Lipocalin-7 Is a Matricellular Regulator of Angiogenesis

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.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-β 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.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

LCN7 is conserved between species.

<p>LCN7 protein sequences from human, rat, zebrafish, cow, and mouse were compared using PRALINE sequence alignment tool. The degree of conservation for each amino acid is indicated by color according to the key and in the “conservation” line of the alignment where * indicates 100% identity. Prosite analysis predicted the location of somatoamedin B (AA 74 to 95), lipocalin (AA 272 to 285), and cathepsin B (AA 402 to 452) and (AA 424 to 443) domains that are outlined with dashed lines. Overall, the alignment showed 79% sequence identity over the entire length of the alignment.</p

Morpholino knockdown in zebrafish embryos.

<p><b>A</b>. LCN7 expression was detected in developing zebrafish embryos by RT-PCR analysis. cDNAs pools were generated from embryos ranging from 2 to 48 hours and subjected to RT-PCR analysis with either LCN7 or GAPDH specific oligonucleotides. <b>B</b>. Freshly fertilized zebrafish embryos between 1 to 8 cells were injected with morpholino vehicle (water +.1% phenol red) or equal volumes containing either 1 ng of LCN7 morpholino, or 1 ng of mismatched LCN7 control morpholino. Microangiogram analysis was performed approximately 28 hours later by detecting endothelial-specific expression of GFP. The accompanying graph depicts average ISV pixel length (converted to µM) as measured along the entire dorsal surface in each of five embryos in each experiment. Results shown are the average +/− SE of five independent experiments. Bar equals 100 µM. <b>C</b>. Freshly fertilized zebrafish embryos were injected with morpholino vehicle (water +.1% phenol red) or equal volumes containing 1 ng of LCN7 morpholinos or 1 ng of LCN7+1.5 ng of p53 morpholinos. ISV vessel length was measured as described above and results are presented as the average +/− SE of five independent experiments consisting of at least five individual fish per injection. For all panels, * indicates p<.05, students t-test. Bar equals 100 µM.</p

Overexpression of LCN7 in endothelial cells.

<p>Murine LCN7 cDNAs were C-terminally FLAG tagged and overexpressed from pMSCV retroviral vectors in MB114 and SVEC murine endothelial cell lines. <b>A</b>. 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>B</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.</p

LCN7 increases endothelial cell angiogenic activities.

<p><b>A</b>. 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 +/− SE of four independent experiments. <b>B</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 +/− SE of three independent experiments. <b>C</b>. 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 +/− SE compared to each cell lines first measurement. For all panels, * indicates p<.05, Students t-test.</p

Recombinant LCN7 increases pro-angiogenic activities.

<p><b>A</b>. Murine His-6 and FLAG-tagged LCN7 cDNA was expressed in <i>E. coli</i> and purified by sequential Ni⁺-agarose (left panel) and anti-FLAG M2 (right panel) affinity columns. Representative coomassie stained gels of the purification process are shown. LCN7 was purified to apparent homogeneity. <b>B</b>. Tissue culture wells were coated overnight with dilute Matrigel, 1 µg/ml of recombinant LCN7 (rLCN7), or 10 µg/ml of BSA, then all wells were blocked with 10 µg/ml BSA. Adherent SVEC and MB114 endothelial cells were stained with crystal violet. Shown are representative images of an experiment that was performed three times. <b>C</b>. MB114 and SVEC cells were cultured on Matrigel coated trans-wells with 1 µg/ml of rLCN7 or an equivalent volume of 1XPBS. Invading cells were stained with crystal violet and quantified by densitometry of stained invasion membranes. Results are depicted as the fold increase +/− SE compared to 1XPBS treated cells from a total of four independent experiments. <b>D</b>. Quiescent MB114 endothelial cells were overlayed with rat tail collagen containing 1 µg/ml of rLCN7 or an equivalent volume of PBS. After 48 hours, endothelial cell sprouting was quantified by manually counting the number of sprouting cells present in 10 high power microscopic fields. Results are depicted as the fold increase +/− SE compared to PBS treated cells from a total of three independent experiments. <b>E</b>. Sections of aorta from a single mouse were embedded into rat tail collagen containing either 1 µg/ml of rLCN7 or an equivalent volume of 1XPBS. Shown are representative images of sprouting cells after 4 days of incubation from a single experiment that was performed three times.</p

LCN7 sensitizes endothelial cells to TGF-β.

<p><b>A</b>. Control and LCN7-overexpressing MB114 endothelial cells were serum starved then stimulated with 5 ng/ml of TGF-β1 for 0, 20, 40, or 80 minutes. Whole cell lysates were fractionated in SDS-PAGE and western blotted with antibodies to detect phosphorylated ERK1/2 MAPK (P-ERK1/2) or β-actin as a loading control. Western blot results from three independent experiments were quantified by densitometry, normalized to β-actin to control for loading differences, and are presented as the fold increase +/− SE of unstimulated control cells. <b>B</b>. Control and LCN7-overexpressing MB114 endothelial cells were stimulated as in A. Whole cell lysates were fractionated and western blotted with antibodies against phosphorylated SMAD-2 (P-SMAD2), phosphorylated SMAD5 (P-SMAD5), or β-actin. Western blot results from three independent experiments were quantified by densitometry, normalized to β-actin to control for loading differences, and are presented as the fold increase +/− SE of unstimulated control cells. <b>C</b>. Control or LCN7 overexpressing MB114 endothelial cells were induced to invade through matrigel coated transwells in the presence of 5 ng/ml TGF-β (TGF) or 10 µM TGF-β antagonist SB431542 (SB) and compared to unstimulated control (C) cells. Invading cells were stained with crystal violet and quantified by densitometry of stained invasion membranes. The experiment was performed three times and the resulting data is presented as the fold invasion of control cells +/− SE. <b>D</b>. Quiescent cultures of control or LCN7 overexpressing MB114 endothelial cells were overlayed with rat-tail collagen containing 5 ng/ml TGF-β (TGF) or 10 uM TGF-β antagonist SB431542 (SB) and compared to unstimulated control (C) cells. Data was collected by manually counting the number of sprouting cells in 10 high power fields and is presented as the average fold increase +/− SE of three independent experiments. * indicates p<.05, student's t-test.</p