Interestingly, both pro- and anti-angiogenic activities have been attributed to TGF-b. However, the mechanistic rationale for this dichotomy are the subject of ongoing debate. One perspective on this debate is that TGF-b activation of SMAD2/3 via the ALK5 receptor is coupled to angiogenesis activation while TGF-b activation of SMAD1/5/8 via the ALK1 receptor mediates angiogenesis resolution [26,27,28,29]. Consistent with this model, we have found that LCN7 increases coupling of TGF-b to SMAD2 but not SMAD5. However, another perspective on this debate claims that it is activation of ALK1 and SMAD1/5/8 by TGF-b that mediates angiogenic activation and moreover that SMAD1/5/8 activation suppresses the angiostatic effects of SMAD2/3 activation [30,31,32]. Although it is beyond the scope of this report, it will be critical in future research to more closely examine the link between LCN7, TGF-b and angiogenesis to gauge the importance of TGF-b signaling to LCN7’s pro- angiogenic activities and to provide insight into the controversy regarding the role of TGF-b in angiogenesis. Our data also determined that LCN7 mediates endothelial cell adhesion. Consistent with this finding, Li et al [9] previously demonstrated that LCN7 promotes vascular smooth muscle cell adhesion by binding to a1, a2, a5 and b1 integrin receptors. Although the identity of integrin receptors involved in endothelial- LCN7 adhesion remain unknown, all of the afore mentioned integrin subunits are expressed in endothelial cells and each of the possible combinations (i.e. a1b1, a2b1, and a5b1) are known regulators of angiogenesis [33]. Therefore it is likely that in addition to sensitizing endothelial cells to TGF-b, LCN7 may also promote angiogenesis by mediating ligation of pro-angiogenic integrins. Future studies will focus on identifying LCN7 binding integrins in endothelial cells and determining the importance of these interactions to LCN7’s pro-angiogenic activities. Finally, LCN7 contains two protein domains that may provide additional hints at the protein’s pro-angiogenic activity. First, LCN7 is a member of the lipocalin family of extracellular matrix proteins which is very diverse and serves a wide range of functions [5]. Of the lipocalins, lipocalin-2 (LCN2, also neutrophil- gelatinase associated lipocalin, NGAL) is most relevant to this study since LCN2 suppresses angiogenesis in pancreatic cancer [34]. Given the small size of the lipocalin domain and the divergence of function between LCN2 and LCN7, it seems unlikely that any general conserved angiogenic function can be assigned to lipocalin proteins. LCN7 also contains cathepsin B-like domains. Cathepsin B is a secreted protease of the cathepsin family which is important for proteolytic processing of extracellular matricies, cellular invasion, migration and are linked to various human pathologies of the cardiovascular and other systems [35]. However, the cathepsin domains of LCN7 are not active [6,10]. This observation has led to the hypothesis that LCN7 may serve as an inhibitor of cathepsin activity and block cell invasion and migration. Our results do not support this hypothesis since we find that LCN7 increases endothelial cell invasion and sprouting. In summary, we have identified and characterized LCN7 as a novel matricellular regulator of angiogenesis. Although we determined that LCN7 promotes endothelial cell invasion, sprouting and angiogenesis in vivo, there remain a significant number of questions that remain to be addressed regarding the biological function of LCN7. For instance, future studies need to be performed to 1.) more precisely define the mechanism by which LCN7 promotes angiogenesis, 2.) describe the impact of LCN7 on additional cell types present in vascular microenvironments, and 3.) determine if LCN7 is an important regulator of angiogenesis in various human diseases characterized by insufficient or excessive angiogenesis. LCN7 Promotes Angiogenesis Materials and Methods Ethics Statement Animal studies were performed in accordance with the animal protocol procedures approved by the Institutional Animal Care and Use Committee of Indiana State University (protocol #1-19- 2008:AA and 11-08-2007:AA). Cell biological assays (proliferation, invasion, sprouting assays) Angiogenic sprouting of MB114 cells in collagen matrices was performed as described previously [36]. Briefly, control- or LCN7- overexpressing cells (400,000 cells/well) were seeded onto 6-well plates and allowed to grow to confluence. Confluent monolayers were overlayed with 2 ml of solidified rat tail collagen containing 10%FBS and 1X MB114 cell media. Sprouting was allowed to proceed for 5 days, whereupon sprouting cells were visualized by carefully focusing a 20X objective just above the plane of quiescent endothelium to identify cells that sprouted from the monolayer and entered the collagen matrix. The number of invading angiogenic sprouts was quantified under a light microscope by determining the average number of sprouts present in 10 independent fields/well. Proliferation assays were performed by seeding 1000 control or LCN7-overexpressing MB114 or SVEC endothelial cells into 96- well plates in 100 ml of complete growth media. Cell proliferation was monitored daily for 3 days by adding 10 ml of WST1 for 2 hours after which absorbance at OD450 in the conditioned media was measured. Invasion assays were performed using a modified Boyden-chamber assay as described previously [36,37]. Briefly, upper chambers (8 mm pore, 24-well format; BD Biosciences) were coated with 100 ml of diluted Matrigel [1:50 in serum-free DMEM (SFM)], which was evaporated overnight at room temperature. Subsequently, control or LCN7-overexpressing MB114 or SVEC cells were washed twice in SFM containing 0.1% BSA (SFM/0.1% BSA) and seeded in upper chambers at a density of 100,000 cells/well. Endothelial cell invasion was stimulated by addition of 2% serum to lower chambers, and was allowed to proceed for 48 h at 37uC. Invading endothelial cells were washed, fixed in 95% ethanol, and stained with crystal violet. The number of invading endothelial cells was determined by densitomet- ric analysis of scanned invasion membranes. Aortic ring assays were performed by first dissecting the aorta from freshly sacrificed C57BL/6 mice and slicing the aorta into approximately 1 mm thick rings. Up to 10 aortic rings isolated from each animal were equally divided and half of each group was imbedded into collagen gels containing 1 mg/ml of recombinant LCN7 while the other half of each group was imbedded into collagen gels containing an equal volume of PBS. Collagen gels were formed by isolating rat tail collagen as previously described [38], mixing purified collagen 1:1 with EGM2 media (clonetics) and overlaying the solidified collagen gels with EGM2 media. Recombinant LCN7 production and purification Murine LCN7 was amplified and prepared for gateway cloning into pDEST42 by a two-stage PCR reaction that was accomplish using oligo sets that were designed to delete the first 18 amino acids and add an ATTB1 site to the 59 end while the FLAG epitope tag and ATTB2 sequences were appended to the 39 end of the protein. First 59 oligo: AAAGCAGGCTTCACCATGGAGGCCCGGCGGAG- TCGT. Second 59 oligo: GGGGACAAGTTTGTACAAAAAAG- CAGGCTTCACCATG. First 39 oligo: CTTATCGTCGTCATC- CTTGTAATCGTGGTGCCCCATGTCCTC. Second 39 oligo: GGGGACCACTTTGTACAAGAAAGCTGGGTCCTTATCG- PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e13905