Lipocalin-7 Is a Matricellular Regulator of Angiogenesis Leslie J. Brown, Mariam Alawoki, Mary E. Crawford, Tiffany Reida, Allison Sears, Tory Torma, Allan R. Albig* Department of Biology, Indiana State University, Terre Haute, Indiana, United States of America Abstract Background: Matricellular proteins are extracellular regulators of cellular adhesion, signaling and performing a variety of physiological behaviors such as proliferation, migration and differentiation. Within vascular microenvironments, matricellular proteins exert both positive and negative regulatory cues to vascular endothelium. The relative balance of these matricellular cues is believed to be critical for vascular homeostasis, angiogenesis activation or angiogenesis resolution. However, our knowledge of matricellular proteins within vascular microenvironments and the mechanisms by which these proteins impact vascular function remain largely undefined. The matricellular protein lipocalin-7 (LCN7) is found throughout vascular microenvironments, and circumstantial evidence suggests that LCN7 may be an important regulator of angiogenesis. Therefore, we hypothesized that LCN7 may be an important regulator of vascular function. Methodology and Principal Findings: To test this hypothesis, we examined the effect of LCN7 overexpression, recombinant protein and gene knockdown in a series of in vitro and in vivo models of angiogenesis. We found that overexpression of LCN7 in MB114 and SVEC murine endothelial cell lines or administration of highly purified recombinant LCN7 protein increased endothelial cell invasion. Similarly, LCN7 increased angiogenic sprouting from quiescent endothelial cell monolayers and ex vivo aortic rings. Moreover, LCN7 increased endothelial cell sensitivity to TGF-b but did not affect sensitivity to other pro-angiogenic growth factors including bFGF and VEGF. Finally, morpholino based knockdown of LCN7 in zebrafish embryos specifically inhibited angiogenic sprouting but did not affect vasculogenesis within injected embryos. Conclusions and Significance: No functional analysis has previously been performed to elucidate the function of LCN7 in vascular or other cellular processes. Collectively, our results show for the first time that LCN7 is an important pro-angiogenic matricellular protein of vascular microenvironments. Citation: Brown LJ, Alawoki M, Crawford ME, Reida T, Sears A, et al. (2010) Lipocalin-7 Is a Matricellular Regulator of Angiogenesis. PLoS ONE 5(11): e13905. doi:10.1371/journal.pone.0013905 Editor: Ludovic Tailleux, Institut Pasteur, France Received July 6, 2010; Accepted October 15, 2010; Published November 9, 2010 Copyright: 2010 Brown et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (# 3R15CA133829-01A1S1), the Indiana State University Promising Scholars Fund, and the Indiana State University Research Council (UNR286) to ARA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: allen.albig@indstate.edu Introduction The control of angiogenesis is highly dependent on proper cues from the vascular microenvironment [1,2]. Vascular microenviron- ments contain a host of factors including growth factors, cytokines and matricellular proteins which serve as critical extracellular integrators between structural and cellular activities [3]. By extension, perturbations of vascular microenvironments provoke major changes in vascular homeostasis including angiogenic activation and resolution [4]. In particular, tumor microenviron- ments are highly angiogenic and are characterized by altered expression of pro-angiogenic matricellular proteins. Understanding how the matricellular composition of tumor microenvironments is manipulated to enhance tumor angiogenesis is of central impor- tance to understanding how to starve tumors of blood thus realizing the potential of anti-angiogenic approaches to cancer therapy. Lipocalin-7 (LCN7, also Tinagl1-Tubulointerstitial nephritis antigen like 1, Tin-ag-RP-Tubulointerstitial nephritis antigen related protein, AZ-1-Adrenocortical zonation factor 1, and ARG1-Androgen regulated gene 1) is a matricellular protein that belongs to the diverse family of lipocalin proteins [5] and is highly conserved between species. LCN7 has been shown to be predominantly localized to vascular tissues in a variety of organ types. For example, LCN7 is abundant in vascular smooth muscle [6], lung capillary endothelium [7], uterine capillaries during the postimplantation period [8], vascular basement membranes of adrenocortical sinusoidal capillaries [9] and glomerular basement membranes [6]. In addition, LCN7 expression has been linked to a variety of physiological stimuli including testosterone signaling in the mouse prostate [10] and Hif1a activation in mouse brain [11]. Most recently it was also discovered that LCN7 is differentially regulated during angiogenesis [12]. Collectively, these findings demonstrate that LCN7 is found within vascular microenviron- ments and is differentially regulated during angiogenesis suggest- ing that LCN7 may be involved with regulation of vascular function. Despite this correlative evidence, there has been no direct attempt to assess the function of LCN7 in vascular function and angiogenesis. Herein we show that LCN7 is a positive regulator of angiogenesis that increases endothelial cell invasion, angiogenic cell sprouting and sensitivity to TGF-b. Moreover, we demonstrate that LCN7 is important for early angiogenesis in zebrafish PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e13905