LCN7 Promotes Angiogenesis Figure 2. LCN7 increases endothelial cell angiogenic activities. A. Control (PURO) and LCN7-overexpressing endothelial cells cultured onto Matrigel coated trans-wells were induced to invade towards a 2% FBS gradient for 48 hours. Invading cells were stained with crystal violet and quantified by densitometry. Data is reported as average fold increase +/2 SE of four independent experiments. B. Quiescent monolayers of control and LCN7-overexpressing MB114 endothelial cells were overlayed with a mixture of rat tail collagen and EGM2 media to induce angiogenic cell sprouting. Data was collected by manually counting the number of sprouting cells in 10 high power fields and is presented as the average fold increase +/2 SE of three independent experiments. C. Control and LCN7-overexpressing cells seeded into tissue culture plates and cell proliferation was monitored daily for three sequential days with WST1. Data is represented as the fold increase +/2 SE compared to each cell lines first measurement. For all panels, * indicates p,.05, Students t-test. doi:10.1371/journal.pone.0013905.g002 LCN7 promotes TGF-b signaling Endothelial cell activation by growth factor stimulation is an early and critical aspect of angiogenesis activation. To begin dissecting the molecular mechanism by which LCN7 might promote angiogenesis, we first examined the activation ERK1/2 MAPK in LCN7-overexpressing cells and control cells after stimulation with various pro-angiogenic growth factors including VEGF, bFGF, and TGF-b. As shown in Figure 3, LCN7 overexpression significantly increased TGF-b induced phosphorylation of ERK1/2 MAPK. However, LCN7 did not have a significant effect on VEGF or bFGF induced ERK1/2 phosphorylation (data not shown). The precise mechanism by which TGF-b regulates angiogenesis remains the subject of much debate, however, it is generally accepted that TGF- b can have both pro- and anti-angiogenic activities and that these activities are mediated by differential activation of the ALK5- SMAD2/3 and ALK1-SMAD1/5/8 signal transduction pathways [18]. Therefore, we monitored TGF-b mediated phosphorylation of SMAD2 and SMAD5 in the presence or absence of LCN7 to further investigate the coupling of LCN7 and TGF-b in endothelial cells. As shown in Figure 3, LCN7 overexpression coupled TGF-b to the phosphorylation of SMAD2 but not to the phosphorylation of SMAD5. These results suggested that LCN7 may promote angiogenesis by enhancing endothelial cell sensitivity to TGF-b mediated ERK1/2 and SMAD2/3 signaling pathways. To determine if enhanced TGF-b sensitivity in LCN7 overexpressing cells was functionally related to the pro-angiogenic activity of LCN7, we performed invasion and angiogenic cell sprouting assays in the presence of TGF-b or the TGF-b inhibitor SB431542. As shown in Figure 3C and 3D, the addition of TGF-b (5 ng/ml) to invading or sprouting cultures of control or LCN7 overexpressing MB114 cells provoked an increase in cellular invasion and sprouting. Consistent with our previous results, LCN7 overexpressing cells were more sensitive than control cells to the pro-invasive and pro-sprouting impact of TGF-b. Importantly, blockade of TGF-b signaling by 10 mM SB431542 reduced the enhanced invasion and sprouting activity of LCN7 overexpressing cells down to control levels. These results provided strong evidence that LCN7 promotes angiogenesis at least in part by sensitizing cells to the pro-angiogenic activity of TGF-b. Recombinant LCN7 mediates endothelial cell adhesion, invasion, and sprouting It was important to confirm that the pro-angiogenic effects of LCN7 were not dependent on retroviral overexpression and to confirm that LCN7 exhibits pro-angiogenic activity in other models of angiogenesis. To accomplish this, we used a combina- tion of Ni+ agarose and anti-FLAG M2 affinity chromatography to purify murine recombinant 6-His and FLAG tagged LCN7 from E. coli cell lysates. As shown in Figure 4A, recombinant LCN7 preparations were free of contaminating proteins, had a molecular mass of approximately 50 kD, and were reactive with anti-FLAG antibodies. Having purified LCN7 protein, we set out to confirm the pro-angiogenic activity of LCN7 on endothelial cell adhesion, invasion and angiogenic sprouting. Integrins serve as critical cell surface proteins that transmit signals from the extracellular matrix to the cell. Ligation of angiogenic integrins is an important aspect by which many matricellular proteins support endothelial cell adhesion and angiogenesis. LCN7 was previously shown to mediate adrenocor- tical, vascular smooth muscle and 293T cell adhesion [9], but endothelial cell adhesion to LCN7 has not previously been demonstrated. Therefore, we compared endothelial cell adhesion to either diluted matrigel as a positive control, BSA as a negative control, or purified LCN7 protein. Both MB114 and SVEC endothelial cells successfully adhered and spread out when cultured on both matrigel and recombinant LCN7 but failed to adhere to BSA (Fig. 4B). Our previous results demonstrated that overexpression of LCN7 increased endothelial cell invasion and sprouting. Therefore, we also tested our recombinant preparations for similar pro-angiogenic activities. As shown in Figure 4C, addition of 1 mg/ml of purified LCN7 increased MB114 and SVEC invasion through matrigel coated boyden chambers. Moreover, inclusion of 1 mg/ml of recombinant LCN7 into collagen overlays also stimulated endothe- lial cell sprouting from quiescent MB114 monolayers. Finally, we tested the ability of recombinant LCN7 to induce angiogenic sprouting from ex vivo sections of mouse aortic rings. As shown in Figure 4E, collagen gels supplemented with 1 mg/ml of rLCN7 provoked more angiogenic sprouting from ex vivo aortic ring sections PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13905