Fig 2: DDAH2 promotes tumor angiogenesis through NO production in lung adenocarcinoma. a Tumor fibroblast-derived DDAH2 increases expression of eNOS in vascular endothelial cells, and enhances NO production, followed by upregulation of the kinase cascade. This pathway stimulates endothelial cell proliferation and migration and results in angiogenesis. b Samples of normal lung (n = 1), AIS (n = 1), and invasive adenocarcinoma (n = 5) were prepared and used for Western blot analysis with antibodies against DDAH2 and eNOS. Immunohistochemistry with anti-eNOS antibody was performed using the same samples as those used for Western blotting. Vascular endothelial cells were subjected to immunohistochemistry with anti-CD31 antibody. c eNOS was expressed strongly in vascular endothelium of the invasive adenocarcinoma. d Immunohistochemistry with anti-CD31 antibody in the same section as c. e The vascular endothelial cells of normal lung were negative for eNOS. f Immunohistochemistry with anti-CD31 antibody in the same section as e

Fig 3: The phenotype maintenance of HUVECs on different Zn surfaces. (a) NO levels of HUVECs cultured on different Zn surfaces for 7 days. (b) Quantification of fluorescence intensity of NO probes. (c) The eNOS staining of HUVECs cultured on different Zn surfaces for 7 days. (d) Quantification of fluorescence intensity of eNOS staining. (e) The VWF staining of HUVECs cultured on different Zn surfaces for 7 days. (d) Quantification of fluorescence intensity of VWF staining. * indicates p < 0.05, compared between two groups. ** indicates p < 0.01, compared between two groups

Fig 4: Immunofluorescence analysis of selected endothelial cell (EC) markers in human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) after differentiation, isolation, and expansion for 2 to 3 passages on fibronectin-coated plates shows expression of (A) CD31, (B) vascular endothelial (VE)-cadherin, (C) von Willebrand factor (vWF), (D) endothelial nitric oxide synthase (eNOS), and (E) Ephrin type-B receptor 4 (EphB4). A functional assay demonstrates that hiPSC-ECs (F) form networks when cultured on Matrigel-coated plates for 24 h. Scale bars = 25 μm. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis of endothelial cells (EC) gene expression in hiPSC-ECs, relative to human umbilical vein endothelial cells (HUVECs), for (G) general EC markers CD31, vascular endothelial (VE)-cadherin and kinase insert domain receptor (KDR; pink), lymphatic marker prospero homeobox protein 1 (Prox-1; green), and (H) arterial markers Notch1, EphrinB2, and Connexin 40 (red) and venous marker chicken ovalbumin upstream promoter transcription factor 2 (Coup-TFII; blue), with asterisks indicating P < 0.05 compared to HUVECs. Western blot showing (I) strong CD31 expression (130 kD band) in isolated hiPSC-ECs compared to HUVECs with HSP90 loading control and (J) expression of intracellular adhesion molecule 1 (ICAM-1; 100 kD band) in hiPSC-ECs after treatment with tumor necrosis factor (left) and in HUVECs (right) with tubulin-loading controls, demonstrating comparable levels of induction. (Error bar indicates ± standard error of mean [SEM] and n = 3 independent experiments. Asterisks indicate P < 0.05 compared to HUVECs).

Fig 5: Effects of RDN on the signaling pathway of NO production. Representative images of Western blots of AMPK/Akt/eNOS pathway protein expression (a) and the corresponding quantitation (b-d); NO (e) and cGMP levels (f) in pig renal arteries. Data are expressed as the mean ± standard deviation. *p < 0.05, **p < 0.01 vs. sham group; n = 5 per group. Abbreviations: RDN, renal denervation; NO, nitric oxide; cGMP, cyclic guanosine monophosphate; AMPK, adenosine 5′-monophosphate (AMP)-activated protein kinase; eNOS, endothelial nitric oxide synthase; Akt, protein kinase B; GAPDH, glyceraldehyde phosphate dehydrogenase