Physiol. Genomics AJP: Regulatory, Integrative and Comparative Physiology
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Physiol. Genomics 27: 20-28, 2006; doi:10.1152/physiolgenomics.00048.2006
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Received 24 March 2006; accepted in final form 11 June 2006.
Physiological Genomics 27:20-28 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Angiopoietin-1 and vascular endothelial growth factor induce expression of inflammatory cytokines before angiogenesis

Alfred C. Aplin1, Maurizio Gelati2, Eric Fogel3, Edvige Carnevale1 and Roberto F. Nicosia1,3

1 Department of Pathology, University of Washington, Seattle, Washington;
2 Laboratory of Neurobiology and Neuroregenerative Medicine-"Carlo Besta" Institute, Milan, Italy; and
3 Division of Pathology and Laboratory Medicine, Veterans Administration Puget Sound Health Care System, Seattle, Washington


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The purpose of this study was to identify novel transcriptional events occurring in the aortic wall before angiogenesis. We used a defined tissue culture system that takes advantage of the capacity of rat aortic rings to generate neovessels ex vivo in response to angiogenic factor stimulation. Total RNA isolated from aortic rings 18 h posttreatment with angiopoietin (Ang)-1 or vascular endothelial growth factor (VEGF) was used to probe oligonucleotide microarrays. Many genes were up- or downregulated by either Ang-1 or VEGF, with a subset being affected by treatment with both growth factors. Grouping of genes by biological function revealed that Ang-1 and VEGF both upregulated a host of immune-related genes including many inflammatory cytokines. A mixture of the Ang-1- and VEGF-induced cytokines stimulated the spontaneous angiogenic response of aortic rings and was synergistic with a low dose of recombinant VEGF. This effect was associated with enhanced recruitment of adventitial macrophages and dendritic cells in the angiogenic outgrowths. Thus Ang-1 and VEGF activate the innate immune system of the vessel wall, stimulating the production of proangiogenic inflammatory cytokines before the emergence of neovessels. This hitherto unreported feature of the angiogenic response might represent an important early component of the cellular and molecular cascade responsible for the angiogenic response of the aortic wall.

aorta; oligonucleotide microarray; gene profiling


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ANGIOGENESIS, THE PROCESS by which new blood vessels develop from preexisting vessels, is characterized by endothelial migration, proliferation, extracellular matrix proteolysis, and capillary tube formation (8). The molecular basis of each of these angiogenic steps has been the focus of intense investigation because of the critical importance of angiogenesis in many human diseases. In addition to its essential role in embryonal development, angiogenesis contributes to the progression of cancer, diabetic retinopathy, macular degeneration, psoriasis, and rheumatoid arthritis (3, 17, 22). Although our knowledge of angiogenic mechanisms has greatly expanded, a gap remains in our understanding of the precise sequence of molecular events that enables adult blood vessels to switch from quiescence to an angiogenic state.

In the present study, we used the ex vivo rat aortic ring model of angiogenesis to identify genes expressed during the early stages of the angiogenic process (32, 35). This system is based on the capacity of the aortic wall to produce neovessels in biomatrix gels after mechanical injury or angiogenic factor stimulation. By making aortic rings "quiescent" before treatment with recombinant angiogenic factors, we defined culture conditions that correspond to angiogenic "on" and "off" switches (59). We used this approach to characterize early transcriptional events induced by angiopoietin (Ang)-1 or vascular endothelial growth factor (VEGF), which are critical regulators of physiological and pathological angiogenesis during embryonal and postnatal life (47, 56). Recent studies have identified VEGF-responsive genes in a variety of culture systems with isolated endothelial cells (6, 16, 23, 46, 54). To our knowledge, there are, however, no reports describing the early effects of angiogenic factor stimulation on gene expression by the intact vessel wall. Because tyrosine kinase receptors for Ang-1 and VEGF are not only expressed in endothelial cells but also in nonendothelial cells (20, 21, 40, 53), studying the gene expression characteristics of the whole aorta may provide new insights into the mechanisms by which these factors initiate angiogenesis. This approach may identify angiogenic factor-induced genes that are not expressed in endothelial cells and yet play a critical role in the induction of angiogenesis.

We examined gene expression profiles of aortic rings treated with Ang-1 and VEGF, applying stringent criteria to screen expression data. This approach identified a host of genes with dysregulated expression after angiogenic factor stimulation. Among these was a broad repertoire of immune-related genes, including many inflammatory cytokines with proangiogenic and macrophage stimulatory activities. These results implicate inflammatory pathways in the angiogenic response of the aortic rings, suggesting that endothelial cells cooperate with the innate immune system during angiogenesis in this system. Our findings support the view that macrophages and immune-related cytokines actively participate in the initiation of angiogenesis, as part of a cascade of cellular and molecular events triggered by angiogenic factor stimulation of the vessel wall (18, 31).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation and treatment of aortic ring cultures.
All animal procedures were performed with approval from the Veterans Administration Puget Sound Health Care System Institutional Animal Care and Use Committee and National Institutes of Health guidelines. Thoracic aortas were dissected from killed 2-mo-old Fischer 344 male rats (Harlan, Indianapolis), cleaned of fibroadipose tissue and blood, and serially cross-sectioned into ~2-mm rings, as described previously (36). Microarray analysis and real-time PCR studies under different treatment conditions (see below) were performed on quiescent rings. Aortic rings were made angiogenically quiescent by a 13-day-long preincubation step in serum-free endothelial basal medium (EBM; Cambrex Bio Science, Walkersville, MD) (32, 33). Briefly, each aortic ring was housed in an 18-mm agarose-coated well of a four-well dish (Nunc, Naperville, IL) containing 0.5 ml of serum-free EBM, which was changed three times a week. Following this step, aortic rings failed to spontaneously produce neovessels when embedded in collagen but remained viable and maintained their angiogenic properties, which could be reactivated with exogenous angiogenic factors, as reported (33).

Array hybridization and data analysis.
Three separate animals were used as the source of aortic rings for the microarray analysis studies. For each animal, quiescent aortic rings floating in 0.5 ml of serum-free EBM were treated with Ang-1 (100 ng/ml) or VEGF (10 ng/ml) or left untreated (4–6 rings/experimental group). Total RNA was isolated from aortic rings from each experimental group 18–20 h after addition of growth factors to the media. Each set of rings was snap frozen in liquid nitrogen and manually pulverized with a model 59012 BioPulverizer (BioSpec Products, Bartlesville, OK). RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA), followed by further purification with the RNeasy Micro Kit (Qiagen, Valencia, CA). RNA was dissolved in water and determined to be intact and free of genomic DNA by examination with a Bioanalyzer 2100 (Agilent, Palo Alto, CA). RNA labeling, array hybridization, washing, and scanning were carried out at the University of Washington’s Center for Expression Arrays. Total RNA (0.5 µg) from each treatment group was labeled via the standard Affymetrix T7 promoter-mediated linear amplification procedure, producing biotinylated target RNA (52). All samples were analyzed using RAE230 A and B oligonucleotide arrays (Affymetrix, Santa Clara, CA), described at http://www.affymetrix.com/products/arrays/specific/hgu95.affx. Image processing and expression analysis were performed using Affymetrix Microarray Suite (MAS 5.1) software. Detailed descriptions of quality control metrics can be found in the Affymetrix Expression Analysis Technical Manual at http://www.affymetrix.com/support/technical/manual/expression_manual.affx. The data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE3355 (5, 14). After normalization of microarray data in each respective sample, further data analysis was handled in Microsoft Excel. For each treatment group, individual data sets from three independent array hybridizations were combined, and ANOVA was performed. Results representing differential expression of genes between treated and untreated controls were considered significant if P < 0.05. Each of the sequences identified as an expressed sequence tag (EST), unidentified cDNA, or generic rat cDNA on the original Affymetrix array was submitted to basic local alignment search tool (BLAST) (4). Sequences that returned >90% identity with a named rat gene were considered a positive match. The entire list of differentially expressed genes, ESTs, and cDNAs is available as Supplemental Table 1 (the online version of this article contains supplemental data).

Real-time PCR analysis.
We used RNA prepared for microarray analysis and from additional aortic explants treated with the same protocol for independent validation of selected expression data by real-time RT-PCR. The two-step quantitative RT-PCR (qRT-PCR) SYBR Green method (Applied Biosystems, Foster City, CA) was employed to compare and confirm the levels of a subset of genes deemed statistically significant by microarray analysis. Random primed RT was carried out with 50 ng of RNA and Superscript III reverse transcriptase (Invitrogen). Reactions lacking enzyme were carried out in tandem for each RNA sample to act as negative controls. One-fiftieth (1/50) of the final RT reaction was used as template in qRT-PCR reactions containing oligonucleotide primers (Invitrogen) specific for selected genes. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), using the following settings: 125-bp amplicon, 20mer, 60°C melting temperatures, and all others as defaults. Gene names, accession numbers, and forward and reverse primer sequences are listed in Table 1. Each primer set produced a single product, as determined by melt-curve analysis.


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  		Table 1. Oligonucleotide primer sequences for real-time RT-PCR

 
Relative quantification was carried out on an ABI 7000 thermal cycler (Applied Biosystems) in 25-µl reactions containing 1x Universal SYBR Green Master Mix (Applied Biosystems), 300 nM forward and reverse primers, and 5 µl of cDNA-diluted template. The PCR cycling profile was as follows: 2 min at 50°C, 10 min at 95°C and 40 cycles of 95°C for 15 s, 60°C for 1 min. Each PCR reaction was carried out in triplicate on one plate, and results presented are combined from separate assays. qRT-PCR data were recorded and analyzed using Prizm software (Applied Biosystems). Relative ratios of fluorescent intensities of products from each treatment group were calculated by use of the 2–{Delta}{Delta}Ct method (where Ct is threshold cycle) (27). The mRNA expression levels of all samples were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ß-actin expression measured in the same RT sample.

ELISA.
ELISA kits for growth-related gene (GRO)-1 (Assay Designs, Ann Arbor, MI), IL-1ß (Pierce Chemical, Rockford IL), TNF-{alpha} (Pierce), and VEGF (R&D Systems) were used to measure the protein levels in conditioned media according to the instructions of the manufacturers. Conditioned media were collected from cultures of quiescent aortic rings treated for 18 h with either 100 ng/ml Ang-1 or 10 ng/ml VEGF and compared with conditioned media from untreated controls. All reactions were carried out in triplicate, and the results presented are representative of multiple experiments.

Collagen gel culture and cytokine treatment of rat aorta.
For angiogenesis studies, freshly cut aortic rings were embedded in collagen gels and cultured in serum-free EBM (35, 59) with or without the following cytokines, which were added to the culture medium individually or together as a cocktail: GRO-1/CXCL1 (10 ng/ml; Cedar Lane Labs, Hornby, ON, Canada), IL-1ß (10 ng/ml; Biosource International, Camarillo, CA), macrophage inflammatory protein (MIP)-1{alpha} (MIP-1{alpha}/CCL3, 10 ng/ml; Biosource International), TNF-{alpha} (10 ng/ml; R&D Systems, Minneapolis, MN), and MIP-2 (50 ng/ml; Serotech, Raleigh, NC). Studies with individual cytokines included dose response experiments. Negative control cultures were incubated in serum-free EBM alone; positive controls were stimulated with VEGF. Synergistic interactions between cytokines and VEGF were tested by treating aortic cultures with both the cytokine mixture and a low dose of VEGF (5 ng/ml) that is otherwise minimally stimulatory when used alone. The angiogenic response was measured in living cultures by counting the number of neovessels over a period of 8–14 days, according to published criteria (35). Images were captured with an Olympus MagnaFire S99800 digital camera (Olympus, Melville, NY) mounted on a Leitz Laborlux K microscope.

Immunofluorescence staining and confocal microscopy.
Expression of leukocyte and macrophage markers in angiogenic outgrowths was evaluated in whole mount preparations of Formalin-fixed thin prep cultures using double immunofluorescence staining followed by confocal microscopy (59). Double staining was obtained by reacting the cultures with the Alexa Fluor 568-conjugated isolectin-B4 endothelial cell marker (Molecular Probes, Eugene, OR) and mouse antibody against CD11b (integrin-{alpha}M; Chemicon, Temecula, CA), CD54 (ICAM-1, BD Biosciences, San Jose, CA), CD45 (common leukocyte antigen, Serotech), or the macrophage marker ED2 (BD Biosciences) followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody. Immunostaining with leukocyte markers was also performed on en face preparations of freshly isolated whole aortas. Samples were mounted in Aqua Polymount (Polysciences, Warrington, PA) on glass slides, and images were taken with either a Leica TCS-SP laser scanning confocal microscope or an Olympus BX41 fluorescent microscope equipped with an Optronics MicroFire SE digital camera. Confocal images were obtained by Z-plane analysis followed by projection and overlay, using Leica software.

Electron microscopy.
For ultrastructural studies, collagen gel cultures were fixed in 2.5% glutaraldehyde 0.1 M Na cacodylate, pH 7.4, and processed for embedding in EPON-812 (Ted Pella, Redding, CA). Thin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (Jeol, Peabody, MA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Microarray analysis reveals upregulation of immune-related genes by Ang-1 and VEGF in the rat aorta before angiogenesis.
To identify genes dysregulated by Ang-1 and VEGF in isolated aortic rings before angiogenesis, we analyzed the relative expression levels of >31,200 transcripts with oligonucleotide microarrays. Our criteria for calling a gene up- or downregulated required a twofold or greater change in expression with statistical significance (P < 0.05) from a series of three independent array measurements for each treatment group. Comparative analysis of Ang-1- and VEGF-treated samples with untreated controls showed that 1,088 cDNAs were differentially regulated between treatment groups using our selection criteria. Of these transcripts, 290 corresponded to known rat genes, and 798 represented cDNAs and ESTs having either limited or no identity to rat sequences. Named genes were categorized according to broad biological functions (Table 2). Many genes were differentially regulated by either Ang-1 or VEGF, whereas some were modulated by both growth factors. The complete set of genes, cDNAs, and ESTs that were either up- or downregulated by Ang-1 and/or VEGF treatment is provided in Supplemental Table 1.


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  		Table 2. No. of named genes differentially expressed in response to Ang-1 and/or VEGF in rat aortic rings categorized by biological function

 
The biological grouping of dysregulated cDNAs included genes involved in apoptosis, cell adhesion, cell growth, intracellular transport, metabolism, neural development, and proteolysis. Surprisingly, the largest group of upregulated genes was related to the immune system. Among the genes overexpressed after Ang-1 treatment, there were 17 immune-related genes including the inflammatory cytokines IL-1{alpha}, MIP-1ß, the leukocyte {alpha}-integrin CD11b, the Toll-like receptor (TLR)-4 co-receptor CD14, major histocompatibility complex (MHC) I, secretory leukocyte protease inhibitor (Slpi), and the acute-phase reactant orosomucoid. VEGF treatment resulted in the upregulation of 15 genes related to the immune system. These genes include the complement proteins C1q and C2, MHC Ib, and TLR-2. Among the six genes upregulated by both Ang-1 and VEGF were five inflammatory cytokines: GRO-1, IL-1ß, MIP-1{alpha}, MIP-2, and TNF-{alpha}. In total, 10 individual cytokines/chemokines were upregulated in response to Ang-1 and/or VEGF treatment, coincident with neovessel formation in cultured aortic rings (Table 3).


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  		Table 3. Immune-related genes upregulated by Ang1 and/or VEGF by microarray analysis

 
Ang-1 and/or VEGF also upregulated 12 named genes related to neural development including astrotactin, meteorin, nestin, and radixin, which are involved in axonal outgrowth (2, 29, 37). Additional genes differentially regulated by Ang-1 and/or VEGF included 755 unidentified cDNAs including ESTs and cDNAs with unknown biological functions. Many of the named genes up- or downregulated by Ang-1 or VEGF have not been associated previously with the angiogenic process.

Validation of selected microarray data with qRT-PCR and ELISA.
To confirm the microarray expression data, we performed qRT-PCR on 10 differentially regulated genes using RNA samples from aortic rings treated with Ang-1 or VEGF. In general, there was a close correlation between microarray analysis and qRT-PCR in both the direction and magnitude of differential gene expression (Table 4). To further corroborate microarray results at the protein level, GRO-1 and TNF-{alpha} secreted from aortic cultures treated with Ang-1 or VEGF were measured by ELISA. This additional study demonstrated a fivefold and threefold increase of GRO-1 and TNF-{alpha}, respectively, in Ang-1-treated cultures (Fig. 1). Similarly VEGF stimulated GRO-1 and TNF-{alpha} production in the same cultures by threefold. Thus our microarray analysis study of differential gene expression in aortic cultures treated with angiogenic factors was validated at both the mRNA and protein levels.


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  		Table 4. Validation of microarray results by qRT-PCR

 

Figure 1
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  		Fig. 1. ELISA of conditioned medium from aortic cultures treated with maximum stimulatory dose of angiopoietin-1 (Ang-1; 100 ng/ml) or vascular endothelial growth factor (VEGF; 10 ng/ml) for 48 h. A: secreted growth-related gene (GRO)-1 levels increased 5-fold in response to Ang-1 and 3.8-fold in response to VEGF treatment. B: TNF-{alpha} secretion increased ~3-fold by both Ang-1 and VEGF treatment. n = 4; **P < 0.01, ***P < 0.001.

 
Ang-1- and VEGF-induced immune cytokines stimulate the ex vivo angiogenic response of the rat aorta.
Our gene expression studies indicated that the aortic wall produces immune cytokines in response to Ang-1 or VEGF stimulation before angiogenesis. To characterize the function of these secreted factors in our system, we tested their activity in the collagen gel aortic ring assay of angiogenesis using recombinant proteins. A cocktail of cytokines induced by both Ang-1 and VEGF (GRO-1, IL-1ß, MIP-1{alpha}, TNF-{alpha}, and MIP-2) significantly stimulated the angiogenic response of the rat aorta, leading to a 100% increase in the number of neovessels (Fig. 2). Interestingly, except for TNF-{alpha}, cytokines were unable to stimulate angiogenesis when used individually, even at higher concentrations than those used in the cocktail (not shown). TNF-{alpha} stimulated angiogenesis but only when administered at a 10-fold higher dose than that used in the cocktail. These findings indicate that Ang-1 and VEGF induce the expression by the aortic wall of immune cytokines that have the capacity to stimulate angiogenesis through synergistic mechanisms.


Figure 2
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  		Fig. 2. Cocktail of Ang-1- and VEGF-induced cytokines stimulates angiogenesis in collagen gel cultures of rat aorta. Shown are aortic ring collagen gel cultures. A: untreated. B: treated with a cocktail of GRO-1, IL-1ß, macrophage inflammatory protein (MIP)-1{alpha}, MIP-2, and TNF. A and B: aortic explants are marked by asterisks; scale bar = 200 µm. Bottom: graph shows increase in no. of neovessels after treatment with cytokines. CTRL, control. n = 4; **P < 0.01.

 
Ang-1-induced cytokines are synergistic with VEGF.
In previous studies, we have demonstrated that the ex vivo angiogenic response of the rat aorta is regulated by endogenous VEGF and can be stimulated further by the addition of recombinant VEGF (33, 34). In angiogenic aortic cultures, levels of secreted VEGF decreased from 17 pg/ml at day 3 to 2 pg/ml at day 5. Interestingly, addition of Ang-1- and VEGF-induced cytokines resulted in an eightfold increase of VEGF to 17 pg/ml at day 5 (Fig. 3), coincident with their stimulatory effect on angiogenesis. This suggested a proangiogenic interaction between the cocktail of immune cytokines and VEGF. To investigate this possibility, we included in the cytokine cocktail a low dose of recombinant VEGF that was otherwise minimally stimulatory when used alone (5 ng/ml). Addition of a minimal dose of VEGF to the cytokine cocktail resulted in a 300 and 400% increase in the angiogenic response over that obtained with VEGF or the cytokine cocktail alone, respectively (Fig. 4). Conversely, the cytokine cocktail had no significant potentiating effect on the capacity of Ang-1 to stimulate angiogenesis (data not shown). These studies indicate that immune-related cytokines produced after stimulation with Ang-1 or VEGF have the capacity to sensitize the aortic wall to angiogenic stimulation by VEGF.


Figure 3
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  		Fig. 3. VEGF ELISA of medium conditioned by aortic rings treated with cytokine cocktail. n = 4; **P < 0.01. Note: cytokine cocktail stimulates VEGF production, maintaining elevated VEGF levels through day 5.

 

Figure 4
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  		Fig. 4. Cocktail of cytokines/chemokines synergistically stimulates angiogenesis with a low dose of VEGF. Shown are aortic ring collagen gel cultures. A: untreated. B: treated with 5 ng/ml VEGF. C: treated with a cocktail of GRO-1, IL-1ß, MIP-1{alpha}, MIP-2, and TNF. D: the VEGF/cytokine cocktail combination. A–D: aortic explants are marked by asterisks; scale bar = 200 µm. E: graph shows maximum no. of neovessels at day 7 posttreatment. n = 4; **P < 0.01, ***P < 0.001.

 
Macrophages and dendritic cells are closely associated with the endothelium of angiogenic outgrowths.
The discovery that Ang-1- and VEGF-treated aortic cultures induced expression and production of immune-related cytokines as well as the induction of several other inflammation-related genes prompted us to investigate whether the cultured aortic rings contained cells of the immune system. Whole mount preparations of freshly dissected rat aorta and collagen gel preparations of rat aortic rings were stained for immune cell markers. These studies demonstrated that aortic ring cultures contained abundant mononuclear leukocytes that were positive for the leukocyte markers CD45 and CD11b. These cells, which had a distinctly granular morphology and rounded appearance in the live cultures, were abundantly present at the cut edges of the explants, the roots of the angiogenic outgrowths, and around individual neovessels growing into collagen gels (Fig. 5, A–C). In addition, dendritic cells immunoreactive for CD54 (Fig. 5D) and CD11c (not shown) were identified around aortic explants and between angiogenic outgrowths. Electron microscopy studies showed a close association of macrophages and dendritic cells with endothelial and mural cells of neovessels (Fig. 5E). Treatment with the cytokine cocktail stimulated the outgrowth of mononuclear leukocytes from the aortic explants by 58%, whereas VEGF increased it by 35%. Interestingly, the combination of VEGF and cytokines had a synergistic effect resulting in a 170% increase in the number of leukocytes (n = 4, P < 0.05).


Figure 5
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  		Fig. 5. Adventitial macrophages participate in the angiogenic response of the rat aorta. A: phase contrast image of aorta-derived neovessel surrounded by macrophages exhibiting typical granular morphology (black arrows); white arrow shows mural cell. B: immunofluorescence of collagen gel cultures of rat aorta double stained for leukoctyes (CD45, red) and endothelial cells (isolectin-B4, green). C and D: confocal images of leukocytes (C; CD11b, red) and dendritic cells (D; CD54/ICAM-1, red) associated with sprouting endothelial cells (isolectin-B4, green). A–D: scale bar = 40 µm. E: electron micrograph demonstrating close association between a macrophage (arrow) showing characteristic lysosomal vacuoles and pseudopods and a neovessel; lumen indicated by asterisk and mural cell by arrowhead. Scale bar = 10 µm.

 
To localize the source of leukocytes associated with angiogenic neovessels, we immunostained whole mount preparations of freshly isolated rat aortas for the tissue macrophage markers ED2 and CD45 . This study revealed a plethora of mononuclear leukocytes including many macrophages throughout the aortic adventitia (Fig. 6).


Figure 6
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  		Fig. 6. En face images of rat aorta adventitia immunostained for leukocyte markers. A: staining for the tissue macrophage-specific marker ED2 shows abundant macrophages. B: staining for the leukocyte common antigen CD45 demonstrates numerous leukocytes throughout the adventitia. Scale bar = 40 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of this study was to characterize changes in gene expression occurring in the aortic wall after angiogenic factor stimulation and before angiogenic sprouting. For this project, we used a modification of the rat aorta model in which aortic rings were made quiescent before angiogenic stimulation. Our results demonstrate that the angiogenic factors Ang-1 and VEGF, which are critical regulators of angiogenesis during embryonal development and postnatal life (9, 13, 47, 56), modulate the expression of a broad variety of genes, many of which were previously not know to be responsive to angiogenic growth factors. Surprisingly, the largest group of genes upregulated by Ang-1 and/or VEGF were related to the immune system and included 10 inflammatory chemokines/cytokines. A mixture of cytokines upregulated by both Ang-1 and VEGF, namely GRO-1, IL-1ß, MIP-1{alpha}, MIP-2, and TNF-{alpha} stimulated the spontaneous angiogenic response of the rat aorta. Experiments with individual cytokines of this group, or different cytokine combinations, showed optimal proangiogenic effects only when all the cytokines were included in the cocktail and identified TNF-{alpha} as a critical member of the cytokine mixture. Interestingly, the cytokine cocktail stimulated endogenous production of VEGF and had synergistic angiogenic effects with a low dose of VEGF (5 ng/ml) that was otherwise minimally angiogenic. The cytokines overexpressed in response to Ang-1 and VEGF are established mediators of the innate immune response and endothelial-leukocyte interactions (24, 25, 44). The observation that TNF-{alpha} is the most critical of the Ang-1-stimulated cytokines is consistent with previous reports indicating that TNF-{alpha} has angiogenic activity and potentiates basic fibroblast growth factor- and VEGF-mediated invasion of fibrin and collagen gels by isolated endothelial cells during angiogenesis in vitro (11).

In addition to its effect on cytokine production, Ang-1 upregulated the expression of the leukocyte integrin CD11b, the TLR-4 co-receptor CD14, and MHC I. Interestingly, Ang-1 also induced expression of anti-inflammatory proteins such as secretory leukocyte protease inhibitor and the acute-phase reactant orosomucoid (42, 58). These findings are consistent with previous reports indicating that Ang-1 effects may be anti-inflammatory or proinflammatory depending on the context in which Ang-1 operates (10, 19, 50). The concurrent upregulation of inflammatory and anti-inflammatory genes may be a mechanism by which Ang-1 modulates the immune response of the vessel wall, potentiating some effects and inhibiting others. Thus Ang-1 may stimulate the proangiogenic effects of the immune response, which is mediated by cytokines, while decreasing others such as vascular permeability (49). As demonstrated for Ang-1, the stimulatory effects of VEGF on the expression of immune-related genes were not limited to cytokines but included the complement proteins C1q and C2, the MHC Ib, and TLR-2. These effects are entirely consistent with the reported proinflammatory activity of VEGF (7, 13, 55, 57).

Taken together, our findings implicate the involvement of the innate immune system in the angiogenic response of the aortic wall to Ang-1 and VEGF. Confocal and electron microscopy studies demonstrate that this system is represented in the aortic ring model by adventitial macrophages and dendritic cells that actively migrate from the aortic explants, becoming associated with the roots and stems of the neovessel outgrowths. Our observations suggest that early upregulation of innate immune response genes may be a basic mechanism by which injured blood vessels respond to angiogenic stimulation. This is consistent with studies by others implicating macrophages as cellular sensors of tissue perturbations and regulators of the early stages of the inflammatory response to injury (18, 28). Ang-1 and VEGF may activate the aortic innate immune system indirectly through cytokine products of endothelial cell origin, but it is also possible that at least some of their effects are due to direct activation of adventitial macrophages. In fact, macrophages express VEGF receptor-1 (40), and the Ang-1 receptor Tie2 has been recently identified in a subpopulation of proangiogenic monocytic leukocytes (12, 26, 45). These receptors are also expressed by periendothelial mural cells and/or mural progenitor cells (20, 21, 53), which may represent additional early mediators of the angiogenic response.

The widespread upregulation of immune-related genes in response to angiogenic factor stimulation resembles activation of TLRs, which are characteristically expressed in macrophages and dendritic cells but can also be found in endothelial cells. Interestingly, the TLR-2 and the TLR-4 co-receptor CD14 are among the genes upregulated by Ang-1 and/or VEGF (Table 3). TLRs are a family of transmembrane proteins that typically regulate the response of the innate immune system to microbial components (48). Recent studies, however, indicate that, in addition to functioning as pattern recognition receptors for microbial ligands, TLRs can become activated in injured tissues by endogenous ligands released from necrotic cells (38, 51) or by fibrinogen, which typically accumulates in the extracellular matrix during wound healing (41). In addition, TLRs have recently been implicated in the angiogenic process (39). By upregulating expression of TLR genes, Ang-1 may activate the innate immune response of the aortic rings, thereby potentiating their ability to respond to angiogenic factor stimulation. Ang-1 and VEGF may amplify TLR responses by synergistic mechanisms through shared intracellular signaling pathways that are activated in the aortic wall after the injury of the dissection procedure. This possibility is corroborated by ongoing studies in our laboratory indicating that activation of TLRs with specific ligands stimulates angiogenesis in the aortic ring model (data not shown).

Many genes aside from those involved in the immune response are differentially expressed in the cultured rat aorta treated with Ang-1 or VEGF. Some of these genes such as IGF-II, placental growth factor, and tissue inhibitor of metalloproteinase-3 are known regulators of the angiogenic process, but most of the others have not been previously associated with angiogenesis. Interestingly, 12 of the named genes upregulated by Ang-1 and/or VEGF have been implicated in neural development and maintenance (Supplemental Table 1), a finding consistent with recent reports showing close developmental links and shared gene products between the neural and vascular systems (30, 43). Our study produced a list of 755 ESTs and cDNAs with unknown function that are dysregulated early in the process of neovessel formation. Whether these hitherto uncharacterized genes may encode for important protein mediators of the angiogenic process remains to be determined.

Previous studies utilizing microarray analysis to identify genes associated with angiogenesis have focused on VEGF-stimulated endothelial cell strains (1, 6, 15, 23, 46, 54). Our experiments instead analyzed the genetic response to angiogenic factor stimulation by the intact aortic wall, with all its cellular components, including endothelial cells, smooth muscle cells, fibroblasts, and macrophages. This approach allowed us to identify genes differentially expressed in response not only to VEGF but also to Ang-1 and to demonstrate an unexpected overlap of Ang-1- and VEGF-induced genes. It is likely that at least some of the changes in gene expression reflect paracrine interactions between the different cells of the vessel wall. Interestingly, though, changes in immune-related cytokine expression such as upregulation of GRO-1 and monocyte chemoattractant protein-1, which we report here, have also been observed in three-dimensional cultures of isolated endothelial cells (46, 54). The reproducibility of these findings in different models points to a common molecular mechanism by which endothelial cells respond to angiogenic factor stimulation.

In summary, we have demonstrated that the aorta model of angiogenesis can be used to dissect out the cascade of genetic events occurring in the vessel wall after stimulation with angiogenic factors. The demonstration that both Ang-1 and VEGF activate the innate immune system of the aortic wall and induce upregulated expression of many proangiogenic inflammatory cytokines provides new insights into the early transcriptional events occurring in the aorta before angiogenic sprouting. Although more studies are needed to characterize the role of the different gene products induced by Ang-1 and VEGF and identify their cell of origin, our observations point toward inflammatory cytokines as important early mediators of the angiogenic process. Further analysis of the immune-related events that precede angiogenic sprouting may unveil important new targets for both pro- and antiangiogenic therapeutic applications.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-52585 and HL-072370 and by a Merit Review Grant from the Department of Veterans Affairs Medical Research Service.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Debbie Jones, Division of Pathology and Laboratory Medicine, VA Puget Sound Health Care System, Seattle, WA, for excellent technical assistance with the electron microscopy studies. In addition, we thank the Center for Expression Arrays at the University of Washington, Seattle, WA, for microarray hybridizations and raw data analysis.


   FOOTNOTES
 
Address for reprint requests and other correspondence: R. F. Nicosia, Division of Pathology and Laboratory Medicine (S-113-Lab), VA Puget Sound Health Care System, 1660 South Columbian Way, Seattle, WA 98108 (e-mail: roberto.nicosia{at}va.gov).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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