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Am J Physiol Cell Physiol 295: C1215-C1229, 2008. First published September 11, 2008; doi:10.1152/ajpcell.00336.2008
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EXTRACELLULAR MATRIX, CELL INTERACTIONS

Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing

Shih-Chi Su, E. Adriana Mendoza, Hyeong-il Kwak, and Kayla J. Bayless

Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, Texas

Submitted 26 June 2008 ; accepted in final form 2 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sprouting angiogenesis is a multistep process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression alongside multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate, basic FGF, and VEGF. Gene expression changes were confirmed by real-time PCR and Western blot analyses. A cohort of cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated, whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, a disintegrin and metalloproteinase with thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt and Notch pathways was observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. This study has enhanced the molecular road map of gene expression changes that occur during endothelial invasion and highlighted the utility of three-dimensional models to study EC morphogenesis.

angiogenesis; extracellular matrix; Notch; endothelial cell; sphingosine-1-phosphate; collagen

ANGIOGENESIS IS THE FORMATION of new blood vessels from preexisting structures and is a key step in development, tumor progression, and wound healing (18, 19, 45). Several key signaling pathways have been defined that mediate angiogenesis. This list includes, but is not limited to, integrins, cell surface receptors, growth factors, lipids, and the extracellular matrix (ECM). Integrins are heterodimeric transmembrane receptors, composed of {alpha}- and β-chains that combine to integrate the cytoskeleton with the ECM. The {alpha}2β1-integrin is an endothelial receptor for collagen type I (38, 129, 144) and has been shown to be required for angiogenesis in vivo and in vitro (33, 137), as have other integrins, including {alpha}1β1, {alpha}vβ3, and {alpha}5β1 (6, 12, 23, 31, 136, 137, 154). Angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are powerful proangiogenic stimuli, and multiple studies have documented the involvement of these growth factors and their receptors in mediating angiogenic events. Transgenic animals lacking VEGF and its receptors fail to form a functional vasculature (44, 46, 138), as do mice lacking the {alpha}v-integrin receptor (2) and the {alpha}5-integrin receptor (166), resulting in an embryonic lethal phenotype. In addition to angiogenic growth factors and integrins, sphingosine-1-phosphate (S1P) is a lysosphingolipid that has been reported to induce angiogenesis under various conditions (39, 58, 78, 79, 143) and is emerging as a novel target in anticancer therapies (96, 157). Cellular responses to S1P are mediated through activation of one or more of their five known high-affinity G protein-coupled receptors, S1P1–S1P5 (143). Knockout of the S1P1 receptor (also known as Edg1) in mice results in vascular defects, attributed to impairment of mural cell association with developing vasculature (85). Within wounded tissues, platelets deposit S1P and VEGF locally, while fibroblasts and other cells secrete bFGF (4, 14, 40, 99, 158, 167). Thus the coordination of S1P, growth factors, and integrin-ECM interaction is critical for mediating angiogenesis in wound healing. The in vitro system used here mimics a wound environment where S1P release would couple with local production of VEGF and FGF at the wound site to stimulate endothelial cell (EC) outgrowth and sprouting angiogenesis.

Because sprouting angiogenesis is a multistep process consisting of EC activation, basement membrane degradation, invasion and proliferation of ECs, lumen formation, and also stabilization, standard two-dimensional cell culture environments cannot completely reflect this dynamic process. One experimental approach to overcome this restriction is to use in vitro three-dimensional (3-D) models in which individual steps of angiogenesis can be reproduced (33, 37, 101, 103, 156). These studies not only allow for characterization of morphological changes, but also allow simultaneous monitoring of differential gene expression (7, 47, 52, 67). We have exploited one such model (5) in which S1P and angiogenic growth factors synergistically induce rapid EC sprouting and invasion in 3-D collagen matrices to mechanistically investigate the changes in gene expression that regulate the process. Data presented here indicate the coordinated regulation of molecules implicated in cell-cell and cell-matrix contacts, as well as in degradation and remodeling of ECM as endothelial invasion proceeds. To demonstrate a functional requirement for one such regulated gene, we knocked down a disintegrin and metalloproteinase with thrombospondin-type repeats-1 (ADAMTS1) using small interfering RNA (siRNA). Silencing of ADAMTS1 resulted in decreased invasion responses, indicating that ADAMTS1 is required for ECs to invade into 3-D collagen matrices. We also demonstrated that several components of signaling pathways, in particular, Wnt and Notch pathways, are systematically regulated. Activation of Notch signaling by enhanced expression of Delta-like 4 (DLL4) and Notch intracellular domains blocked EC invasion, while inhibition of Notch signaling by {gamma}-secretase inhibitor increased invasion density. Collectively, these data demonstrate that this system of EC invasion in 3-D collagen matrices closely resembles angiogenic events in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs), passages 3–6 (Lonza, Cambridge, MA), were passaged once weekly and cultured on gelatin-coated (1 mg/ml) tissue culture flasks in medium 199 (M199) containing 100 µg/ml heparin (Sigma), 0.4 mg/ml lyophilized bovine hypothalamic extract (Pel-Freeze Biologicals, Rogers, AK) (88), and 15% fetal bovine serum (Lonza). Collagen Type I was isolated from tendons of one rat tail by incubation with gentle agitation in 150 mL 0.1% acetic acid for 1 wk. Supernatant was lyophilized, weighed, and resuspended in 0.1% acetic acid at 7.1 mg/ml and stored at 4°C. Collagen matrices were prepared as reported previously (5). S1P (Avanti Polar Lipids, Alabaster, AL) was added at a final concentration of 1 µM (5). Gels (25 µl) were added to half area 96-well plates (Corning-Costar) and allowed to equilibrate for 45 min at 37°C with 5% CO2. HUVECs were fed 24 h before the beginning of each experiment. For invasion assays, confluent flasks of ECs were washed with 1x HEPES-buffered saline, trypsinized, and counted. After pelleting, cells were washed once in 10 ml M199. The final cell pellet was resuspended at a density of 40,000 cells per 50 µl in M199 containing reduced serum II (RSII) and allowed to attach for 30 min. The RSII was added from a sterile 250x stock containing 500 µg/ml BSA (Sigma), 5 µg/ml human holo-transferrin (Sigma), 5 µg/ml insulin (Sigma), 4.28 µg/ml sodium oleate (Sigma), and 5 ng/ml sodium selenite (Sigma). Growth media contained RSII, 50 µg/ml ascorbic acid (Sigma), and 40 ng/ml VEGF and bFGF (R&D Systems). No S1P or growth factors were included in zero-hour time point cultures. For each replicate, 40 wells were prepared.

Gene profiling studies and data analysis. Three replicate experiments were performed, in which invading cells were collected at 0, 6, 12, and 18 h. Collagen matrices were digested with 50 µg collagenase (Sigma) each at 37°C for 5 min before being transferred into 8 ml of 1x M199 and centrifuged at 350 g for 5 min. RNA was extracted using an RNA extraction kit according to the manufacturer's instructions (Qiagen). RNA was eluted with 50 µl nuclease-free water (Ambion, Austin, TX). Total RNA was submitted to the Texas A&M University Center for Environmental and Rural Health for quality analysis using an Agilent Technologies 2100 Bioanalyzer. RNA was generated into biotin-labeled cRNA via a modified Eberwine RNA amplification protocol. The labeled cRNA was applied to the bioarray (GE Amersham CodeLink Human Whole Genome) and incubated for 18 h, then washed, stained, and scanned. The array images were processed using CodeLink's system software. Three replicates for each time point (0, 6, 12, and 18 h) were performed. Data were averaged and compared with time 0 data. Each data entry was analyzed by GeneSpring software at the Texas A&M Laboratory for Functional Genomics and considered for further analysis only if a 1.7-fold or greater elevation was observed.

Invasion quantification and image analysis. For quantifying the average numbers of invading cells per standardized field, conditioned media were removed and invasion samples were fixed with 3% glutaraldehyde (Sigma) in PBS overnight at 4°C. Cultures were stained with 0.1% toluidine blue (Sigma) containing 30% methanol (Fisher). Alternatively, cultures were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min and stained with 1.09 µM DAPI (Molecular Probes). Eyepieces mounted with a reticle displaying a 10 x 10 grid, which covers an area of 6.25 mm2, 1 mm2, and 0.25 mm2 at 4x, 10x, and 20x, respectively, were used for quantifying average numbers of invading cells per standardized field. For each data set, three to four separate fields from each treatment were recorded and averaged. For quantifying invasion distance and lumen diameter, photographs of invading cells were taken from a side view with an Olympus CKX41 microscope equipped with a Q color 3 Olympus camera. Measurements of invasion distance and lumen diameter were measured digitally using QCapture software (Olympus/Leeds), and pixel values were converted to micrometers. For immunofluorescence imaging, top-viewed photographs (whole mount) were taken under a Nikon Eclipse TE2000U fluorescence inverted microscope equipped with a CCD camera and MetaMorph software (Universal Imaging), and side-viewed photographs were captured by Stallion digital imaging workstation at the Image Analysis Laboratory, Texas A&M University.

Real-time PCR. An AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Stratagene) was used to prepare cDNA isolated from invading ECs. Primers were designed using Beacon Designer software such that amplicons were 180–220 bp. Primer sequences are listed in supplemental Table S1 (the online version of this article contains supplemental data). Primers were validated by real-time PCR using a fivefold serial dilution (125–0.04 ng/reaction) of untreated HUVEC cDNA and water as a nontemplate control, followed by agarose gel electrophoresis, to determine the amplification efficiency, specificity, and to rule out primer dimers. Reactions were analyzed on a Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection system using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Each real-time PCR reaction contained 0.5 ng/µl of cDNA and 400 nM of each primer in a 25-µl reaction volume. The reaction was initiated at 94°C for 1.5 min, followed by 40 two-step amplification cycles consisting of 15 s of denaturation at 95°C and 45 s of annealing/elongation at 60°C. A final dissociation stage was run to generate a melting curve for verification of amplicon specificity. Assays were performed in triplicate against three independent preparations of cDNA. For each reaction, a threshold cycle was observed in the exponential phase of amplification, and the quantification of relative expression levels was achieved using standard curves for both the target and a constitutively expressed gene, GADPH, whose expression changed <1.15-fold.

Immunoblotting and immunofluorescence. For immunoblotting, cell lysates were prepared by transferring collagen gels containing invading ECs into boiling 1.5x Laemmli sample buffer containing 2% β-mercaptoethanol at 95°C for 10 min. Samples were separated using SDS-PAGE gels and transferred to polyvinylidene difluoride (Millipore). Antibodies against the following proteins were used for detection: claudin-5 (35-2500, Zymed), integrin-{alpha}2 (611016, BD Biosciences), integrin-{alpha}v (611012, BD Biosciences), ADAMTS1 (ab39194, Abcam, Cambridge, MA), Dkk2 (ab38594, Abcam), DAAM1 (M05, clone 5D3, Novus Biologicals, Littleton, CO), Delta-like 4 (NB600-892, Novus Biologicals), actin (CP01, Calbiochem), anti-FLAG M2 (F3165, Sigma), and G3PDH (ab8245, Abcam). For immunofluorescence, cell cultures were fixed in 4% paraformaldehyde and permeabilized in PBS containing 0.5% Triton X-100. Nonspecific binding was blocked by incubation with PBS containing 1% goat serum, 1% BSA, 0.2% sodium azide, and 0.1% Triton X-100. Following incubation with an anti-FLAG M2 monoclonal antibody (1:100 dilution; Sigma) for 2 h and FITC-conjugated goat anti-mouse IgG (1:50 dilution; Jackson ImmunoResearch) for 1 h, samples were mounted on glass slides with an antifading, aqueous mounting medium (Biomeda, Foster City, CA).

siRNA transfection. siGENOME SMARTpool human ADAMTS1, ADAMTS4, ADAM17, and GAPDH control siRNAs were obtained from Dharmacon (Lafayette, CO). Cells were seeded into 25-cm2 flasks at 40–50% confluency. The following day, cells were washed three times with serum- and antibiotic-free DMEM and transfected with 200 nM siRNA in 2.7 ml antibiotic-free DMEM using 20 µl siPORT Amine (Ambion). Flasks were aspirated and supplemented with antibiotic-free growth media at 8 h after transfection. This transfection procedure was repeated 2 days after the first transfection. After the second transfection, cells were fed and allowed to recover for 30 h before testing in invasion assays and verification of gene knockdown.

{gamma}-Secretase inhibition. Cells were resuspended at a density of 40,000 cells per 50 µl in M199 containing RSII and indicated concentrations of {gamma}-secretase inhibitor IX (565770, Calbiochem) or vehicle control (DMSO) and were incubated at 37°C with 5% CO2 for 20 min before being seeded on collagen gels with 100 nM S1P. For {gamma}-secretase inhibition, growth media contained RSII, ascorbic acid, VEGF, FGF-2, and indicated concentrations of {gamma}-secretase inhibitor. Cultures were fixed at 24 h to quantify invasion.

Plasmid constructs and gene transfer. Recombinant lentiviral vector encoding an enhanced green fluorescent protein (EGFP) was previously described (132) and was a kind gift from Dr. George E. Davis (Columbia, MO). Notch1 intracellular domain (N1ICD) (amino acids 1770-2556), Notch4 intracellular domain (N4ICD) (amino acids 1476-2003), and DLL4 were amplified by PCR from HUVEC cDNA and subcloned into pFLAG-CMV-5a (Sigma). The constructs were sequenced and tested for expression of the COOH-terminal FLAG-tagged N1ICD, N4ICD, and DLL4 in human embryonic kidney (HEK)-293 cells using anti-FLAG M2 antibodies (Sigma). FLAG-N1ICD, FLAG-N4ICD, and FLAG-DLL4 were amplified by PCR using pFLAG-CMV-5a clones as the templates and subcloned into pENTR4 (Invitrogen). Subsequently, genes with FLAG sequences were subcloned into pLenti6/V5-DEST using the Gateway system (Invitrogen). HUVECs were transduced with indicated constructs using the ViraPower Lentiviral Expression System (Invitrogen) according to the manufacturer's instructions. Invasion assays were conducted 3–7 days after infection.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of an in vitro endothelial cell invasion assay system. The studies performed here use a 3-D model of endothelial invasion that mimics angiogenesis (5). In these experiments, primary human ECs are seeded as a monolayer onto the surface of 3-D collagen matrices (Fig. 1A). No appreciable invasion occurred under control conditions, while S1P alone or growth factors (GF) alone stimulated modest invasion. However, S1P, a platelet-derived lipid, combined with bFGF and VEGF (GF) to stimulate rapid invasion within 16 h of treatment (Fig. 1B). Quantification of these data (Fig. 1C) illustrates that the combination of S1P, VEGF, and bFGF potently stimulates invasion. On the basis of these data, all subsequent experiments were carried out in the presence of S1P, VEGF, and bFGF. Further quantification with time revealed that ECs migrate or invade into the collagen matrices, forming sprouting structures containing lumens. Photographs taken of fixed cultures viewed from the top (Fig. 1D) and side (Fig. 1E) illustrate that invasion initiated at 6 h, with sprouts increasing at 12 h and extending further by 18 h. The invading structures recapitulate angiogenesis, forming lumens (Fig. 1F) lined by multiple cells (Fig. 1G). Quantification of the distance invaded over time reveals that ECs migrate through the collagen matrix at a linear rate from 0 to 48 h (Fig. 1H). In addition, quantification of lumen formation in these assays shows that lumen formation within these structures does not initiate with sprouting morphogenesis, but is delayed, initiating between 12 and 16 h (Fig. 1I).


Figure 1
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  		Fig. 1. Assay system to study primary endothelial cell (EC) invasion in three-dimensional (3-D) collagen matrices. A: illustration of the invasion system. B: photographs illustrating that sphingosine-1-phosphate (S1P) and angiogenic growth factors (GF) synergize to stimulate invasion. Cultures were allowed to proceed for 24 h under control conditions (No S1P or GF), 1 µM S1P alone, GF alone (40 ng/ml VEGF and basic FGF), or S1P + GF. C: quantification of invasion responses observed in B. Data presented are average numbers of invading cells per standardized field ± SE.(n = 3 fields). **P < 0.01 versus all other conditions using Students t-test. D and E: photographs depicting EC invasion over time. Cells were seeded on the surface of 3-D collagen matrices and allowed to attach for 30 min before addition of growth factors as described in MATERIALS AND METHODS. Cultures were fixed at 0, 6, 12, and 18 h and were stained with toluidine blue before imaging from surface (D) and side view (E). F and G: photographs illustrating that invading structures are multicellular. Cultures were fixed at 24 h and stained with toluidine blue (F) or DAPI (G). Phase contrast and DAPI images were overlaid (G). White arrowheads, nuclei; black arrowheads, areas of lumen formation (light open areas); black arrows, extended peripheral processes at the leading edge of invading structures. H and I: quantification of EC invasion rates. Cells were allowed to invade for the times indicated and were fixed, stained, and quantified for invasion distance (H) and lumen diameter (I). Data are presented as means ± SD; n = 100 (cells). White arrows (E, F, and G) indicate original monolayer where primary ECs were seeded.

 
Microarray analysis of EC gene expression during S1P and angiogenic growth factor-induced invasion in 3-D collagen matrices. To gain a more complete understanding of the mechanisms that direct the transformation of adherent ECs on the surface of a collagen matrix to multicellular invading structures, gene expression profiling studies were performed at various times. Total RNA from invading cells was isolated at 0, 6, 12, and 18 h and subjected to microarray analyses as described in MATERIALS AND METHODS. The zero-hour time point contained cells that were seeded onto collagen matrices lacking S1P and growth factors, thus our microarray analyses monitor gene expression changes with the advancement of invading sprouts. Of the 30,000 human genes screened, 440 were significantly upregulated and 742 were downregulated at least 1.7-fold. A partial listing of these genes is shown in supplemental Tables S2 and S3, respectively. Although multiple and diverse genes were differentially regulated, the focus of this report is on the expression of genes related to ECM interactions, proteolysis, cell-cell communication and regulation of morphogenesis, or cell shape changes.

Differential expression of cell adhesion molecules and ECM genes during EC invasion. Our data reveal that a series of genes that control endothelial cell-cell junctions and ECM recognition are systematically regulated during invasion. The invasion cultures consist of two populations of ECs, those that invade into the collagen matrix and those that stay behind. The decision to leave the monolayer and invade is likely associated with alterations in signaling between cell-cell contacts. Fitting with this idea, a set of genes that regulate tight junction (TJ) integrity was observed (supplemental Table S3). These downregulated genes include connexin 40 (CX40), connexin 45 (CX45), zona occludens 2 (ZO2), claudin5 (CLDN5), claudin11 (CLDN11), and claudin23 (CLDN23). Among them, the reduction of claudin11 transcript during invasion was confirmed by real-time PCR (Fig. 2A). TJs are intercellular ring structures that seal adjacent cells to one another. The TJ of ECs is mainly responsible for regulating paracellular permeability and maintaining planar cell polarity (139). TJs are composed of transmembrane proteins, which act as a fence to restrict the diffusion of lipids and proteins within the plane of the membrane. These data suggest that breakdown of TJ is associated with, and likely indispensable for, changes in cell polarity that occur during EC invasion.


Figure 2
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  		Fig. 2. Differential expression of cell adhesion molecule and extracellular matrix genes during EC invasion. A: verification of expression profiling at 0, 6, 12, and 18 h (n = 3 experiments). Invading cells were harvested at each time point, and total RNA was purified. The expression of mRNA was assessed by real-time RT-PCR. The data represent means ± SE (n ≥ 3) normalized as described in MATERIALS AND METHODS. *P < 0.05, **P < 0.01 compared with time 0 using Students t-test. B: time course of protein expression for claudin 5, integrin-{alpha}2, and integrin-{alpha}v during EC invasion. Invading cells within collagen matrices were collected at indicated times, placed in boiling Laemmli sample buffer, and heated at 95°C for 10 min before Western blot analysis using antibodies against indicated proteins. The expression of actin was used as a loading control. Data shown are representative of 3 independent experiments. PODXL, podocalyxin-like protein; CLDN11, claudin11; PCDH10, protocadherin10; ITGA2, integrin-{alpha}2; ITGAV, integrin-{alpha}v; SELE, E-selectin; ANTXR2, anthrax toxin receptor 2; SPP1, secreted phosphoprotein 1; NID2, nidogen 2; NOX4, NADPH oxidase 4.

 
In contrast to TJ components, a series of cell adhesion molecule (CAM) genes associated with adherens junctions and cell-matrix interactions were upregulated. These include protocadherin10 (PCDH10), protocadherin17 (PCDH17), mucosal addressin cell adhesion molecule-1 (MADCAM1), integrin-{alpha}2 (ITGA2), integrin-{alpha}v (ITGAV), and E-selectin (SELE) (supplemental Table S2). Protocadherin10 and protocadherin17, cadherin-related receptors belonging to the {delta}2-protocadherin subfamily, not only exhibit cell-cell adhesion activities but also are believed to possess other functions, such as signal transduction and growth control (115). Our real-time PCR analysis revealed an increase in the expression levels of mRNA for PCDH10 and PCDH17 as ECs undergo invasion, increasing approximately two- and threefold, respectively (Fig. 2A). Moreover, ITGA2 and ITGAV were induced approximately threefold at the mRNA level (supplemental Table S2), and these data were confirmed by real-time PCR analysis (Fig. 2A). Another CAM gene with enhanced expression confirmed by real-time PCR analysis is E-selectin (Fig. 2A).

Strikingly, also induced are some transmembrane receptors, which are involved in adhesion in some respects. These induced molecules are CUB domain-containing protein 1 (CDCP1), anthrax toxin receptor 2 (ANTXR2), anthrax toxin receptor 1 (ANTXR1), transcript variant 3, and podocalyxin-like (PODXL) protein. CDCP1, which represents a putative transmembrane protein containing three CUB domains in its extracellular part, is found to be overexpressed in human colon and lung cancer (133). Intriguingly, upregulation of ANTXR1 and ANTXR2 occurred during EC invasion. ANTXR1 is also known as a tumor-specific endothelial marker (20). ANTXR2 has been reported to bind collagen type IV and laminin directly (7), suggesting an important role for basement membrane matrix assembly. In addition, podocalyxin-like protein is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34 (69, 70). It has been reported to be a novel sinusoidal endothelial cell marker in hepatocellular carcinoma (22, 56). The induction of ANTXR2 and PODXL transcripts during invasion was verified by real-time PCR (Fig. 2A). Overall, these findings indicate that augmented expression of these CAM genes is consistent with stimulation by S1P and angiogenic growth factors. These factors would be expected to foster adequate adhesions with adjacent cells and ECM, as well as initiate diverse signaling events that mediate EC invasion.

In addition, multiple ECM genes are induced, such as secreted phosphoprotein 1 (SPP1), statherin (STATH), decorin (DCN), transcript variant B, laminin-{gamma}2 (LAMC2), collagen, type I, {alpha}2 (COL1A2), and nidogen 2 (NID2) (supplemental Table S2). The ECM serves as a scaffold in which mechanical forces are established among distal ECs, thereby providing guidance cues in the absence of cell-cell contact (34). Moreover, ECs require adhesions to ECM for migration, invasion, proliferation, and survival, all of which are critical for the process of angiogenesis. We used real-time PCR analysis to further confirm the increased expression of mRNA for secreted phosphoprotein 1, nidogen 2 and NADPH oxidase 4 (Fig. 2A). Secreted phosphoprotein SPP1, also known as osteopontin, is a matricellular protein that is upregulated during vascular injury and wound healing (48, 83). In addition, multiple studies have demonstrated upregulation of SPP1 in endometrial tissue where angiogenesis occurs during pregnancy (63–66). Nidogen 2 is a basement membrane-associated molecule that binds collagen I, collagen IV, perlecan, and laminin-1 (73, 93). Such induction of diverse ECM components in this system suggests an important role for the ECM as well as basement membrane components to precisely regulate the invasion process.

Induction of genes encoding ECM proteinases and their inhibitors. Cumulative evidence has shown that an intricate balance between ECM proteinases and their inhibitors is critical for mediating diverse physiological events such as lineage decisions during embryogenesis, wound repair, cell migration, vascular stabilization, and survival (28, 132, 146, 159). Based on distinct domain structures, ECM proteolytic enzymes are divided into several protein families (148), many of which also display an augmented expression in our invasion system (supplemental Table S2). Microarray data indicated that ADAMTS1, a secreted metalloproteinase with thrombospondin type I motifs, was induced at the mRNA and protein level during invasion (Fig. 3, A and B). As shown in Fig. 3A and supplemental Table S2, another upregulated ADAMTSs is ADAMTS5, which is the major proteinase responsible for cartilage degradation in vivo and in vitro (49, 145). Interestingly, {alpha}2-macroglobulin, an endogenous inhibitor of ADAMTS5, was also induced (supplemental Table S2).


Figure 3
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  		Fig. 3. Induction of selected genes encoding extracellular matrix proteases and protease inhibitors during EC invasion. A: verification of mRNA expression for a disintegrin and metalloproteinase with thrombospondin-type repeats-1 (ADAMTS1), ADAMTS5, and latexin (LXN) observed from microarray analysis. Experiments were performed as described in Fig. 2. Data represent means ± SE (n ≥ 3). *P < 0.05, compared with time 0 using Students t-test. B: time course of protein expression for ADAMTS1 during EC invasion. Protein samples were collected as in Fig. 2. Antibodies against ADAMTS1 and actin, which was used a loading control, were used in Western blot analyses. Data shown are representative of 3 independent experiments.

 
Other serine proteases including tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), together with one of their inhibitors, neuroserpin (SERPINI1), were upregulated (supplemental Table S2). Both tPA and uPA are involved in angiogenesis and tumor progression (24, 62, 114, 118), whereas neuroserpin is seldom reported to possess endothelial-related functions due to its abundance in the nervous system. In vitro studies with cultured hippocampal neurons demonstrated that neuroserpin mRNA is increased by depolarization with elevated extracellular KCl (9). Interestingly, we have observed an intriguing upregulation of many voltage-gated potassium channels as well (supplemental Table S2). Other upregulated genes encoding ECM proteinases and protease inhibitors include trypsin 2, matrix metalloproteinase-10 (MMP-10), cathepsin K (CTSK), and latexin (LXN) (supplemental Table S2). MMP-10, a soluble proteinase, has been reported to facilitate human capillary tube regression (131), and its repression is essential in maintaining vascular integrity (21). Cathepsin K, a cysteine protease that exhibits strong degradative activity against the ECM, is involved in prostate cancer progression (16). In addition, the increased gene expression of latexin was validated by real-time PCR analysis (Fig. 3A). Latexin, the endogenous protein inhibitor of the A/B subfamily of metallocarboxypeptidases, is discovered as a primary regulator of hematopoietic stem cells (82). These findings suggest that a delicate protease/antiprotease balance orchestrates the process of EC invasion into 3-D collagen matrices.

ADAMTS1 is required for EC invasion in 3-D collagen matrices. The data in Fig. 3 demonstrate that ADAMTS1 is upregulated at the mRNA and protein level. Because ADAMTS1 is known to associate with the plasma membrane and has been reported to promote collagen degradation (116), we next investigated whether ADAMTS1 is functionally required for EC invasion in collagen matrices. To accomplish this, ECs were treated with siRNA directed to G3PDH, ADAM17, ADAMTS1, and ADAMTS4. Western blot analyses of extracts collected from invading cultures revealed selective knockdown of ADAMTS1 and G3PDH controls with respective siRNAs (Fig. 4A). Photographs (side view) of invading cultures revealed that ECs treated with ADAMTS1 siRNA before being placed in invasion assays invaded shorter distances and contained larger lumens (Fig. 4B). ECs were tested in invasion assays in the presence and absence of phorbol ester (TPA), which enhanced invasion of ECs placed in 3-D matrices (72, 100, 102). Quantification of the invasion density revealed decreased numbers of invading cells with ADAMTS1 siRNA treatment (Fig. 4C). Invasion distance in ADAMTS1 siRNA-treated ECs was also significantly decreased compared with controls in the presence and absence of phorbol ester (Fig. 4D). Also, quantification of lumen diameter in invading structures revealed ECs treated with ADAMTS1 siRNA assembled into structures containing larger lumens (Fig. 4E). Thus, silencing of ADAMTS1 with siRNA confirms the functional involvement of ADAMTS1 in S1P and growth factor-stimulated EC invasion in 3-D collagen matrices.


Figure 4
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  		Fig. 4. Knockdown of ADAMTS1 interferes with EC invasion. A: verification of ADAMTS1 protein suppression. Custom pooled small interfering RNA (siRNA) were delivered to Human umbilical vein endothelial cells (HUVECs) before seeding on the surface of collagen matrices. Cells were allowed to invade for 24 h before cultures were fixed and cell extracts were prepared. Antibodies against ADAMTS1 and G3PDH, as an experimental control, were used for Western blot analyses. B: photographs illustrating the invasion responses (side view). Cultures were fixed at 24 h and stained with toluidine blue. White arrows indicate areas of lumen formation, and black arrowheads indicate monolayer of endothelial cells. C–E: quantification of EC invasion responses. Cells were allowed to invade for 24 h in the absence (control) or presence of 50 ng/ml of a phorbol ester, tumor-promoting antigen (TPA). Cultures were fixed, stained, and quantified for invasion density (C), distance (D), and lumen diameter (E). HPF, high-powered field. For invasion density, data represent average numbers of invading cells per standardized field (n = 3 fields). For invasion distance and lumen diameter, data from 100 cells were averaged and are presented as means ± SE. *P < 0.05 using Students t-test.

 
Regulated expression of genes implicated in cell signaling pathways. Numerous signaling pathways have been reported to modulate angiogenesis. Here, we demonstrated the differential expression of multiple members of the Wnt and Notch pathways. Multiple Wnt signaling pathway members were identified, including dickkopf homolog 2 (DKK2) and disheveled associated activator of morphogenesis (DAAM1), which were upregulated (supplemental Table S2). In contrast, AXIN1 upregulated 1 (AXUD1) and Frizzled homolog 4 (FZD4) were downregulated (supplemental Table S3). These data were confirmed by real-time PCR (Fig. 5A). Although the Wnt signaling pathway has a well-defined role in development, its role in angiogenesis and the exact components involved have not been completely delineated. Unlike DKK1, a pure inhibitor of the Wnt/β-catenin signaling pathway, DKK2 can function either as an agonist or antagonist of Wnt signaling, depending on the cellular context (13, 91, 164). DAAM1, a member of the mammalian diaphanous-related formins, is shown to communicate with Rho GTPases and Src to regulate the Wnt/planar cell polarity signaling pathway (1). Beyond the transcriptional influences, we showed that Dkk2 and Daam1 proteins were induced as invasion proceeds (Fig. 5D). In addition, consistent with a recent finding that the expression of AXUD1, a downstream target of the Wnt regulator AXIN1, was decreased in lung, kidney, liver, and colon cancers (61), AXUD1 is downregulated in our EC invasion system, suggesting an inhibitory role in vascular morphogenesis. Surprisingly, also downregulated is FZD4, a putative Wnt receptor implicated in retinal angiogenesis (121). These findings raise the possibility that both canonical and noncanonical Wnt pathways may contribute to EC invasion.


Figure 5
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  		Fig. 5. Genes involved in various signaling pathways are regulated during EC invasion. Gene expression profiling of members of Wnt (A), Notch (B), and hedgehog and NF{kappa}B (C) signaling pathways. Experiments were conducted as described in Fig. 2. The data represent means ± SE (n ≥ 3). *P < 0.05, **P < 0.01 compared with time 0 using Students t-test. D: time course of protein expression for Delta-like 4 (Dll4), Daam1, and dickkopf homolog 2 (Dkk2) during EC invasion. Protein samples were collected as in Fig. 2. Antibodies against Delta-like 4, Daam1, Dkk2, and actin, which was used as a loading control, were used in Western blot analyses. Data shown are representative of 3 independent experiments. FZD4, frizzled homolog 4; AXUD1, AXIN1 upregulated 1; HEY1, hairy and enhancer of split (HES)-related with YRPW motif 1; HIP, hedgehog-interacting protein.

 
Next, a set of genes involved in Notch signaling was downregulated as invasion proceeds. These include Delta-like 4 (DLL4), jagged 1 (JAG1), hairy and enhancer of split 1 (HES1), and HES-related with YRPW motif 1 (HEY1) (supplemental Table S3). DLL4 and JAG1 are Notch ligands, while HEY1 and HES1 are target genes of Notch. In this experimental system, we confirmed the reduction of DLL4 at the mRNA and protein level (Fig. 5, B and D) and a dramatic decrease in HEY1 mRNA by real-time PCR analysis during EC invasion (Fig. 5B). In addition, ligand of numb-protein X (LNX), a RING-type E3 ubiquitin ligase, was found to be upregulated (supplemental Table S2). Intriguingly, LNX can cause proteasome-dependent degradation of Notch inhibitor, Numb, thereby enhancing Notch signaling (104).

Additional genes involved in cellular signaling include hedgehog-interacting protein (HIP) and NF-{kappa}B inhibitor-{alpha} (NFKBIa), both of which exhibit downregulation (Fig. 5C and supplemental Table S3). Hedgehog-interacting protein is a ligand for all three members of the hedgehog family, Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). HIP is expressed at high levels in ECs and can antagonize Shh responses in epithelial cells. Its expression is reduced on matrigel during chord formation, as well as in human cancers of the lung, liver, and gastrointestinal tract (108), which is consistent with downregulation in our model. Thus, HIP may function as an angiogenic suppressor, suggesting that activation of the hedgehog pathway may allow invasion to proceed. In addition, tumor invasion and angiogenesis are associated with NF-{kappa}B-mediated gene products, such as MMPs, uPA, and numerous chemokines (10, 43, 105), many of which are also induced during invasion. The signals that activate NF-{kappa}B during metastasis or angiogenesis remain incompletely understood, but they may be related to mutation of NF-{kappa}B inhibitor-{alpha} gene and enhanced degradation of NF-{kappa}B inhibitor-{alpha} protein (98, 162). Our findings suggest that a transcriptional regulation of NF-{kappa}B inhibitor-{alpha} may be involved in controlling EC invasion, along with modulation of Wnt, hedgehog, and Notch signaling pathways.

Notch signaling negatively regulates EC invasion. Because several members of the Notch pathway were downregulated during EC invasion, we theorized that Notch inhibition would stimulate EC invasion. Treatment with {gamma}-secretase inhibitor, which prevents Notch processing, stimulated EC invasion in a dose-dependent manner (Fig. 6A). To confirm these data, we induced Notch activation in ECs using recombinant lentiviruses to express DLL4 and two constitutively active forms of Notch, Notch1 intracellular domain (N1ICD) and Notch4 intracellular domain (N4ICD), fused to a COOH-terminal Flag epitope, along with GFP control. Expression of DLL4 and activated Notch4 markedly blocked the EC invasion in the presence and absence of TPA, whereas activation of Notch1 modestly but significantly reduced invasion (Fig. 6B). In the presence of TPA, the ability of N4ICD and DLL4 to inhibit invasion was less pronounced, but N1ICD, N4ICD, and DLL4 all similarly reduced invasion compared with controls. Moreover, the invading structures formed by activated Notch4- or DLL4-expressing cells were markedly shorter and exhibited less branching than those derived from cells transduced with a GFP expression vector (Fig. 6, C and E), suggesting that activation of Notch signaling may alter the decision of EC for invading but also impair the ability to migrate in collagen matrices. Incorporation of COOH-terminal FLAG epitopes resulted in an ability to distinguish recombinantly expressed from endogenous proteins. We typically achieved lentiviral transduction efficiencies from 50% to 80% (data not shown). Figure 6D shows the expression of FLAG-tagged N1ICD, N4ICD, and DLL4 protein by immunoblotting. It is reported that Notch intracellular domain is targeted for degradation via proteasome pathway (106, 163). We have observed similar results in HUVECs, where treatment with proteosome inhibitors stabilized activated Notch protein expression (data not shown). Localization of Notch recombinant proteins within invading ECs was examined by immunofluorescence (top view Flag-FITC, Fig. 6E; supplemental Fig. S1). Photographs illustrate that GFP distributed throughout the cytoplasm, whereas N1ICD and N4ICD proteins localized to nucleus, which is typical of constitutively active Notch proteins (135, 147), and DLL4 protein localized on the plasma membrane (supplemental Fig. S1). Furthermore, GFP-expressing cells remained in the monolayer and invaded (Fig. 6E, top right). N1ICD- and N4ICD-positive cells, in contrast, did not invade and remained in the monolayer. DLL4-positive cells, like GFP, were detectable in invading cells as well as those that remained in the monolayer.


Figure 6
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  		Fig. 6. Notch signaling negatively regulates EC invasion. A: {gamma}-secretase inhibition promotes EC invasion. Cells were resuspended with indicated concentrations of {gamma}-secretase inhibitor IX or vehicle control (DMSO) for 20 min before being seeded on collagen matrices containing 100 nM S1P for 24 h. Data represent average numbers of invading cells per standardized field (n = 3 fields). B and C: activation of Notch signaling inhibits EC invasion. Recombinant lentiviruses were generated that express green fluorescent protein (GFP), Notch1 intracellular domain (N1ICD), Notch4 intracellular domain (N4ICD), and DLL4 fused to a COOH-terminal FLAG epitope. Three to seven days after transduction, ECs were allowed to invade for 24 h with 50 ng/ml phorbol ester (TPA) or without (control) and were fixed, stained, and quantified for invasion density (n = 3 fields) (B) and distance (n = 30 cells) (C). *P < 0.05, **P < 0.01 vs. GFP control using Students t-test. D: expression of FLAG-tagged N1ICD, N4ICD, and DLL4 in HUVECs. Cells were treated with TPA and proteasome inhibitor I for 12 h before collection of cell extracts. Western blot analyses were conducted with anti-FLAG monoclonal antibodies. E: photographs depicting the inhibition of invasion responses in Notch-activated ECs. Left to right: top-viewed and side-viewed images of 24-h toluidine blue-stained cultures are shown in first two columns. Black arrows indicate invading ECs and black arrowheads indicate EC monolayer. In the third column, localization of N1ICD, N4ICD, and DLL4 proteins is shown by immunofluorescence of top-viewed 12-h cultures stained with an anti-FLAG monoclonal primary antibody and FITC-conjugated secondary antibodies. The fourth column shows a side view of invading ECs immunofluorescently labeled with FLAG antibodies in 24-h cultures. White arrowheads indicate monolayer of endothelial cells. Differential interference contrast (DIC) and FITC images were overlaid in the fifth column. Note that N1ICD- and N4ICD-positive cells remain in the monolayer.

 

   DISCUSSION
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 ABSTRACT
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A variety of studies have used genome-wide screening and differential gene analysis to identify regulated genes in 3-D models of in vitro angiogenesis (7, 47, 52, 67). Despite minor variations in the experimental conditions and the way ECs are seeded, a high degree of consistency emerges from these data, and numerous findings are in accordance with in vivo angiogenic events. In the present study, we have examined the gene expression profile of primary EC invasion stimulated by angiogenic growth factors and S1P.

The three-dimensional model used in the present study incorporates the lysosphingolipid S1P, which is present in serum and is released by activated platelets. To our knowledge, this is the first study to investigate S1P- and growth factor-induced gene expression changes in primary endothelial cells. The assay system used here mimics a wound environment where S1P release would be coupled with local production of VEGF and bFGF to stimulate granulation tissue formation and new blood vessel growth. The combination of these factors not only stimulates EC invasion but likewise upregulates endothelial expression of the surface markers E-selectin and mucosal addressin cell adhesion molecule-1 (MADCAM1) that are involved in directing leukocyte extravasation (3, 8). Interestingly, NADPH oxidase 4 and cytochrome p450 (CYP450) are upregulated significantly at 6 h of invasion (Fig. 2A and supplemental Table S2), which suggests that this pathway may be initiated to activate the endothelium following exposure to S1P and angiogenic growth factors. CYP450 monooxygenases and NADPH oxidase can induce E-selectin, ICAM-1, and VCAM-1, whereas MADCAM1 induction may depend exclusively on CYP450-derived oxidants (130). Furthermore, multiple chemokines and chemokine receptor mRNA expression were observed to be significantly regulated with time. These are CCL1, CXCR, and CCRL1. Other relevant molecules include THY1, TNFSF11, TNFSF18, and COX-1. A recent study has shown that stable knockdown of one S1P receptor, S1P1, in ECs decreased the expression of several CAMs and influenced the inflammatory response of ECs (75). These data suggest that exposing the endothelium to S1P and angiogenic growth factors not only stimulates new blood vessel growth, but also alters EC expression of several inflammatory proteins that promote leukocyte extravasation and wound healing. These data are consistent with the well-known link between inflammatory cells and angiogenesis not only during tissue healing, but in many other contexts as well, including pregnancy (68, 149, 153), choroidal neovascularization (41, 51, 107, 127, 151), and tumor angiogenesis (26, 90, 113). These data also agree with the recognized role for S1P as an important inflammatory mediator that regulates lymphocyte egress, graft rejection, and recurrent multiple sclerosis (11, 35, 92, 123).

Cell-cell and cell-matrix contacts are integral in regulating angiogenesis. We have observed the downregulation of a set of genes associated with tight junctions, including CX40, CX45, ZO2, CLDN5, CLDN11, and CLDN23 (Fig. 2 and supplemental Table S2). Connexins have been recognized as tumor suppressors, whose expression is relevant to tumor progression in breast, lung, skin, liver, bladder, esophagus, and prostate cancers (95). Among members of connexin family, connexins 26 and 43 are the most extensively studied and found to be downregulated in various cancers and tumor angiogenesis (95). In a recent study, decreased expression of connexin 40 is detected in conditional phosphatase and tensin homolog (PTEN) knockout mice, which display enhanced tumorigenesis (53). Similarly, alterations in the expression levels of claudins (CLDNs) occur in various cancers. A thorough survey of CLDN expression in normal and neoplastic tissues indicates that the expression of most CLDN genes is decreased in cancers, while CLDN3, CLDN4, and CLDN7 are elevated in many malignancies (57). These data suggest that a decrease in endothelial tight junctions is a prerequisite for the onset of angiogenesis. In addition, our findings regarding the increased expression of integrin-{alpha}2 and -{alpha}v fits the well-defined roles for integrin receptors in sprouting angiogenesis (31). These integrins not only provide the adequate and necessary interactions with ECM but also emit intracellular signals to control cell shape and contractility. Integrin-{alpha}2β1 modulates VEGF-induced angiogenesis in vivo by a notable induction of actin polymerization through inhibition of PKA (cAMP-dependent protein kinase A) activity (136, 137, 160). Like {alpha}2β1 in collagen matrices (33), {alpha}vβ3 in combination with {alpha}5β1 control EC shape changes, including vacuolation and lumen formation in fibrin matrices (6). Therefore it seems plausible that multiple integrins may act together to create combinations of signals for neovascularization. Another intriguing finding in the present study is the induction of two members of {delta}2-protocadherin gene family, PCDH10 and PCDH17 (Fig. 2). So far, little is known about the expression and function of these two protocadherins. However, it is clear that protocadherins may be more involved in intracellular signaling than in adhesive functions. One member of {delta}2-protocadherin subfamily, PCDH8/PAPC, is found to interact with the Frizzled 7 receptor and can regulate the activity of the small GTPase, Rho and JNK, indicating a role in modulating the Wnt/planar cell polarity signaling pathway (94, 152). Perturbing PCDH8 activity in mice leads to the disruption of somite epithelialization during development (119), yet mice defective in PCDH8 not only possess normal skeleton but also are viable and fertile (165). These results suggest that functional redundancy among protocadherins exists.

ECM remodeling is crucial for all aspects of vascular biology and tumor progression. The ECM can be remodeled by biosynthesis and proteolytic degradation. We have demonstrated that the expression of several ECM genes is regulated during EC invasion (Fig. 2 and supplemental Table S2). Many ECM components identified in our microarray analysis were likewise upregulated in other studies using 3-D models of in vitro angiogenesis (7, 67) and proven as regulators of angiogenesis or vascular regression events (34, 142). Decorin, a proteoglycan that plays an important role in regulating collagen fibril organization (29), was induced in our invasion system (supplemental Table S2). Whereas decorin can inhibit EC migration and tube formation (30, 71), a line of evidence showed that decorin promotes endothelial tube formation in collagen gels (134). These conflicting results may be due to the complexity of ECM.

In addition to matrix molecules, a set of genes associated with the proteolytic processes of ECM was upregulated, including ADAMTS1, ADAMTS5, A2M, tPA, uPA, SERPINI1, MMP-10, CTSK, PRSS2, and LXN (Fig. 3 and supplemental Table S2). These molecules function as proteases and endogenous protease inhibitors. Cumulative evidence implies that the balance between proteases and their inhibitors is critical for angiogenesis. Most of these proteases are expressed in the extracellular milieu as inactive forms, which can be activated upon proteolytic cleavage by several families of proteases. To date, we have become aware that those proteases can target many non-ECM proteins, including growth factors, growth factor receptors, cell-associated molecules, and cytokines. Because several clinical trials using ECM proteases and their inhibitors as targets unfortunately appear inefficacious (25, 36, 111), a full understanding of the involvement and function of these molecules will be helpful for future treatments.

Gene expression profiling of the 3-D cultures revealed that S1P and angiogenic growth factor stimulation upregulated ADAMTS1 expression, which has been correlated with the highly vascularized ovulation cone in ovulation (15, 97, 120). Furthermore, targeted disruption of mouse ADAMTS1 gene results in abnormal adrenal medulla architecture without capillary formation, suggesting a role in angiogenesis (141). ADAMTS1 can cleave the proteoglycans aggrecan and versican (77, 128), which regulates ovulation (125). Inactivation of the gene by homologous recombination in mice resulted in decreased growth and renal defects (140). ADAMTS1 has been demonstrated to promote collagen degradation (116), fitting with our data that decreasing ADAMTS1 expression levels resulted in decreased invasion density and distances. ADAMTS1 was first cloned from a colon adenocarcinoma (76) and has been reported to be silenced by hypermethylation in colorectal tumors (84). Of note, the addition of recombinant ADAMTS1 to corneal pocket and chick chorioallantoic angiogenic assays (155) resulted in inhibition of angiogenic responses. ADAMTS1 can diminish VEGFR2 phosphorylation by binding and sequestering VEGF. This binding occurs via the COOH-terminal thrombospondin (TSP) motifs (87). Interestingly, ADAMTS1 is processed in this region to release the last two TSP domains. The cleavage event requires metalloproteinases, which may include MMP-2, MMP-8, and MMP-15 (122). It has been proposed that ADAMTS1 could function as an anti-angiogenic molecule through the release of TSP repeats (60) as do other proteins that harbor anti-angiogenic potential, such as collagen XVIII and plasminogen (17, 109, 110). Thus, presentation of ADAMTS1 on the cell surface or in soluble form may be critical for determining its mechanism of action. Thus far, we have been unable to detect cleavage or liberation of ADAMTS1 from the endothelial surface, consistent with its role as a collagenase during EC invasion.

Notch signaling has long been implicated in angiogenic events. It is well documented that the activation of Notch signaling plays an important role in vascular development (54, 59, 74, 124). Leong and colleagues (80) have described that expression of constitutively active Notch4 in human dermal microvascular endothelial cells inhibits endothelial sprouting and migration through collagen but not fibrinogen. In addition, Sainson and colleagues (126) have studied Notch inhibition in ECs resuspended in fibrin matrices. By using {gamma}-secretase inhibitors, a dominant-negative form of Notch1 or antisense for Notch1 and Notch4, Notch inhibition resulted in enlarged sprout diameter but not length through enhanced cell proliferation and extensive sprout branching. In contrast, another study indicated that constitutive activation of Notch1 enhanced network and cord formation of human iliac artery ECs (86). Here, we showed that several members of Notch signaling pathway, in particular target genes of Notch, were downregulated with time in our 3-D invasion system (Fig. 5 and supplemental Table S3), suggesting that Notch signaling is inhibited during EC invasion. Inhibition of Notch signaling using {gamma}-secretase inhibitor promoted increased numbers of invading ECs, and overexpression of a Notch ligand, DLL4, and two activated Notch receptors, N1ICD and N4ICD, all inhibited the EC invasion responses (Fig. 6). Even though all the above Notch data emerged from in vitro 3-D models, minor variations in the experimental conditions, such as matrices used, stimuli added, cell types involved, and the way Notch signal is manipulated, are present among those studies. Paradoxically, the activation of Notch signaling can exhibit both inhibitory effects on angiogenesis (55, 150, 161, 169) as well as promotive (27, 89, 112, 168). There are several variables that may account for these contradictory results. First, signals emitted from cis-interaction (cell-autonomous activation) could be different from those from trans-interaction (non-cell-autonomous manner) (50, 81). Next, different Notch intracellular domains may contribute to different transcriptional functions and target different genes for expression (42, 117). This, therefore, could be the reason that we observed differential effects between activated Notch1 and Notch4 on EC invasion. Another possible explanation is that a conventional two-dimensional cell culture environment fails to accurately reflect how Notch signaling is regulated during EC morphogenesis since sprouting angiogenesis is a 3-D multistep process. All of above may account for the similarities or discrepancies between our results and other's (80, 86, 126).

In conclusion, following stimulation by S1P and angiogenic growth factors, bFGF, and VEGF, gene expression profiles during EC invasion in 3-D collagen matrices were analyzed. Consistencies exist between our data and findings reported from other in vitro models and in vivo studies. The validation of gene expression in this study is focused on cell adhesion molecules, ECM, proteases/antiproteases, Wnt and Notch signaling pathways, and potential regulators of angiogenesis at mRNA or protein levels. In addition, EC invasion requires ADAMTS1 and is regulated following manipulation of Notch signaling. This knowledge will serve as a valuable resource for exploring molecular mechanisms underlying sprouting angiogenesis.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The work was supported by American Heart Association Scientist Development Grant 0530020N (to K. J. Bayless).


   ACKNOWLEDGMENTS
 
The authors thank Dr. George Davis (University of Missouri) for the kind gift of GFP lentiviral expression plasmid, Dr. David Zawieja (Texas A&M HSC) for use of the Bio-Rad real-time PCR cycler, and Natasha Popovic for helpful advice. We thank Dr. Farida Sohrabji for the gift of claudin-5 antisera and Dr. Greg Bix for review of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. J. Bayless, Assistant Professor, Dept. of Molecular & Cellular Medicine, Texas A&M Univ. HSC, College Station, TX 77843-1114 (e-mail: kbayless{at}medicine.tamhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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