Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins

Monica Iurlaro1,2, Marta Scatena3, Wen-Hui Zhu1, Eric Fogel4, Susan L. Wieting4 and Roberto F. Nicosia1,4,*

1 Department of Pathology, University of Washington, Seattle, WA 98195, USA
2 IFOM, Institute FIRC for Molecular Oncology, Milan 20139, Italy
3 Department of Engineered Biomaterials, University of Washington, Seattle, WA 98195, USA
4 Veterans Administration Puget Sound Health Care System, Seattle, WA 98108, USA

* Author for correspondence (e-mail: roberto.nicosia{at}med.va.gov)

Accepted 22 April 2003


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 Materials and Methods
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Recent studies have implicated the Tie2 tyrosine-kinase receptor and its main ligands - angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) - as crucial regulators of mural cell recruitment during angiogenesis. Angiopoietin-mediated activation of Tie2 promotes perivascular mural cell assembly, but the mechanisms regulating this process are poorly understood because differentiated mural cells do not have the Tie2 receptor, which is reportedly expressed only in endothelial cells. There is also no direct evidence that Tie2 activation results in production of mural cell chemoattractants by the endothelium. In the rat aorta model of angiogenesis, developing microvessels recruit mural cells from the intimal/subintimal layers of the aortic wall. Ang-1 and Ang-2 promote angiogenesis in this system, stimulating branching morphogenesis and mural cell assembly. Mural precursor cells (MPCs) isolated with a nonenzymatic method from the intimal aspect of the rat aorta were positive for smooth muscle cell markers ({alpha}-smooth muscle actin and calponin) and negative for endothelial markers (factor-VIII-related antigen and CD31). These cells responded chemotactically to Ang-1 and Ang-2, and secreted MMP-2 when treated with these factors. Western-blot analysis, immunocytochemistry and RT-PCR demonstrated that MPCs express the Tie2 receptor. Immunoprecipitation showed phosphorylation of MPC Tie2 on tyrosine residues upon stimulation with Ang-1 or Ang-2. Surface expression of Tie2 was further demonstrated by isolating Tie2+/{alpha}-smooth muscle actin+ MPCs from primary aortic outgrowths with anti-Tie2-IgG-coated magnetic beads. Immunostaining of the rat aorta confirmed expression of Tie2 not only in endothelial cells but also in nonendothelial mesenchymal cells located in the aortic intimal/subintimal layers, which are the source of MPCs. These data indicate that the aortic wall contains Tie2+ nonendothelial mesenchymal cells and suggest that Tie2-related recruitment of mural cells during angiogenesis may occur through angiopoietin-mediated direct stimulation of these cells.

Key words: Angiogenesis, Angiopoietins, Tie2, Mural cells, Pericytes


    Introduction
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 Introduction
 Materials and Methods
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 Discussion
 References
 
Angiogenesis, the formation of new blood vessels from the endothelium of pre-existing vessels, plays an important role during developmental, physiological, reactive and pathological processes (Carmeliet and Jain, 2000Go; Nicosia and Villaschi, 1999Go). Neovessels form in response to growth factors that induce endothelial migration, proliferation, proteolytic activity and capillary tube formation. Neovessel survival following angiogenesis depends on multiple factors including the assembly of a stabilizing layer of mural cells (smooth muscle cells/pericytes) around the endothelium (Benjamin et al., 1999Go; Hirschi and D'Amore, 1996Go). This process is regulated by paracrine interactions between sprouting endothelial cells and surrounding mesenchyme (Nicosia and Villaschi, 1995Go; Villaschi and Nicosia, 1994Go).

The angiopoietins, a newly discovered family of angioregulatory polypeptides, play a crucial role in endothelial sprouting, vessel wall remodeling and mural cell recruitment. Angiopoietin-1 (Ang-1) binds to and induces phosphorylation of Tie2 (Sato et al., 1995Go), a tyrosine kinase receptor expressed by endothelial cells (Sato et al., 1995Go; Davis et al., 1996Go). Angiopoietin-2 (Ang-2) has different effects on Tie2 phosphorylation depending on the context in which it operates. It can function as an agonist or an antagonist depending on its concentration and duration of treatment (Teichert-Kuliszewska et al., 2001Go) or the extracellular matrix environment (Kim et al., 2000aGo; Maisonpierre et al., 1997Go). Ang-2 binds to endothelial Tie2 without inducing its phosphorylation but can also activate Tie2 in nonendothelial mesenchymal cells genetically engineered to express this receptor (Maisonpierre et al., 1997Go). Ang-3 and Ang-4 bind to Tie2 and are believed to be the mouse and the human counterparts, respectively, of the same gene. Ang-3 acts as an antagonist, whereas Ang-4 appears to function as an agonist (Valenzuela et al., 1999Go).

Ang-1 is produced predominantly by mural and perivascular mesenchymal cells (Davis et al., 1996Go; Kim et al., 2001Go). Ang-2 is produced by endothelial, mural and mesenchymal cells (Holash et al., 1999Go; Mandriota et al., 1998). Genetic ablation of the Ang-1 or Tie2 genes results in lethal defects in vascular remodeling, maturation and stabilization (Sato et al., 1995Go; Suri et al., 1996Go). Vessels formed in the absence of a functioning Ang-1/Tie2 system do not acquire a properly assembled layer of mural cells and fail to mature into an arborizing network of small and large vessels (Sato et al., 1995Go; Suri et al., 1996Go). Point mutations of the gene encoding the Tie2 receptor resulting in constitutive Tie2 phosphorylation cause arteriovenous malformations characterized by abnormal layering of smooth muscle cells around the endothelium (Vikkula et al., 1996Go). Overexpression of Ang-2 in transgenic mice causes abnormal vascular development similar to that observed in Ang-1 and Tie2 knockout mice (Maisonpierre et al., 1997Go). Overexpression of Ang-1 in mice genetically engineered to express this molecule in the skin induces formation of a dense vascular network composed of vessels that are larger and more branched than those of control animals (Suri et al., 1998Go).

Although there is compelling evidence that the angiopoietin/Tie2 system is involved in mural cell recruitment, the mechanisms of this process are poorly understood. To gain further insight into the mechanisms by which mural cells are recruited by sprouting neovessels, we established a method to isolate mural precursor cells (MPCs) from the rat aorta. MPCs were isolated from the aortic intimal/subintimal layers, which have the capacity to generate mural cell-coated neovessels in collagen gel culture. Isolated MPCs are polygonal in shape, have a high proliferative rate and produce large amounts of laminin- and type-IV collagen-rich extracellular matrix (Villaschi et al., 1994Go). In addition, they respond to endothelial cues by transforming into a dendritic phenotype and becoming incorporated as pericytes in capillary networks bioengineered from isolated endothelial cells in a collagen gel overlay assay (Nicosia and Villaschi, 1995Go).

Tie2 is reportedly expressed in endothelial cells but not in mural cells (Sato et al., 1995Go; Kim et al., 2001Go; Suri et al., 1996Go). In this paper, we report that aorta-derived MPCs express the Tie2 receptor, respond chemotactically to Ang-1 and Ang-2, and produce matrix metalloproteinase-2 (MMP-2) when stimulated by these factors. These observations suggest that mural cells of angiogenic neovessels might arise from a precursor subpopulation of less differentiated mesenchymal cells capable of responding directly to angiopoietins produced by the sprouting endothelium.


    Materials and Methods
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recombinant proteins
Ang-1 and Ang-2 were provided by Regeneron Pharmaceuticals (Tarrytown, NY) or purchased from R&D Systems (Minneapolis, MN). Soluble Tie2 (Tie2Fc) was obtained from Regeneron Pharmaceuticals or Immunex (Seattle, WA). Platelet-derived growth factor BB (PDGF-BB) was purchased from R&D Systems.

Rat aorta model of angiogenesis
Rings of rat aorta were prepared and cultured in floating collagen gels as previously described (Nicosia and Ottinetti, 1990Go). Each ring was embedded in approximately 300 µl of interstitial collagen purified from rat tail tendons as reported (Elsdale and Bard, 1972Go). Collagen gel cultures of rat aorta were kept at 35.5°C in serum-free MCDB 131 culture medium (Clonetics, San Diego, CA) with or without Ang-1 and/or Ang-2, in the presence or absence of soluble Tie2. The medium was changed three times a week. Branching morphogenesis was evaluated by counting branch points at the peak of angiogenic growth (day 7). Mural cell recruitment was scored by counting the number of mural cells visible under phase contrast microscopy in the ten longest microvessels of each culture, as previously reported (Nicosia and Villaschi, 1995Go).

Isolation of MPCs
MPCs were isolated nonenzymatically from everted rat aortas, as previously described (Villaschi et al., 1994Go) and grown at 37°C in MCDB 131 growth medium supplemented with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO) and 50 µg ml-1 gentamicin (Sigma). MPCs were {alpha}-smooth muscle actin ({alpha}-SMA) positive and factor-VIII-related antigen (FVIII-RA) negative by immunocytochemistry. The capacity of MPCs to transform into pericytes was evaluated in a collagen gel overlay coculture assay with rat aorta endothelial cells (Nicosia and Villaschi, 1995Go). MPC were periodically reisolated to insure a constant supply of early passage cells (passages 7-12, at 1:3 split ratio).

MPCs were also isolated with an immunomagnetic method by incubating an endothelial-depleted suspension of aortic outgrowth cells with anti-Tie2-coated magnetic beads (Alessandri et al., 2001Go). A mixed population of aorta-derived cells grown on gelatin-coated dishes were trypsin treated and first incubated with magnetic beads (anti-mouse-IgG-coated beads, cell:bead ratio 1:4; Dynal Biotech, Oslo, Norway) coated with an anti-CD31 mouse monoclonal antibody (BD Biosciences Pharmingen, San Diego, CA). Endothelial cells, which selectively express CD31, bound to the beads coated with anti-CD31 antibody and were removed with a magnetic particle concentrator (Dynal Biotech). The remaining CD31-negative cells were then recovered by centrifugation and incubated with magnetic beads coated with mouse monoclonal antibody to Tie2 (BD Biosciences Pharmingen; cell:bead ratio 1:4). The CD31-/Tie2+ cells were recovered with a magnetic particle concentrator, seeded on a gelatin-coated 60 mm dish and cultured at 37°C in MCDB 131 growth medium supplemented with 10% FBS and gentamicin. Detachment of beads from cells was facilitated by gently trypsinizing the isolated cells after a few days in culture.

Endothelial cell lines
Rat aortic endothelial cells (RAECs) were isolated and subcultured as reported (Nicosia et al., 1994Go). The human-endothelium-derived permanent cell line EAhy 926 (Edgell et al., 1983Go) was kindly provided by C. J. S. Edgell (Department of Pathology, University of North Carolina, Chapel Hill, NC). These cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, Rockville, MD) containing 10% FBS (Sigma) and HAT growth supplement (Life Technologies).

Immunocytochemistry and immunohistochemistry
For immunocytochemical studies, MPCs were cultured in 35 mm dishes in endothelial basal medium (EBM) supplemented with 10% FBS and gentamicin. EAhy 926 cells and RAECs were used as positive controls of endothelial cell differentiation. Subconfluent monolayers were fixed in cold methanol or ethanol and stained, according to previously published procedures (Villaschi and Nicosia, 1994Go), with biotinylated Griffonia Simplicifolia isolectin-B4 (Vector Laboratories, Burlingame, CA), rabbit polyclonal anti-FVIII-RA antibody (DAKO, Carpinteria, CA) or mouse monoclonal antibodies against CD31 (BD Biosciences, Pharmingen), {alpha}-SMA (Sigma) or calponin (Sigma). Tie2 protein expression in isolated cells was demonstrated in separate experiments with two rabbit polyclonal anti-Tie2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Alpha Diagnostics International, San Antonio, TX). Biotinylated secondary antibodies of the appropriate animal species were used after the primary antibody, as described (Villaschi and Nicosia, 1994Go). The cultures were overlaid with ABC solution (Vectastain Elite Kit, Vector Laboratories) for 10 minutes, reacted with diaminobenzidine (DAB), rinsed in distilled water and mounted in Gelvatol (Monsanto, St Louis, MO). Negative controls included cultures incubated with non-immune IgG of the same species as the primary antibody of interest and cultures reacted only with secondary antibody. For the immunohistochemical localization of Tie2 in the aortic wall, the native rat aorta was fixed in buffered formalin, embedded in paraffin, cross-sectioned and mounted on Fisherbrand Superplus slides (Fisher Scientific, Pittsburgh, PA). Histological sections were deparaffinized, rehydrated and blocked according to standard protocols. They were then immunostained by the ABC immunoperoxidase procedure with a rabbit polyclonal anti-Tie2 antibody (Alpha Diagnostics International) and mounted in Gelvatol (Monsanto).

Confocal microscopy and double immunofluorescence
Double immunofluorescence followed by confocal microscopy was used to localize Tie2 and {alpha}-SMA in MPCs. MPCs cultured in gelatin-coated Lab-Tek chamber slides (Nalge Nunc, Naperville, IL) were fixed in formalin, washed twice with PBS, permeabilized with 0.2% Triton X-100 solution and reacted for 1 hour with a cocktail of rabbit polyclonal anti-Tie2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse antibody against {alpha}-smooth-muscle actin (Sigma). They were then rinsed in PBS, incubated for 30 minutes with a cocktail of AlexaFluor-488-labeled goat anti-rabbit antibody (green fluorochrome) and AlexaFluor-568-labeled goat anti-mouse antibody (red fluorochrome) (Molecular Probes, Eugene, OR, USA), rinsed twice with PBS, transferred onto histology glass slides, mounted in aqueous mounting medium (Gelvatol) and examined by confocal microscopy using a Leica TCS-SP laser scanning microscope. Image analysis and z-plane sectioning were performed using proprietary Leica software.

Chemotaxis analysis
Chemotaxis assay was carried out in a 48-well Boyden chamber (Neuro Probe, Cabin John, MD) as reported (Albini et al., 1993Go). MPCs were harvested in trypsin 0.05% NaHCO3 (Life Technologies), incubated in MCDB 131 plus 10% FBS to neutralize trypsin, centrifuged, resuspended in serum-free MCDB 131 (Clonetics) and seeded in the upper compartment of the chemotaxis chamber (5x104 cells per 50 µl). Each experimental group comprised six wells, whose lower and upper compartments were separated by a gelatin-coated polycarbonate filter of 3-µm-pore-size (Nucleopore/Costar, Cambridge, MA). The lower compartment of the wells was filled with PDGF-BB, Ang-1, Ang-2, the Ang-1/Ang-2 combination, the Ang-1/Tie2 combination, albumin or serum-free medium. To rule out chemokinesis (random migration), the Ang-1 effect was further evaluated by adding Ang-1 to both lower and upper compartments. After a 4-hour incubation at 37°C in a humidified 5% CO2 incubator, the filters were removed from the Boyden chamber. Nonmigrating cells were scraped off the upper side of the filter with a rubber spatula. Filters were then stained with a HEMA 3 kit (Biochemical Sciences, Swedesboro, NJ) to demonstrate cells that had migrated toward the lower compartment of the chamber. Cell migration was measured as optical density (OD) with a GS-710 densitometer (Bio-Rad Laboratories, Hercules, CA).

Zymography and MMP gelatinase activity assay
MMP secretion in cultures of MPC treated with Ang-1, Ang-2, the Ang-1/Ang-2 combination or serum-free medium was evaluated by gelatin zymography (Kleiner and Stetler-Stevenson, 1994Go). Equal volumes of medium conditioned by MPC (300,000 cells per well) for 48 hours were electrophoresed onto a 7.5% SDS-polyacrylamide gel containing 0.6 mg ml-1 type A gelatin from porcine skin (Sigma). After electrophoresis, the gel was washed in 2.5% Triton X-100 for 2 hours to remove SDS, incubated for 18 hours at 37°C and stained with 0.1% Coomassie brilliant blue. Regions of gelatinolytic activity appeared as white bands against a blue background. Gelatinase bands were quantitatively evaluated with a Biorad GS710 imaging densitometer. The optical density value of each band was normalized by the total protein content of each sample. Total protein was measured with the Micro BCA assay (Pierce, Rockford, IL).

Proliferation assay
Thymidine incorporation was used to evaluate MPC proliferation in response to stimulation by angiopoietins. Cells were trypsin treated, suspended in MCDB 131 growth medium supplemented with 10% FBS (Life Technologies) and seeded in a 24 well plate (5x103 in 0.5 ml in each well). Two days later, the cells were washed with serum-free MCDB 131 and incubated with or without Ang-1, Ang-2 or the Ang-1/Ang-2 combination under serum-free conditions. FBS (10%) was used as a positive control. After a 24-hour treatment, triplicate cultures were pulsed with 2 µCi ml-1 [3H]-thymidine (NEN Life Science Products, Zaventum, Belgium) for 2 hours and treated with 10% trichloroacetic acid (TCA). The TCA precipitates were solubilized in 0.5 N NaOH for 30 minutes and evaluated in a liquid scintillation counter.

Western-blot analysis
Proteins were extracted from MPCs or EAhy 926 monolayers in RIPA buffer containing protease inhibitors (Tris-HCl 50 mM, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 mM NaVD, 1 mM NaF). After centrifugation and boiling, protein concentration was measured with the Micro BCA assay (Pierce, Rockford, IL). 50 µg total protein per lane was resuspended in Laemmli buffer and loaded onto 6% SDS-polyacrylamide gel. After electrophoresis, the gel was transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and probed with monoclonal mouse anti-Tie2 antibody (Upstate Biotechnology, Lake Placid, NY) or polyclonal rabbit anti-Tie2 antibody (Santa Cruz Biotechnology). A peroxidase-conjugated donkey anti-mouse or goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as secondary antibody. Immunoreactive bands were visualized with the ECL chemiluminescence detection system (Amersham, Piscataway, NJ). In some experiments protein samples were immunoprecipitated with a polyclonal rabbit anti-Tie2 antibody (Santa Cruz Biotechnology) prior to western-blot analysis, as described below for the Tie2 phosphorylation study.

Tie2 phosphorylation study
MPCs or EAhy 926 were grown in gelatin-coated 100 mm dishes until confluence, rinsed with PBS pH 7.4 and exposed for 2.5 minutes to Ang-1 or Ang-2 in serum-free EBM. Treated cells and untreated controls were collected with a cell scraper in RIPA buffer. Protein concentration was measured with the Micro BCA assay (Pierce). 200 µg of each sample were then incubated on ice with 100 µl protein-G-coated agarose beads (Sigma) for 10 minutes and centrifuged at 10,000 g for 5 minutes. The supernatant was collected and immunoprecipitated overnight with anti-Tie2 rabbit polyclonal antibody (Santa Cruz Biotechnology) at 4°C. The immunoprecipitated samples were washed three times in RIPA buffer, boiled for 3 minutes to release the agarose beads, resuspended in Laemmli buffer and run onto 6% SDS-polyacrylamide gel. The gel was then blotted onto a nitrocellulose membrane (Bio-Rad Laboratories), which was probed with a mouse monoclonal anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). A peroxidase-conjugated donkey polyclonal anti-mouse antibody (Jackson ImmunoResearch Laboratories) was used as secondary antibody. Immunoreactive bands were visualized with the ECL luminescence detection system (Amersham, Piscataway, NJ). The membrane was incubated in stripping buffer according to recommended protocol in the Amersham ECL manual and re-exposed to ECL detection reagents to ensure absence of residual signals. It was then probed with rabbit polyclonal anti-Tie2 antibody (Santa Cruz Biotechnology) followed by a secondary peroxidase-conjugated goat anti-rabbit antibody to demonstrate that equal amounts of receptor protein were present in each lane. Immunoreactive protein bands were visualized by chemiluminescence as described above.

Reverse transcriptase polymerase chain reaction
RNA was extracted from MPCs and EAhy 926 cells with the TRIZOL method (Life Technologies), according to the manufacturer's protocol. 2 µg of total RNA was reverse transcribed with random hexamer primers. The reaction mixture included 100 pmoles random hexamers (Pharmacia, Peapack, NJ) previously incubated at 70°C for 10 minutes, 20 units RNase inhibitor (Life Technologies), 1 µM dNTPs (Pharmacia), 0.1 M DTT (Life Technologies), 5x first strand buffer (Life Technologies), 200 Units Superscript II (Life Technologies) and the RNA of interest. After a 1-hour incubation at 42°C, the reaction was inactivated at 70°C for 15 minutes. For PCR, the following reagents were mixed to a final volume of 5 µl tube-1: 10x polymerase chain reaction buffer (Life Technologies), 2.5 mM MgCl2 (Life Technologies), 0.2 µM dNTP mix, 1 µM reverse (R) primer and 1 µM forward (F) primers, 2 µl cDNA template and sterile distilled water. The reaction mixture was heated to 94°C for 5 minutes before adding 2.5 U Taq polymerase (Life Technologies). 35 cycles were performed under the following conditions: denaturation, 94°C for 45 seconds; primer annealing, 55°C for 1 minute; primer extension, 72°C for 1 minute; final extension at 72°C for 2 minutes. The Tie2 primers were designed from consensus sequences of human, mouse and bovine Tie2/Tek to amplify a region of 307 bp. Tie2 F primer: 5'-GAGGACAGGCAATAAGGATACG-3'; Tie2 R primer: 5'-GGGTGAAGAGGTTTCCTCCTAT-3'. PCR products purified with a PCR purification kit (Qiagen) were cloned into the TA vector PCR 2.1-TOPO (Life Technologies) according to the manufacturer's recommendations. Two colonies with the proper size insert were sequenced in both directions at the Veterans Administration Puget Sound Health Care System (Seattle, WA). Complete sequences of the amplified PCR products were compared to GenBank using the BLAST program.

Enzyme-linked immunosorbent assay
The effect of Tie2 activation on endothelial PDGF-BB production was tested by treating EAhy 926 cells with Ang-1. The EAhy 926 cell system was chosen because the Tie2 of these cells is unphosphorylated under control conditions and can be activated by exogenous Ang-1 (data not shown). PDGF-BB was measured by performing ELISA (R&D Systems) on medium conditioned by EAhy 926 cells in the presence or absence of stimulatory doses of Ang-1. The production of PDGF-BB was measured in duplicate using a commercial ELISA kit.

Statistical analysis
Data were analysed with GraphPad Prism statistics software (GraphPad Software, San Diego, CA). One-way ANOVA followed by Newman-Keuls multiple comparison test was used to evaluate whether differences between groups were significant. Statistical significance was set at P<0.05.


    Results
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 Materials and Methods
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 References
 
Ang-1 and Ang-2 promote angiogenesis and mural cell recruitment in the rat aorta model
Aortic rings cultured in collagen gels under serum-free conditions generated a self-limited angiogenic response. Neovessels were composed of endothelial cells and surrounding mural cells, as reported (Nicosia and Ottinetti, 1990Go). The effect of Ang-1 and Ang-2 in this system was evaluated by treating the cultures with increasing doses of these factors. Both angiopoietins stimulated angiogenesis in a dose-dependent and saturatable manner (Fig. 1A). Similar results were observed with angiopoietins obtained from two different sources (Regeneron Pharmaceuticals and R&D Systems). Treated cultures exhibited increased endothelial sprouting and branching morphogenesis compared with untreated controls. Angiopoietin-stimulated neovessels were thicker than control neovessels because of an enhanced recruitment of mural cells around the sprouting endothelium (Fig. 1B). The proangiogenic effects of both Ang-1 and Ang-2 were abrogated by soluble Tie2, which blocked the stimulation of mural cell recruitment by these molecules (Fig. 1B).



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Fig. 1. Effect of Ang-1 and Ang-2 on angiogenesis (A) and mural cell recruitment (B) in the rat aorta model. (A) Angiogenesis was measured by counting branch points under light microscopy in the living cultures. Notice that Ang-1 and Ang-2 promoted branching morphogenesis of aorta-derived microvessels in a dose-dependent fashion. Bars represent the SEM (n=4). *, P<0.05; **, P<0.01. (B) Mural cell recruitment was evaluated by counting the mural cells of the ten longest microvessels of each culture at day 7, under phase-contrast microscopy. Notice that Ang-1 (0.67 µg ml-1), Ang-2 (0.67 µg ml-1) and the Ang-1/Ang-2 combination stimulated mural cell recruitment. Soluble Tie2 blocked stimulation of mural cell recruitment by Ang-1 and Ang-2 used alone or in combination. Bars represent the SEM (n=40). *, P<0.01; **, P<0.001.

 

Isolation and characterization of MPCs from the rat aorta
We have previously reported that mural cells of rat aorta-derived neovessels originate from the intimal/subintimal layers of the aortic wall (Villaschi and Nicosia, 1994Go). These cells can be isolated nonenzymatically from everted aortic tubes. When co-cultured with branching capillaries formed by endothelial cells in collagen gel overlay co-cultures, MPCs migrate towards the endothelium, becoming incorporated as pericytes around the capillary tubes (Nicosia and Villaschi, 1995Go). To evaluate the molecular mechanisms regulating the recruitment of MPCs by endothelial cells, we isolated and characterized MPCs based on their morphologic, immunohistochemical and behavioral characteristics using published protocols (Villaschi and Nicosia, 1994Go; Nicosia and Villaschi, 1995Go). Confluent MPCs displayed a polygonal shape (Fig. 2A), grew mainly as a monolayer, had a high proliferative rate and produced large amounts of laminin- and type-IV-collagen-rich extracellular matrix, as described previously (Villaschi and Nicosia, 1994Go). Immunostaining demonstrated that these cells were positive for calponin (Fig. 2B) and {alpha}-SMA, (Fig. 2C) and negative for the endothelial markers FVIII-RA (Fig. 2D) and CD31 (data not shown).



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Fig. 2. Photomicrographs of living cultures (A) and immunoperoxidase preparations of MPC stained for the smooth muscle cell markers calponin (B) and {alpha}-SMA (C) or the endothelial cell marker FVIII-RA (D). Notice that MPCs are positive for calponin and {alpha}-SMA, and negative for FVIII-RA. Scale bar, 100 µm.

 

Ang-1 fails to induce the production of MPC chemoattractants by endothelial cells
To evaluate whether the Ang-1/Tie2 system induced mural cell migration indirectly through endothelial activation, cultures of EAhy 926 cells were treated for 24 hours with Ang-1 under serum-free conditions. Immunoprecipitation of the EAhy 926 Tie2 receptor followed by western-blot analysis with an anti-phosphotyrosine antibody confirmed activation of the endothelial Tie2 by Ang-1, as previously reported (Davis et al., 1996Go) (data not shown). Medium conditioned by endothelial cells in the presence or absence of Ang-1 was tested in a modified 48-well Boyden chamber for its capacity to promote MPC chemotaxis. Treatment of endothelial cells with Ang-1 failed to stimulate MPC migration above control values. In addition, ELISA studies showed no evidence that Ang-1 induces endothelial production of PDGF-BB (data not shown).

Ang-1 and Ang-2 directly stimulate MPC chemotaxis
While performing the chemotaxis experiments with endothelium-conditioned medium, we noticed that MPCs migrated in response to Ang-1-containing medium not exposed to endothelial cells. This suggested that Ang-1 might function as a direct chemoattractant for MPCs. Dose/response experiments confirmed this observation, showing a dose-dependent and saturable chemotactic effect by Ang-1 (data not shown). Interestingly, a similar chemotactic response was also observed with Ang-2. Ang-2 had no antagonistic effects when administered together with Ang-1 and the chemotactic effect of the Ang-1/Ang-2 combination was actually higher than that obtained with the single molecules (Fig. 3A). To rule out a nonspecific chemokinetic effect of Ang-1, the experiment was repeated by placing Ang-1 at the same concentration in both the lower and upper compartments of the Boyden chamber, a condition that eliminates chemotactic gradients in the system. MPCs exposed to Ang-1 under these conditions did not migrate (Fig. 3B). The Ang-1 effect was also significantly reduced by preincubating Ang-1 with soluble Tie2 for 15 minutes prior to the chemotaxis assay (Fig. 3B). This observation supports the hypothesis that Ang-1 stimulates MPC migration through a Tie2-mediated chemotactic mechanism.



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Fig. 3. Effect of Ang-1 and Ang-2 on chemotaxis of MPC in a Boyden chamber assay in which PDGF-BB was used as positive control. (A) Ang-1 (0.67 µg ml-1) and Ang-2 (0.67 µg ml-1) used alone or in combination stimulated the migration of MPCs. The stimulatory effect obtained with the Ang-1/Ang-2 combination was higher than but not significantly different from that obtained with the single molecules. Bars represent the SEM (n=6). *, P<0.0001; **, P<0.02; ***, P<0.03. (B) Ang-1-mediated stimulation of MPC migration (0.67 µg ml-1) was inhibited by soluble Tie2 (13.4 µg ml-1). Cells were responsive to the PDGF-BB positive control and showed no random migration (chemokinesis) when Ang-1 (0.67 µg ml-1) was loaded in both the upper and lower compartments of the Boyden chamber. Bars represent the SEM (n=6). *, P<0.001; **, P<0.01; ***, P<0.05. Values are expressed as optical density (O.D.) in both A and B.

 

Ang-1 and Ang-2 stimulate MMP-2 production by MPCs
It has recently been reported that Ang-1 stimulates production of MMPs by endothelial cells (Kim et al., 2000aGo). To evaluate whether angiopoietins have similar effects on MPCs, these cells were exposed for 48 hours under serum-free conditions to Ang-1, Ang-2 or the Ang-1/Ang-2 combination. Zymographic analysis of MPC-conditioned medium showed that MMP-2, which was detectable at low levels in the untreated control, was significantly increased in cultures treated with Ang-1, Ang-2 or the Ang-1/Ang-2 combination (Fig. 4). MPC-conditioned medium showed no detectable MMP-9 activity in either controls or angiopoietin-treated cultures.



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Fig. 4. Zymographic analysis of medium conditioned by MPCs (300,000 cells per culture well) without (control) or with Ang-1 (0.5 µg ml-1), Ang-2 (0.5 µg ml-1) or the Ang-1/Ang-2 combination (A). Bar graph shows quantitative evaluation of gelatinase bands by densitometry (B). Optical density values were normalized for the total protein content of each sample. Notice that Ang-1 and Ang-2 used alone or in combination stimulated MMP-2 production by MPCs.

 

Ang-1 has no effect on DNA synthesis by MPCs
DNA synthesis by MPCs cultured in the presence or absence of Ang-1, Ang-2 or the Ang-1/Ang-2 combination was studied by incubating the cultures with [H3]-thymidine. Evaluation of thymidine uptake showed that angiopoietins had no effect on MPC proliferation (data not shown), as previously reported for endothelial cells (Huang et al., 1999Go).

MPCs express Tie2 protein and Tie2 mRNA
The observation that MPCs respond directly to angiopoietin stimulation suggested that these cells might express Tie2. Western-blot analysis of MPC extracts immunoreacted for Tie2 demonstrated a 140-kDa band that migrated with the corresponding band of an endothelial Tie2 control (Fig. 5), consistent with the reported molecular weight of Tie2 (Wong et al., 1997Go). RT-PCR confirmed expression of Tie2 in MPCs (Fig. 5). The PCR product was of 282 bp, as expected from published human, mouse and bovine Tie2 sequences, and was confirmed to be Tie2 by sequence analysis. The predicted amino acid sequence from this region was 90%, 88% and 87% homologous to the mouse, human and bovine Tie2 receptors, respectively. A control RT-PCR reaction using RNA from the endothelial cell line EAhy 926 produced a product that was 100% identical to the human sequence, as expected. Immunoprecipitation of MPC extracts with anti-Tie2 antibody followed by western-blot analysis with an anti-phosphotyrosine antibody demonstrated marked induction of Tie2 phosphorylation upon stimulation with both Ang-1 and Ang-2 (Fig. 5). Immunocytochemical staining of MPCs showed peripheral membrane-type localization of Tie2 (Fig. 6A,B), as also demonstrated in control endothelial cells (Fig. 6F). Tie2 expression on the surface of MPCs was further confirmed by isolating these cells with anti-Tie2-antibody-coated magnetic beads from a mixed population of rat aortic cells that had been previously depleted of endothelial cells with anti-CD31-coated beads. Confocal microscopy showed co-localization of {alpha}-SMA and Tie2 in isolated MPCs (Fig. 6C-E).



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Fig. 5. Tie2 expression in MPCs. (Top) Western-blot analysis (WB) of MPC protein extract immunoreacted for Tie2 demonstrated a 140 kDa band that migrated with the Tie2 band of the endothelial cell control (EC). RT-PCR confirmed expression of Tie2 in MPCs at the mRNA level. Endothelial cells (EC) were used as positive control. No PCR bands were detected in the absence of reverse transcriptase (data not shown). Specificity of the PCR products was confirmed by sequence analysis. (Bottom) Analysis of MPC protein extracts immunoprecipitated (IP) with an anti-Tie2 antibody and reacted with an anti-phosphotyrosine antibody ({alpha}-pY) showed phosphorylation of the MPC Tie2 receptor upon stimulation with both Ang-1 and Ang-2. Stripping and reprobing of the membrane with an anti-Tie2 antibody ({alpha}-Tie2) showed that equal amounts of Tie2 were present in each lane.

 


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Fig. 6. Photomicrographs of planar cultures of isolated MPCs (A-E) and endothelial cells (F), and of histologic sections of native rat aorta (G,H) immunostained for Tie2 by immunoperoxidase (A,B,F-H) or immunofluorescence (C-E). Planar cultures: (A) shows a group of confluent MPCs whereas (B) shows a single MPC at higher magnification. Immunoperoxidase staining of cultured cells demonstrated Tie2 in both MPCs (A,B) and control endothelial cells (F). The positive staining reaction for Tie2 was predominantly localized at the cell periphery (arrows). Confocal images of MPCs double stained for Tie2 (C, green fluorescence) and {alpha}-SMA (D, red fluorescence) showed coexpression of Tie2 and {alpha}-SMA in the same cells (E, green and red fluorescence overlay). Cultured cells reacted with nonimmune IgG were negative (data not shown). Scale bars, 50 µM (A,C-E), 30 µM (B,F). Histological sections of native rat aorta: the intimal and subintimal layers of the aorta contain Tie2+ nonendothelial mesenchymal cells (G,H, arrows). Arrowheads highlight the endothelial lining of the aortic intima, which serves as a positive internal control. Scale bar, 100 µm.

 

Tie2 is expressed in intimal/subintimal nonendothelial cells of the native rat aorta
To evaluate whether MPCs express Tie2 in their native environment (i.e. in the intimal/subintimal layers of the aortic wall from which they originate), we immunostained histological sections with an anti-Tie2 antibody. Tie2 was found not only in endothelial cells but also in mesenchymal cells located in the intimal/subintimal layers of the aortic wall (Fig. 6G,H), as predicted by the MPC isolation and characterization studies.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our results demonstrate that MPCs express the tyrosine kinase receptor Tie2 and respond directly to the Tie2 ligands Ang-1 and Ang-2 by migrating and secreting MMP-2. Developmental studies in knockout mice have shown that the angiopoietin/Tie2 system plays a crucial role in angiogenic sprouting, vascular remodeling and neovessel maturation (Sato et al., 1995Go; Suri et al., 1996Go). Ablation of the Ang-1 or Tie2 genes causes major defects in the development of the mural component of the vessel wall (Sato et al., 1995Go; Suri et al., 1996Go). Hereditary defects in the Tie2 gene cause arteriovenous malformations characterized by absence or abnormal layering of mural cells (Vikkula et al., 1996Go). The mechanisms regulating the angiopoietin/Tie2-mediated recruitment of mural cells have remained elusive because Tie2 has been found in endothelial cells (Sato et al., 1995Go; Kim et al., 2001Go) but not in mural cells. It has been proposed that sprouting endothelial cells respond to Ang-1 stimulation by secreting PDGF-BB, which is required for mural cell migration and recruitment during embryonic angiogenesis (Folkman and D'Amore, 1996Go). This indirect mechanism, however, has not been confirmed by experimental data. Using isolated cell strains, we were unable to demonstrate induction of mural cell chemotactic activity or PDGF-BB secretion in endothelial cells treated with Ang-1.

We previously reported that the rat aortic intima has the capacity to form neovessels composed of endothelial and mural cells (Nicosia et al., 1992Go). Subsequent isolation experiments led to the identification of rat aortic intima-derived {alpha}-SMA-positive and FVIII-RA-negative cells with mural cell precursor properties (Villaschi et al., 1994Go). Collagen gel coculture experiments demonstrated that these cells, which we termed MPCs, have a marked endothelial tropism and become incorporated into microvascular networks formed by isolated endothelial cells (Nicosia and Villaschi, 1995Go). MPCs stabilize the microvessels, which are unable to survive in the absence of a nonendothelial mesenchymal cell support.

Our observation that MPCs express Tie2 and respond directly to angiopoietin stimulation provides the basis for a novel understanding of how mural cells are recruited by developing neovessels during angiogenesis. Of particular interest is the observation that MPCs respond not only to Ang-1 but also to Ang-2. Initial reports indicated that Ang-1 was the main activating ligand of Tie2, whereas Ang-2 (which bound to this receptor without inducing its phosphorylation) was considered an antagonist (Maisonpierre et al., 1997Go). Recent studies, however, have shown that Ang-2 might act as an agonist in the context of a three-dimensional matrix (Teichert-Kuliszewska et al., 2001Go) and when used at high concentration (Kim et al., 2000bGo) or for prolonged periods of time (Teichert-Kuliszewska et al., 2001Go), conditions that might all occur at sites of angiogenesis (Kim et al., 2001Go; Goede et al., 1998Go). The cellular context in which Tie2 is expressed has also a profound influence on the response of the receptor to Ang-2. Maisonpierre et al. (Maisonpierre et al., 1997Go) found that Ang-2 was able to induce Tie2 activation when the receptor was transgenically expressed in NIH 3T3 fibroblasts. Our results confirm and expand these observations demonstrating that Ang-2 functions as a Tie2 agonist in MPC, which spontaneously express this receptor. Ang-2 promotes both MPC migration and MMP-2 production and does not inhibit the stimulatory effect of Ang-1 on these cells. In some experiments we actually observed greater MPC migration and MMP2 production when the cells were exposed to both Ang-1 and Ang-2. Ang-2 produced by the endothelium during the early stages of angiogenesis might provide crucial migratory and proteolytic signals to Tie2+ MPCs. Ang-1 produced by the mural cells themselves and surrounding fibroblasts may further potentiate this process. According to this model, Tie2+ MPCs would eventually differentiate into Tie2- mural cells, a process suggested by the observation in our laboratory that Tie2 expression is eventually lost in MPCs after repeated passages in culture (M.I. and R.F.N., unpublished).

The finding of Tie2+ MPCs in the rat aorta confirms recent observations that Tie2 expression is not restricted to the endothelium and can also be demonstrated in nonendothelial mesenchymal cell types such as stromal cells of pyogenic granulomas (Yuan et al., 2001), vascular smooth muscle cells of breast cancer xenograft tumors (Tian et al., 2002Go), vascular smooth muscle cells of reactive synovial tissue (Shahrara et al., 2002Go), pericytes associated with venous malformations (Calvert et al., 1999Go) and fibroblast-like cells of choroidal neovascular membranes (Otani et al., 1999Go). Tie2 is also expressed by hematopoietic stem cells (Hattori et al., 2001Go; Takakura et al., 1998Go) and has been reported in cytotrophoblastic cells (Dunk et al., 2000Go).

Establishing the origin of the MPCs might be crucial for understanding how vessels develop and mature. One possibility is that MPCs originate through transdifferentiation of endothelial cells into a mesenchymal cell type, which maintains the capacity to express Tie2. An endotheliummesenchyme transformation has been reported previously in other systems such as endocardial cushion development (Nakajima et al., 1997Go). Alternatively, Tie2+ MPCs might arise from a vascular progenitor stem cell capable of both endothelial and mural cell differentiation. Recent studies have identified a common Flk1+ progenitor cell from which both endothelial and mural cells originate in response to vascular endothelial growth factor (VEGF) or PDGF-BB, respectively (Yamashita et al., 2000Go; Carmeliet, 2000Go). These immature cells can be found in bone marrow, circulation and peripheral tissues during angiogenic responses (Asahara et al., 1997Go; Asahara et al., 1999Go; Harraz et al., 2001Go). They have been identified in the human embryonic aorta (Alessandri et al., 2001Go), raising the possibility that the ex vivo angiogenic response of the postnatal aorta might be, at least in part, the result of de novo formation of neovessels from mesenchyme, as previously suggested by others (Akita et al., 1997Go).

In conclusion, our results provide a novel mechanistic explanation for the angiopoietin/Tie2-mediated recruitment of mural cells during angiogenesis. Although more studies are needed to determine whether this model can be universally applied to angiogenic responses in different tissues, our observations raise the intriguing possibility that mural cell recruitment during angiogenesis can be genetically promoted or blocked by targeting the Tie2 receptor of MPCs. Possible applications of this approach include stimulation of neovessel survival and arteriogenesis in diseases characterized by inadequate vascularization, induction of neovessel regression in angiogenesis-dependent disorders such as cancer and bioengineering of vascularized tissue constructs for in vivo implantation. The implications of these observations go beyond the angiogenesis field because of the crucial role molecular mechanisms of mural cell recruitment play in the pathogenesis of a number of vascular disorders including atherosclerosis and restenosis of bypass grafts.


    Acknowledgments
 
This work was supported by a fellowship from the American-Italian Cancer Foundation (to MI) and by grants from NIH (HL52585, to RFN), Medical Research Service, Department of Veterans Affairs (to RFN) and NSF-Engineering Research Center Program (EEC-9529161, to RFN). We thank R. Green for his excellent technical assistance with confocal microscopy.


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