Fig 1: Changes in the expression of TNF-alpha (1./a) IL-6, (1./b) IL-8 (1./c), ET-1 (1./d), ECE (1./e), and NOS (1./f) genes in endothelial cells in a hyperglycaemia in an in vitro model system of endothelial cell model after treatment with 30 mM glucose for 24 h. Each experiment was performed in triplicate. Data are expressed as the mean ± SEM of three individual experiments. *, and *** mark significant (p < .05, .0005, respectively) differences compared with control (modified t-test).
Fig 2: VEGF-A signaling through the Shc/MAP kinase pathway in primary endothelial cells. (A) Endothelial cells were isolated from the hearts of 2-month-old mice (n = 5/genotype) and treated with (+) or without (–) VEGF-A (40 ng/mL) for 5 min. (i) Lysates were subjected to immunoprecipitation (Ip) with a-Shc antibody followed by immunoprobing with antibodies against Shc, phospho-CEACAM1 (pCC1), and phospho-VEGFR2 (pVEGFR2) to evaluate the level of these proteins in the a-Shc immunopellet. (ii–iii) Lysates were subjected to immunoblotting with a-phospho-antibodies against phosphorylated MAPK and NF-?B and in parallel gels, against MAP Kinase (MAPK), and NF-?B protein for normalization. (iv) Endothelin-1 (ET-1) was assayed in the media of isolated endothelial cells stimulated with VEGF-A for 20 min. Values were expressed as mean ± SEM. * p < 0.05 vs. no VEGF-A (solid bars)/same genotype; † p < 0.05 vs. (–) VEGF-A in all 3 controls; § p < 0.05 (+) VEGF-A (vertical lines) in all 3 controls. (B) Retro-orbital venous blood was drawn from overnight fasted mice fed HC for 3 months (n = 5/genotype) to analyze plasma (i) ET-1, (ii) PGE2, and (iii) PDGF-B. Values were expressed as mean ± SEM. * p < 0.05 vs. Ldlr-/-VECad–Cc1+/+ (a, white bar); † p < 0.05 vs. Ldlr-/-VECad+Cc1+/+ (b, grey bar); and § p < 0.05 vs. Ldlr-/-VECad–Cc1fl/fl (c, teal bar).
Fig 3: Paeonol improved the cardiac function and ameliorated the cardiac damage of CHF rats. (A–E) The parameters underlying the cardiac function of the rats, including LVSP (A), LVEDP (B), +dp/dtmax (C), –dp/dtmax (D), and HR (E), were detected using RM6240 series multichannel physiological signal acquisition apparatus. (F–K) The concentration of several specific markers related to the myocardial damage in the rats, including BNP (F), LDH (G), RE (H), Ang II (I), ALD (J), and ET-1 (K), were evaluated using a Synergy H1 Hybrid Multi-Mode Reader. ??P < 0.01, ???P < 0.001, vs. Sham; *P < 0.05, **P < 0.01, ***P < 0.001, vs. DOX. DOX, doxorubicin; LVSP, left ventricular systolic pressure; LVEDP, left ventricle end diastolic pressure; +dp/dtmax, +left ventricle dp/dtmax; –dp/dtmax, –left ventricle dp/dtmax; HR, heart rate; BNP, brain natriuretic peptide; LDH, lactate dehydrogenase; RE, renin; Ang II, angiotensin II; ALD, aldosterone; ET-1, endothelin 1.
Fig 4: Effects of liraglutide on ET-1 concentration changes in rat plasma obtained by cardiac puncture and cGMP concentration changes in rat lung tissue.Plasma ET-1 and rat lung tissue cGMP concentrations were decreased after liragltuide treatments compared to saline. Bars represent the mean ± SEM of n = 5 per group. *p < 0.05 versus controls; #p < 0.05 versus saline prevention group; +p < 0.05 versus saline treatment group. MCT = monocrotaline; cGMP = cyclicguanosine monophosphate; ET-1 = endothelin-1; Pv saline = saline prevention group; Pv 75 = liraglutide 75 µg·kg-1 prevention group; Pv 200 = liraglutide 200 µg·kg-1 prevention group; Tx saline = saline treatment group; Tx 75 = liraglutide 75 µg·kg-1 treatment group; Tx 200 = liraglutide 200 µg·kg-1 treatment group.
Fig 5: ET-1 is a downstream effector of TGF-ß1, and blockade of either Smad or ERK1/2 activities attenuates profibrotic effects of TGF-ß1. (A–C) Serum-starved adult HCFs were pretreated with LY2109761 (TGF-ß inh; 5 µM), bosentan (1 µM), or valsartan (valsar; 1 µM) for 1 h before treatment with 1 ng/mL TGF-ß1 for 6 h (A) or 24 h (B,C). (D–F) Serum-starved adult HCFs were pretreated with 1 µM Smad inhibitor (Smad inh), 1 µM FR180204 (ERK inhibitor; ERK inh), 1 µM SB203580 (p38 MAPK inhibitor; p38 inh), or Smad inh plus ERK inh for 1 h before treatment with 1 ng/mL TGF-ß1 for 6 h (D) or 24 h (E,F). (A,D) Relative mRNA levels of fibrotic markers, a-SMA and collagen I, were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). After treatment, immunofluorescence staining was used to determine a-SMA expression (green) (B,E) and stress fiber formation (red) (C,F). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. Data are expressed as the mean ± SEM (n = 3). * p < 0.05 vs. vehicle; # p < 0.05 vs. TGF-ß1.
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