Fig 1: DDAH1 mediates acute and chronic VEGF-mediated signaling pathways in ECs(A) HUVECs were infected with lentivirus expressing of control shRNA and DDAH1 shRNA, cultured in low-serum EMB for 4 h and then exposed to stimuli of acute (single stimulus of VEGF at 10 ng/mL) and chronic VEGF (three stimuli of VEGF at 10 ng/mL with 30 min intervals), respectively. Proteins were collected 10 min after VEGF stimulation and analyzed with western blotting (A).(B) Control and DDAH1 knockdown HUVECs were labeled with DAF-FM (2.5 μM) and then exposed to acute and chronic VEGF stimulation, respectively. DAF-FM fluorescence was measured at Excitation/Emission of 495/515 nm. N numbers are presented in the scatter plot. Data is expressed as mean ± SD. ∗∗∗, p < 0.001.(C) HUVECs were infected with retrovirus expressing of LacZ and DDAH1, cultured in low-serum EMB and then exposed to acute and chronic VEGF stimulation. Proteins were collected 10 min after VEGF stimulation and analyzed with western blotting.
Fig 2: Knockdown of DDAH1 reduces VEGF-mediated acute and chronic vascular hyperpermeabilityDouble transgenic zebrafish Tg(pKTol2H70-mC-hVEGF-gcG);Tg(fli:EGFP) were injected with Cre mRNA and DDAH1 or control morpholino (100 μM, 4.5 nL) at one-cell stage and then microinjected Texas Red-dextran (70 kDa) to the pericardium at 3-dpf.(A) Alignment of ISVs was analyzed.(B) Zebrafish embryos were collected and subjected to western blotting to confirm the decrease of DDAH1 protein levels.(C–F) Control and DDAH MO-injected zebrafish embryos were exposed to a single stimulus of heat shock at 37°C for 30 min (C and D) and three times of incubation at 37°C for 30 min separated by 30-min intervals at 28.5°C (F–G). Zebrafish embryos were imaged (D, G) and the vascular permeability of anterior and posterior trunk regions were quantified with our algorithm and compared (E and H). ∗∗∗, p < 0.001. Zebrafish numbers in each group are presented in the scatter plot. Data is expressed as mean ± SD. Scale bar, 200 μm, in (A, D, and G).
Fig 3: Vascular leakiness was enhanced in DDAH1 transgenic mice but reduced in DDAH1 knockout mice(A–C) Sex- and age-matched wild type (WT), DDAH1 transgenic (TG) and DDAH1 knockout (KO) mice were subjected to Miles assay. Evans blue (1%) were intravenously injected according to body weight and then intradermal injection of VEGF (50 ng) and PBS was performed. Skin was dissected and imaged (A) and Evans blue was quantified after extraction with formamide and normalized to tissue weight (B). Plasma was also collected at the endpoint to show comparable level of Evans blue in the circulation (C). ∗, p<0.05. Animal numbers in each group are presented in the scatter plot. Data is expressed as mean ± SD. Scale bar, 1 mm, in (A)
Supplier Page from Sino Biological, Inc. for Human DDAH1 Gene ORF cDNA clone in cloning vector