genomic database to identify the zebrafish LCN7 homolog. The zebrafish LCN7 gene encodes a 471 aa protein with predicted molecular weight of 53.9 kD. We used the PRALINE alignment tool (http://www.ibi.vu.nl/programs/pralinewww/) to construct a multiple sequence alignment of LCN7 proteins from human, rat, mouse, cow and fish (D. rerio). Multiple sequence comparison revealed that LCN7 was highly conserved between species over the entire length of the protein (Fig. 5). Features of the conserved alignment included N-terminal Somatomedin-B domain(s), inactive pro-cathepsin B-like domains and the signature lipocalin domain. Overall, the multiple sequence alignment revealed 79% sequence identity between all species over the length of the entire alignment. Before performing anti-sense morpholino knockdown of LCN7, it was first important to determine if LCN7 mRNA is present during early zebrafish development. To accomplish this, we used RT-PCR to monitor LCN7 mRNA in cDNA pools generated from 2 to 48 hour old embryos. As shown in Figure 6A, LCN7 mRNA was detectable within 8 hours after embryo fertilization and continued to be present over the remainder of the time course. Sequencing of the DNA fragments confirmed amplification of LCN7 cDNA (data not shown). We designed a splice blocking anti-sense morpholino to transiently knockdown LCN7 expression in developing Fli1-GFP transgenic zebrafish embryos. Control LCN7 morpholinos were also designed with five mismatched bases and used as to control for possible non-specific affects caused by LCN7 morpholino injections. Fli1-GFP embryos (1 to 8 cells) were injected with 1 to 8 ng of either LCN7 antisense morpholino, LCN7 mismatched morpholino, or with equivalent volumes of morpholino vehicle alone (water +0.1% phenol red). As shown in Figure 6B, injection of 1 ng of LCN7 morpholino significantly delayed the develop- ment of intersegmental vessels (ISV) compared to embryos injected with either mismatched morpholinos or morpholino vehicle alone. Overall, approximately 24 hours after injection, ISV length in vehicle injected embryos was 70+/23.7 mm. Injection of 1 ng of LCN7 MO reduced average ISV length to 51+/24.8 mm. Injection of 1 ng of mismatched MO did not reduce ISV development compared to vehicle injected embryos. Moreover, and most likely as a consequence of delayed ISV development, dorsal longitudinal anastomatic vessel (DLAV) development was also delayed in LCN7 injected embryos. However, development of the aorta and the cardinal vein was unaffected indicating that angiogenesis, not vasculogenesis, was implicated with LCN7 deletion. Importantly, no defects in body patterning were obvious in embryos injected with up to 2 ng of morpholino solutions. Collectively, these results suggested that delayed ISV development was directly linked to LCN7 knockdown rather than to off-target effects of LCN7 morpholinos. Although LCN7 knockdown appeared to produce specific effects on ISV development, injection of 4 ng or more of either LCN7 morpholinos or LCN7 mismatched control morpholinos also caused defects in body patterning and development (data not shown). Off-target morpholino effects are p53 dependent and simultaneous knockdown of p53 reverses these non-specific effects on zebrafish development [22]. Therefore, as an additional control for possible non-specific effects of LCN7 morpholino injections, we examined ISV development in LCN7 + p53 double knockdown embryos. To accomplish this, we injected 1 ng LCN7 morpholino together with 1.5 fold excess p53 specific morpholino as previously described [22]. As shown in Figure 6C, p53 co-knockdown failed to rescue the delay in ISV development produced by LCN7 knockdown. This result confirms that vascular defects induced by LCN7 knockdown are vascular-specific and not related to off target effects of LCN7 morpholinos. Collectively, these results LCN7 Promotes Angiogenesis indicate that LCN7 is essential for proper vascular function during angiogenesis in vivo. Discussion Growth factors such as VEGF, bFGF, and TGF-b are some of the most potent regulators of angiogenesis. However, growth factor stimulation of endothelial cells alone is not sufficient to produce blood vessels with the physiological properties required for proper capillary function. This is illustrated by the leaky blood vessels observed in tumors that are stimulated by abnormally high secretions of pro-angiogenic growth factors such as VEGF [23]. Development and maintenance of a normal vascular system also requires input from structural and matricellular proteins of the vascular microenvironment such as LCN7. In fact, remodeling of structural and matricellular proteins within vascular microenvi- ronments during angiogenesis or angiostasis serve a major role in controlling vascular function [2]. Because these proteins are so vital for proper vascular development, function and maintenance, their identification and characterization are necessary for pro- and anti-angiogenic therapies to achieve the great potential predicted by Judah Folkman and others [17,24]. LCN7 has been shown to be expressed within vascular smooth muscle [6] and the endothelial cell lining of blood vessels [7,9]. Furthermore, LCN7 was previously linked to Hif1a activation [11] and is differentially expressed during angiogenesis in vitro [12]. Such circumstantial observations spatially and temporally position LCN7 to be an important regulator of angiogenesis. Despite these observations, no functional analysis of LCN7 in angiogenesis or any other cellular process has previously been performed. In this study, we have used overexpression, recombinant protein and knockdown approaches combined with both in vitro and in vivo angiogenesis models to investigate the role of LCN7 in angiogenesis. Our results indicate that LCN7 mediates endothelial cell adhesion and is essential to angiogenesis in developing zebrafish embryos. Mechanistically, we found that LCN7 promotes angiogenesis by increasing endothelial cell invasion, sprouting and sensitivity to TGF-b. Angiogenesis consists of a series of endothelial cell activities including response to pro-angiogenic growth factor signals, stimulation of cell proliferation, degradation of the vascular basement membrane, endothelial cell sprouting, migration and invasion of activated cells, formation of lumenized endothelial stacks and eventually, the development of anastomoses and capillary networks [25]. In this manuscript, we analyzed the function of LCN7 in several of these angiogenic stages. Our data demonstrates that both LCN7 overexpression and administration of highly purified recombinant LCN7 proteins increases invasion of two distinct endothelial cell lines. Moreover, we have also shown that overexpression of LCN7 and recombinant LCN7 promote endothelial cell sprouting from quiescent endothelial cell mono- layers and from dissected ex vivo aortic rings. The precise mechanism by which LCN7 promotes angiogenic activities remains largely unknown. However, our results provide the first evidence that LCN7 promotes angiogenesis by increasing endothelial cell sensitivity to TGF-b, a major determinant of pro- angiogenic signaling [18]. In support of this, we have shown that LCN7 increases ERK1/2 phosphorylation in TGF-b treated cells but not in cells treated with other angiogenic growth factors (VEGF or bFGF). We also demonstrated that the pro-angiogenic activity of TGF-b is enhanced in LCN7 overexpressing cell lines and that blockade of TGF-b signaling eliminates the increased invasion and angiogenic sprouting observed in LCN7 overex- pressing MB114 endothelial cells. PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e13905