embryos. Collectively, these results indicate that LCN7 is a proangiogenic matricellular protein. Results Overexpression of LCN7 in endothelial cell lines LCN7 is consistently localized to vascular tissues [7,8,9,13], has been linked to Hif1a activation in hypoxic brain damage [11] and was previously shown to be differentially expressed during endothelial cell angiogenesis on Matrigel matrices [12]. However, the functional role of LCN7 in vascular function and/or angiogenesis has not been addressed. Therefore, we set out to determine if LCN7 may be important for angiogenesis. To undertake this project, we first used a retroviral overexpresson strategy to drive LCN7 protein expression in mouse brain microcapillary endothelial (MB114) cells [14] and tumor derived SVEC4-10 (SVEC) endothelial cells [15]. The murine LCN7 cDNA was PCR amplified with oligonucleotides engineered to append a C-terminal FLAG epitope and subsequently cloned into a retroviral vector (pMSCV). MB114 and SVEC cell lines were transduced with LCN7 encoding pMSCV viral particles or empty, control viral particles. Polyclonal populations of cells harboring integrated sequences were selected with puromycin and tested for overexpression of FLAG-tagged LCN7. Conditioned media from the resulting overexpressing and control cell lines was collected, precipitated with TCA/DOC and analyzed by western blot with anti-FLAG M2 monoclonal antibodies. As shown in Figure 1A, both MB114 and SVEC cells expressed and secreted FLAG-tagged LCN7 protein. Overexpressed LCN7 migrated as a single 50 kD band suggesting that the overexpressed protein was not glycosylated as previously observed [16]. We attempted to compare the degree of LCN7 protein overexpression to basal LCN7 expression using commercially available LCN7 antibodies, however these antibody preparations demonstrated significant non-specific binding and failed to detect overexpressed protein much less endogenous LCN7 (data not shown). Instead, we used RT-PCR analysis to compare relative levels of LCN7 mRNA in control and overexpressing cells. LCN7 Promotes Angiogenesis We found that SVEC cells but not MB114 cells express low levels of endogenous LCN7 mRNA and that overexpression significantly increased LCN7 mRNA over basal levels in both cell lines (Fig. 1B). Collectively these results demonstrated that we successfully overexpressed LCN7 and allowed us to next examine the effect of LCN7 on several in vitro models of angiogenesis. Overexpression of LCN7 promotes pro-angiogenic endothelial cell behaviors Angiogenesis is characterized by several distinct endothelial cell activities including endothelial cell proliferation, invasion through vascular basement membranes and sprouting from quiescent monolayers [17]. To measure the effect of LCN7 overexpression on these processes, we used a series of in vitro models that recapitulate these angiogenic activities. Transwell assays were first used to monitor the effect of LCN7 on endothelial cell invasion through artificial basement membranes (i.e. matrigel). As shown in Figure 2A, overexpression of LCN7 significantly increased both MB114 and SVEC endothelial cell invasion through Matrigel coated Boyden chambers as compared to respective control counterpart cells. We next examined the effect of LCN7 on anigogenic sprouting from quiescent MB114 endothelial mono- layers that had been overlayed with rat tail collagen. This assay is designed to recapitulate the events that occur when quiescent vasculature encounters collagen I outside the vascular basement membrane during a wound healing process. As shown in Figure 2B, LCN7 overexpression significantly increased MB114 endothelial cell sprouting from quiescent monolayers into the collagen gel as compared to control cells. Finally, we used WST1 to measure the proliferation rates of control or LCN7-overex- pressing MB114 and SVEC cells over a timecourse of three days in complete growth media. As shown in Figure 2C, overexpression of LCN7 did not have a significant impact on either MB114 or SVEC endothelial cell proliferation. Collectively, these results demonstrated for the first time that LCN7 promotes endothelial cell invasion and sprouting and suggest that LCN7 is a pro- angiogenic protein of the extracellular matrix (ECM). Figure 1. Overexpression of LCN7 in endothelial cells. Murine LCN7 cDNAs were C-terminally FLAG tagged and overexpressed from pMSCV retroviral vectors in MB114 and SVEC murine endothelial cell lines. A. Conditioned media from empty retroviral control cells (PURO) and LCN7- overexpressing cells was precipitated with TCA/DOC, fractionated through SDS-PAGE gels and western blotted with anti-FLAG monoclonal antibodies. Shown are representative images from a single experiment that was conducted twice with identical results. B. RT-PCR analysis was performed on either reverse transcribed (+RT) or non-reverse transcribed (-RT) RNA pools with oligonucleotides designed to amplify LCN7 (469 bp fragment) or GAPDH (237 bp fragment) as a loading control. Shown are representative images from a single experiment that was conducted three times with identical results. doi:10.1371/journal.pone.0013905.g001 PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13905