A5L rabbit monoclonal antibodies; number 32027), phospho- eNOS (Ser1177; Cell D ow nloaded from http://ahajournals.org by on January 26, 2024 Signaling [C9C3] rabbit monoclonal antibodies, 9570), iNOS (Proteintech, number 18985- 1- AP), anti- COX2 (Cayman; aa 584– 598, number 160126), NLR family pyrin domain containing 3/Nacht leucine- rich repeat protein 3 (Novus Biotech; number NBP2- 12446), X- box binding protein 1 (Abcam; number ab238456), anti– CCAAT/enhancer- binding protein (Cell Signaling; L63F7 mouse monoclonal antibodies number 2895), IRE1 (Abcam; number ab37073, lot GR3354835- 4), activating transcription factor 6 (Abcam; number ab37149, lot GR3353621- 3), and beta- actin (Santa Cruz; number SC47778).

Techniques: Western Blot, Isolation

Journal: PLoS ONE

Article Title: Jujuboside B Reduces Vascular Tension by Increasing Ca 2+ Influx and Activating Endothelial Nitric Oxide Synthase

doi: 10.1371/journal.pone.0149386

Figure Lengend Snippet: (A)Jujuboside B increased NO generation in HAECs concentration-dependently. * P < 0.05, ** P < 0.01 compared with Control, n = 3. (B) Jujuboside B increased NO generation in HAECs negative time-dependently. * P < 0.05, ** P < 0.01 compared with 15 min group, n = 3. (C) L-NAME significantly inhibited Jujuboside B-induced NO generation. * P < 0.05, ** P < 0.01, n = 3. (D) L-NAME significantly inhibited Jujuboside B-induced eNOS activation. ** P < 0.01, ## P < 0.01, n = 4. (E) Western blot analysis of eNOS and p-eNOS at Serine-1177 in HAECs showed that Jujuboside B significantly increased p-eNOS Serine-1177 expression. (F) Densitometric analysis showed that Jujuboside B significantly increased p-eNOS Serine-1177 expression. * P < 0.05 compared with Control, n = 3.

Article Snippet: The residual protein bands were blocked with 5% milk and the membrane was incubated with rabbit p-eNOS (Serine-1177) antibody and rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, USA) dilution overnight.

Techniques: Concentration Assay, Control, Activation Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Jujuboside B Reduces Vascular Tension by Increasing Ca 2+ Influx and Activating Endothelial Nitric Oxide Synthase

doi: 10.1371/journal.pone.0149386

Figure Lengend Snippet: (A)EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS activation. ** P < 0.01 compared with Control, # P < 0.05, ## P <0.01 compared with Jujuboside B, n = 4. (B) Western blot analysis showed that EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS phosphorylation at Serine-1177. (C) Densitometric analysis showed that EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS phosphorylation at Serine-1177. # P < 0.05 compared with Control, * P < 0.05 compared with Jujuboside B, n = 3.

Article Snippet: The residual protein bands were blocked with 5% milk and the membrane was incubated with rabbit p-eNOS (Serine-1177) antibody and rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, USA) dilution overnight.

Techniques: Activation Assay, Control, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: Activated ROCK/Akt/eNOS and ET-1/ERK pathways in 5-fluorouracil-induced cardiotoxicity: modulation by simvastatin

doi: 10.1038/s41598-020-71531-8

Figure Lengend Snippet: Effect of 5-FU and Sim treatments on cardiac tissue contents of ( A ) p -Akt and ( B ) p -eNOS. Values are presented as the mean of 6–7 experiments ± SEM. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post-hoc test ; as compared to normal (*), Sim (#), and 5-FU (@)-treated groups (p < 0.05). 5-FU 5-fluorouracil, p-Akt (Ser473) phosphorylated protein kinase B at Ser 473, p-eNOS (Ser1177) phosphorylated endothelial nitric oxide synthase at Ser 1177, p-ERK1/2 (Thr202/Tyr204) phosphorylated extracellular signal-regulated kinase 1/2 at Thr 202 & Tyr 204, Sim simvastatin.

Article Snippet: The corresponding ELISA kits were used to determine the cardiac contents of p -NF-κB p65 (Ser536) (Abcam, Cambridge, UK; Cat# ab176647), p -ERK1/2 (Thr202/Tyr204) (Invitrogen, CA, USA; Cat # EMS2ERKP), p -Akt (Ser473) (Elabscience, Wuhan, China; Cat # E-EL-R1135) and p -eNOS (Ser1177) (Cell Signaling Technology, MA, USA; Cat# 7980C).

Techniques:

Journal: American Journal of Translational Research

Article Title: PKCβ increases ROS levels leading to vascular endothelial injury in diabetic foot ulcers

doi:

Figure Lengend Snippet: Expression changes in PKCβ/p66shc and inflammation-related proteins in diabetic foot ulcer. A, B. Western blot analysis showed significantly increased expression levels of p-PKCβ and p-P66shc in the DFU group vs. the CON group (P<0.01). C, D. Western blot analysis showed decreased protein levels of eNOS and significantly increased expression levels of ICAM-1 and p-NF-κB in the DFU group (P<0.01). n≥3 per group, with the experiment repeated three times. Values are the means and standard errors (*P<0.05 and **P<0.01 versus CON).

Article Snippet: Antibodies against endothelial nitric oxide synthase (eNOS) (35362), ICAM-1 (4915), nuclear factor-kB (NF-κB) (49D7), p-NF-κB (108D2), and β-actin (13E5) were bought from Cell Signaling Technology (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: American Journal of Translational Research

Article Title: PKCβ increases ROS levels leading to vascular endothelial injury in diabetic foot ulcers

doi:

Figure Lengend Snippet: LY333531 inhibited changes in the activity of the PKCβ-p66shc pathway caused by high glucose in HUVECs. A, B. LY333531 rescued the increased levels of p-PKCβ and p-P66shc in HUVECs treated with glucose. C, D. LY333531 rescued the increased levels of ICAM-1 and p-NF-κB and the inhibition of eNOS in HUVECs treated with glucose. E, F. Immunofluorescence showed that LY333531 could restore the expression levels and distribution of p-P66shc and restore the eNOS level in HUVECs treated with high glucose. G, H. The mean fluorescence intensity of p-P66shc and eNOS was quantified and is presented as the means ± SDs. Each group was a mixture of cells collected three times (n = 3), and the experiment was repeated three times. Values are the means and standard errors (*P<0.05 and **P<0.01 versus CON).

Article Snippet: Antibodies against endothelial nitric oxide synthase (eNOS) (35362), ICAM-1 (4915), nuclear factor-kB (NF-κB) (49D7), p-NF-κB (108D2), and β-actin (13E5) were bought from Cell Signaling Technology (Shanghai, China).

Techniques: Activity Assay, Inhibition, Immunofluorescence, Expressing, Fluorescence

Journal: Experimental Physiology

Article Title: Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide

doi: 10.1113/ep089064

Figure Lengend Snippet: FIGURE 4 (a) Left ventricular cytosolic p-eNOS-Ser1177 expression. (b) Left ventricular cytosolic endothelial nitric oxide synthase (eNOS) expression. (c) Left ventricular cytosolic inducible nitric oxide synthase (iNOS) expression. (d) Left ventricular cytosolic neuronal nitric oxide synthase (nNOS) expression. Abbreviations: HH, acute hypobaric hypoxia group; N, normoxic group. Values are means ± SD. *P < 0.05 versus N group

Article Snippet: Blots were probed with a 1:1000 dilution of primary antibody specific for p-eNOS (rabbit monoclonal, #9570; Cell Signaling Technology, Danvers, MA, USA), eNOS (rabbit polyclonal, #sc-654; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nNOS (rabbit, amino terminus, H-299; Santa Cruz Biotechnology), iNOS (mouse monoclonal, #sc-7271; Santa Cruz Biotechnology) or βtubulin (mouse monoclonal, #ab131205; Abcam, Cambridge, United Kingdom).

Techniques: Expressing

Journal: Experimental Physiology

Article Title: Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide

doi: 10.1113/ep089064

Figure Lengend Snippet: FIGURE 6 Hypothetical main myocyte differential molecular response activated during acute hypobaric hypoxia (HH) related to age. (a) In prepubertal rats, HH increased nitric oxide (NO) production by vascular endothelial cells through their own endothelial NOS (eNOS), triggering, by negative feedback over the nucleus of the surrounding myocytes, a reduction of mRNA and nitric oxide synthase (NOS) synthesis (dashed boxes). That reduction in NO production by NOS might be compensated by xanthine oxidoreductase (XOR), which would produce NO from the nitrite–nitrate (Nx) reservoir, coming largely from the diet (Moretti et al., 2019) and possibly also from the previously oxidized vascular NO mentioned above. This maintains normal intracellular NO levels, resulting in a maintenance of mitochondrial and contractile function [left ventricular develop pressure (LVDP)] as in normoxic conditions, preventing the development of endogenous cardioprotection (LVDP dashed box). The increased heat (Ht) detected could be attributed to a major relative participation of active transport mechanisms, such as the Na+–Ca2+

Article Snippet: Blots were probed with a 1:1000 dilution of primary antibody specific for p-eNOS (rabbit monoclonal, #9570; Cell Signaling Technology, Danvers, MA, USA), eNOS (rabbit polyclonal, #sc-654; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nNOS (rabbit, amino terminus, H-299; Santa Cruz Biotechnology), iNOS (mouse monoclonal, #sc-7271; Santa Cruz Biotechnology) or βtubulin (mouse monoclonal, #ab131205; Abcam, Cambridge, United Kingdom).

Techniques: