Enhanced CXCL1 production and angiogenesis ... - The FASEB Journal

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Enhanced CXCL1 production and angiogenesis in adenosine-mediated lung disease Amir Mohsenin,*,† Marie D. Burdick,‡ Jose G. Molina,* Michael P. Keane,‡ and Michael R. Blackburn*,†,1 *Department of Biochemistry and Molecular Biology and †The Graduate School of Biomedical Sciences, University of Texas Houston Medical School, Houston, Texas, USA; and ‡Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles, California, USA Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling molecule that has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The current study utilized adenosine deaminase (ADA)deficient mice to determine whether chronic elevations in adenosine in vivo result in pulmonary angiogenesis. Results demonstrate substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. ADA replacement enzyme therapy resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the angiogenic chemokine CXCL1 (mouse functional homologue of human IL-8) were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its receptor, CXCR2, resulted in the inhibition of angiogenic activity, which suggests that CXCL1 signaling through the CXCR2 receptor mediated the observed increases in angiogenesis. Our findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis.— Mohsenin, A., Burdick, M. D., Molina, J. G., Keane, M. P., Blackburn, M. R. Enhanced CXCL1 production and angiogenesis in adenosine mediated lung disease. FASEB J. 21, 1026 –1036 (2007)

ABSTRACT

Key Words: chemokine 䡠 asthma 䡠 macrophage 䡠 adenosine deaminase Asthma is a chronic lung disease that affects 20 million Americans and results in over 11 billion dollars per year in direct health care expenditures [www. aaai.org]. It is characterized by an immune response that results in chronic inflammation, airway remodeling, and a progressive loss of lung function. One identified aspect of tissue remodeling in asthma is angiogenesis (1–3). Angiogenesis is defined as the growth of new blood vessels from preexisting vascula1026

ture and can occur as a result of chronic inflammation. Researchers have demonstrated angiogenesis in both the tracheas and lungs of asthmatic patients (1– 4). It has been hypothesized that increases in vasculature may contribute to the airway obstruction and airway hyperresponsiveness seen in asthmatic patients (5). In addition, new vessels may also serve as novel routes for inflammatory cell entry and as nutrient sources for the chronic inflammation and tissue remodeling. Angiogenesis is not a unique feature of asthma but has also been noted in other chronic lung diseases. Biopsies from patients with idiopathic pulmonary fibrosis (IPF) have shown increased lung angiogenic potential (6, 7), and vascular remodeling has been demonstrated to be important in bronchiolitis obliterans syndrome (BOS) following lung transplantation (8). In addition, bronchoalveolar lavage cells from patients with pulmonary sarcoidosis have an enhanced capacity to induce angiogenesis as compared to nonaffected individuals (9). Although angiogenesis is a feature of numerous chronic lung disorders, the pathways that regulate angiogenesis in the diseased lung are not completely understood. The CXC chemokine family consists of a group of chemotactic cytokines, which contain four conserved cysteines in their amino acid sequence with the first two separated by an intervening amino acid (10). The presence of the ELR (Glu, Leu, Arg) motif on the NH2 terminus of these molecules separates this family into two subsets. CXC chemokines containing the ELR motif have proangiogenic capabilities, whereas those lacking it act as inhibitors of angiogenesis (10). This chemokine family has been shown to play an important role in regulating angiogenesis during ischemia, wound healing, and cancer growth (11–15). CXC chemokine signaling has also been found to be important in regulating the angiogenic activity of BOS and IPF (6, 8). However, little is known about the factors that 1 Correspondence: Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston Medical School, 6431 Fannin St, Houston, TX 77030, USA. E-mail: [email protected] doi: 10.1096/fj.06-7301com

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regulate the production of ELR⫹ chemokines in injury situations associated with angiogenesis. Adenosine is a molecule that has a well-documented history of stimulating blood vessel growth. Many studies have shown that elevations in extracellular adenosine result in the stimulation of blood vessel formation both in vitro and in vivo (16 –21). Adenosine is a potent and ubiquitous signaling molecule, which accumulates in times of cellular stress and damage. At sites of inflammation, adenosine is generated by the catabolic breakdown of ATP and functions to regulate the inflammatory response through engagement of putative adenosine receptors. Consistent with this, asthmatic patients have increased adenosine levels in their BAL fluid and exhaled breath condensates, and the degree of adenosine elevations correlate with disease severity (22, 23). These findings suggest that adenosine may be involved in the disease pathogenesis of chronically inflamed asthmatic lungs, including angiogenesis. To address the consequences of elevated adenosine levels in vivo, we utilized mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) (24). By lacking the degradation pathway for adenosine, these mice develop systemic accumulations of this nucleoside. In the lungs of ADA-deficient mice, adenosine levels increase markedly in association with progressive pulmonary inflammation and airway remodeling (25). However, the status of angiogenesis in this model was not known. The current study utilized ADA-deficient mice to determine whether chronic elevations in adenosine result in pulmonary angiogenesis and to identify factors that could potentially mediate this process. We found angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. ADA replacement enzyme therapy resulted in a lowering of adenosine levels and also reversed the tracheal angiogenesis indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the proangiogenic chemokine CXCL1, the mouse functional homologue to CXCL8 (IL-8), were significantly elevated in the lungs of ADA-deficient mice. Interestingly, CXCL8 has been shown to be regulated by adenosine (26) and is elevated in the lungs of asthmatics (27). Our findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis. These are the first studies to demonstrate a functional connection between elevations in endogenous adenosine levels, ELR⫹ chemokine expression and angiogenesis in the diseased lung.

MATERIALS AND METHODS Mice ADA-deficient mice were generated and genotyped as described (24). Mice homozygous for the null Ada allele were designated ADA-deficient (ADA⫺/⫺), while mice heterozygous (⫾) or wild-type (⫹/⫹) for the null Ada allele were designated as ADA control mice (ADA⫹). All mice were on a mixed 129sv/C57BL/6J background, and all phenotypic comCXCL1 MEDIATED ANGIOGENESIS IN ADA-DEFICIENT MICE

parisons were performed among littermates. All experiments were repeated at least 3 times. Animal care was in accordance with the Animal Care Committees at the University of Texas Health Science Center at Houston and University of California at Los Angeles, and NIH guidelines. All mice and rats were housed in ventilated cages equipped with microisolator lids and maintained under strict containment protocols. No evidence of bacterial, parasitic, or fungal infection was found, and serological reports on cage littermates were negative for 12 of the most common murine viruses. Whole-mount staining of airway vasculature Mice were anesthetized and the trachea exposed. A longitudinal incision was made down the length of the trachea and then the trachea was excised. Surrounding tissue was removed from the trachea after which the trachea was flattened down using insect pins. Tracheas were then fixed using zinc fixative (BD Pharmingen, Franklin Lakes, NJ, USA) for 24 h and then washed 3 times in 1⫻ PBS for 5 min per wash. Tracheas were then permeabilized using PBS containing 0.1% Triton X-100 for 30 min at room temperature. Endogenous peroxidase activity was blocked by incubating tissues in 0.6% hydrogen peroxide for 30 min at room temperature. Immunohistochemistry for CD31 was performed according to the manufacturer’s guidelines with an antirat Ig HRP Detection Kit (BD Pharmingen). CD31 localization was achieved by incubating the tissues overnight at room temperature with a 1:250 dilution of rat anti-mouse CD31 antibody (BD Pharmingen). Tracheas were dehydrated and mounted using Permount (Fisher Scientific, Pittsburgh, PA, USA) to prepare for morphometric measurements. Morphometric measurements of vessels Morphometric measurements of blood vessels were made in whole mount tracheas stained with anti-CD31 antibody. Images were taken at ⫻40 using a Spot Camera (Diagnositic Instruments, Sterling Heights, MI, USA). The number of vessels traversing a cartilage ring was used as representative index of the total number of vessels in the trachea (28). This index was determined by placing a line of known length parallel to the long axis of the cartilage ring and counting the number of vessels intersecting the line. At least 12 cartilage rings from multiple mice per group were examined. Results were expressed as the number of vessels per unit length of the line drawn. ADA enzyme therapy Polyethylene glycol modified-ADA (PEG-ADA) was generated by the covalent modification of purified bovine ADA with activated polyethylene glycol as described previously (29). ADA⫺/⫺ mice received 5 U of PEG-ADA on postnatal day 18 via intraperitoneally (i.p.) injection. Mice were sacrificed 72 h after injection. PEG-ADA effects on tracheal angiogenesis were performed as follows: On day 18, ADA⫹ and ADA⫺/⫺ mice received 5 U of PEG-ADA via i.p. injection and every fourth day thereafter for a total of 3 injections. Mice were sacrificed on the tenth day after the initiation of enzyme therapy for assessment of tracheal angiogenesis. Bronchoalveolar lavage Mice were anesthetized with avertin, and lungs were lavaged 4⫻ with 0.3 ml PBS; 0.95–1 ml of pooled lavage fluid was recovered. Total cell counts were determined using a hema1027

cytometer, and aliquots were cytospun onto microscope slides and stained with Diff-Quick (Dade Behring, Deerfield, IL, USA) for cellular differentials. Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR Total RNA was isolated from whole-lung tissue using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Total RNA was treated using RNase-free DNase (Invitrogen Corp.). Lung RNA (1 ␮g) was used in a Superscript One-Step RT-PCR (Invitrogen Corp.) reaction with CXCL1 or ␤-actin–specific primers following manufacturer’s instructions. One-Step RT-PCR primer pairs used were as follows: CXCL1 (sense 5⬘-ATGAGCTGCGCTGTCAGTGC-3⬘, antisense 5⬘- CACCAGACGGTGCCATCAGA-3⬘). Alternatively, transcript levels were quantified using real-time quantitative RT-PCR. CXCL1 and ␤-actin transcripts were analyzed using Taqman probes on a Smart Cycler (Cepheid, Sunnyvale, CA, USA). Specific quantitative primers for mediators were developed using Primer Express software (Applied Biosystems, Foster City, CA, USA), following the recommended guidelines based on sequences from GenBank. The following Taqman primer and probe sequences were used to track transcript levels: CXCL1 (sense: 5⬘-CTGCACCCAAACCGAAGTC-3⬘, antisense 5⬘AGCTTCAGGGTCAAGGCAAG-3⬘, Probe 5⬘-CACTCAAGAATGGTCGCGAGGC-3⬘). ␤-actin (sense 5⬘-GCTCTGGCTCCTAGCACCAT-3⬘, antisense 5⬘-CCACCGATCCACACAGAGTAC-3⬘, probe ATCAAGATCATTGCTCCTCCTGAGCGC-3⬘). CXCR2 transcript levels were quantitated using Taqman primers and probes from Assays on Demand (Applied Biosystems). Specific transcript levels were determined using Smart Cycler analysis software through comparison to a standard curve generated from the polymerase chain reaction (PCR) amplification of template dilutions. Protein analysis Whole lung protein lysates were made, and protein concentrations were determined by use of a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded onto a sandwich ELISA kit specific for CXCL1 (R&D Systems, Minneapolis, MN, USA). Data are expressed as amount of CXCL1 protein per milligram of total lung protein. BAL fluid CXCL1 content was obtained by loading equal volumes of lavage fluid onto the CXCL1 specific ELISA plate and then multiplying the readout by total lavage fluid recovered. The data are expressed as total CXCL1 protein in the BAL fluid recovered.

Paraffin-embedded tissues were sectioned (5 ␮m), exposed to two changes of histoclear, and rehydrated in a series of graded alcohols to water. Antigen unmasking was performed before CXCL1 localization using target retrieval solution following the manufacturer’s guidelines (Dako, Copenhagen, Denmark). Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide. CXCL1 immunohistochemistry and blocking procedures were followed according to the manufacturer’s guidelines using goat IgG VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). CXCL1 localization was performed by incubating slides overnight at 4°C with a 2 ␮g/ml concentration of goat anti-mouse CXCL1 Ab (R&D Systems). After incubation with appropriate biotinylated secondary antibodies, the slides were incubated with avidin-biotinylated peroxidase complex (Vector Laboratories) for 30 min. The slides were developed using 3,3⬘-diaminobenzidine-tetrachloride (DAKO) for 7–10 min, Vol. 21

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Quantification of adenosine Mice were anesthetized, the thoracic cavity exposed, and the lungs rapidly removed and frozen in liquid nitrogen. Adenine nucleosides were extracted from frozen lungs using 0.4 N perchloric acid (PCA) as described, and adenosine was quantified using reversed-phase HPLC (30). Adenosine levels were normalized to protein content, and values are given as nmol/mg protein. Corneal micropocket assay of angiogenesis Angiogenic activity of lung homogenates was assayed in vivo in the avascular cornea of hooded Long-Evans rat eyes, as described previously (31–34). Briefly, equal volumes of lyophilized lung tissue specimens normalized to total protein were combined with sterile Hydron (IFN Sciences, New Brunswick, NJ, USA) casting solution. Aliquots (5 ␮l) were pipetted onto the flat surface of an inverted sterile polypropylene specimen container and polymerized overnight in a laminar flow hood under UV light. Before implantation, pellets were rehydrated with normal saline. Animals were anesthetized with ketamine (150 mg/kg) and atropine (250 ␮g/kg) i.p. Rat corneas were anesthetized with 0.5% proparacaine hydrochloride ophthalmic solution followed by implantation of the Hydron pellet into an intracorneal pocket (1–2 mm from the limbus). Six days after implantation, animals received 1000 U of heparin and ketamine (150 mg/kg) i.p., followed by a 10 ml perfusion of colloidal carbon via the left ventricle. Corneas were harvested and photographed. No inflammatory response was observed in any of the corneas treated with the above specimens. Positive neovascularization responses were recorded only if sustained directional growth of capillary sprouts and hairpin loops toward the implant was observed. Negative responses were recorded when either no growth was observed or only an occasional sprout or hairpin loop displaying no evidence of sustained growth was detected. For neutralization studies, lung extracts were preincubated with either a goat anti-mouse CXCL1 antibody (R&D Systems) or a rabbit anti-mouse antibody against CXCR2 (8).

RESULTS Adenosine-dependent tracheal angiogenesis in ADAⴚ/ⴚ mice

Immunohistochemical localization of CXCL1

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counterstained with methyl green for 5 min, dehydrated, and mounted.

Increased tracheal vascularity is a feature of chronic lung disease (1, 6, 8). To assess whether the adenosineinduced pulmonary phenotype observed in ADA⫺/⫺ mice also results in tracheal angiogenesis, immunohistochemistry was performed on whole mount tracheas (Fig. 1). Tracheas from 18 d old mice were excised, flattened, fixed, and stained with an anti-CD31 antibody in order to visualize blood vessels. Angiogenesis, when present, can be observed in the capillary beds traversing cartilage rings (28). Blood vessel vascularity was quantified by counting the number of vessels intersecting a line of known length drawn lengthwise down a cartilage ring. ADA⫺/⫺ mice demonstrated increased vessel numbers as compared to ADA⫹ mice

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(Fig. 1A, B) indicating that chronic elevations in adenosine result in new blood vessel formation. To determine if the increased vasculature observed in the tracheas was adenosine mediated, mice were treated with PEG-ADA enzyme therapy at a stage when angiogenesis was already established. Injections of PEGADA enzyme were initiated on postnatal day 18 and were given every fourth day for a total treatment period of 10 d. Analysis of tracheal vascularity after PEG-ADA treatment revealed that enzyme therapy was able to reverse established angiogenesis (Fig. 1A, B) Specifically, ADA⫺/⫺ mice receiving PEG-ADA showed vessel numbers similar to ADA⫹ mice, indicating that adenosine levels directly correlate with the degree of vascularity (Fig. 1B). To verify that the decreased vascularity seen was associated with decreased adenosine levels, adenine nucleosides were extracted and quantified in the lungs of mice treated with PEG-ADA as described above. Results demonstrated that treatment with PEGADA was associated with significant reductions in lung adenosine levels (Fig. 1C). Together, these results suggest that adenosine-dependent angiogenesis in the tracheas of ADA⫺/⫺ mice is reversible with ADA enzyme replacement therapy. CXCL1 RNA and protein are increased in the lungs of ADAⴚ/ⴚ mice

Figure 1. Effects of ADA enzyme therapy on tracheal angiogenesis. A) Day 18 ADA⫹ and ADA⫺/⫺ mice were kept on 10 d of PEG-ADA enzyme therapy. On day 10, tracheas were excised, fixed, and stained with anti-CD31 antibody using whole mount immunohistochemistry. ADA⫺/⫺ mice not treated with PEG-ADA enzyme therapy were analyzed on postnatal day 18. Images are representative of 3 to 5 samples per group. B) Tracheal vascularity was quantified in the samples shown in A by counting the number of vessels intersecting a line down the length of the cartilage ring. At least 12 cartilage rings were analyzed per sample. Data are represented as fold changes ⫾ sem; n ⫽ 3 for ADA⫹ and n ⫽ 4 for ADA⫺/⫺, n ⫽ 5 for ADA⫹ ⫹ PEG-ADA, n ⫽ 5 for ADA⫺/⫺ ⫹ PEG-ADA. C) Whole lung adenosine levels were quantified in lung samples from mice treated as described in (A) above. Data are presented as mean nmoles adenosine per mg protein ⫾ sem; n ⫽ 6 for ADA⫹ and ADA⫺/⫺, n ⫽ 6 for ADA⫹ ⫹ PEG-ADA and ADA⫺/⫺ ⫹ PEG-ADA. *P ⱕ 0.05 compared to ADA⫹ mice. **P ⱕ 0.05 compared to ADA⫺/⫺ mice.

CXCL1 MEDIATED ANGIOGENESIS IN ADA-DEFICIENT MICE

To begin to ascertain what factors may be responsible for the angiogenesis observed in ADA⫺/⫺ mice, we examined the expression of the angiogenic chemokine CXCL1 (Fig. 2). Semiquantitative RT-PCR was performed on total lung RNA extracts from day 18 ADA⫹ and ADA⫺/⫺ mice using CXCL1 specific primers (Fig. 2A). RNA extracts from two separate cohorts of animals showed increased message in the lungs of ADA⫺/⫺ mice. These findings indicate that CXCL1 levels are elevated at a stage when there is increased adenosine (25) and angiogenesis (Fig. 1) in the lungs of ADA⫺/⫺ mice. ADA enzyme replacement therapy is an effective means of lowering lung adenosine concentrations in mice (25). To more accurately quantify the levels of CXCL1 and monitor their dependence on elevations in adenosine, real-time RT-PCR was performed on total lung RNA extracts were made from the lungs of day 18 ADA⫹ and ADA⫺/⫺ mice along with lungs of ADA⫺/⫺ mice treated with PEG-ADA. ADA⫺/⫺ mice demonstrated a significant increase in CXCL1 transcripts that was reversible on PEG-ADA administration (Fig. 2B). Whole lung CXCL1 protein levels were assayed with a CXCL1 specific ELISA that demonstrated a similar pattern of increased CXCL1 protein levels in ADA⫺/⫺ lungs that was reversible on PEG-ADA treatment (Fig. 2C). In addition, CXCL1 protein in the BAL fluid of ADA⫺/⫺ mice was elevated and was lower following PEG-ADA treatment (Fig. 2D). These results indicate that in the setting of elevated adenosine there is elevated CXCL1 RNA and protein. Furthermore, once adenosine levels are elevated and then acutely lowered 1029

with PEG-ADA, CXCL1 RNA and protein are reduced to normal levels. CXCL1 expression is elevated in alveolar macrophages of ADAⴚ/ⴚ mice Bronchoalveolar lavage was performed on day 18 ADA⫹ and ADA⫺/⫺ mice, and cell differentials were obtained. Analysis revealed a significantly increased number of alveolar macrophages in the airways of ADA⫺/⫺ mice (Fig. 3A). To determine if these cells were a source of the increased CXCL1 in ADA⫺/⫺ lungs, real-time RTPCR was performed on BAL cell pellets. Results demonstrated that CXCL1 transcripts are only present in BAL cell pellets form ADA⫺/⫺ mice, while BAL cell pellets from ADA⫹ mice showed undetectable levels (Fig. 3B). Given that the alveolar macrophage is the most prominent cell type in BAL cell pellets of ADA⫺/⫺ mice, we hypothesized that this cell is a source of CXCL1 transcripts. To validate this observation, lung sections from day 18 ADA⫹ and ADA⫺/⫺ mice were stained with a CXCL1 specific antibody (Fig. 3C, D). Lung section from ADA⫺/⫺ mice showed increased CXCL1 protein present in alveolar macrophages and to a lesser degree airway epithelium. Thus, alveolar macrophages and airway epithelial cells are sources of CXCL1 in the lungs of ADA⫺/⫺ mice. ADAⴚ/ⴚ mice have increased lung angiogenic activity Next, the corneal micropocket assay of angiogenesis (8) was utilized to directly examine the angiogenic activity of the lungs of ADA⫺/⫺ mice. Lung extracts from day 18 ADA⫹ and ADA⫺/⫺ mice ⫾ PEG-ADA enzyme therapy were assayed for angiogenic activity (Fig. 4). Results demonstrated that extracts from the lungs of ADA⫺/⫺ mice have angiogenesis in 6/6 (100%) of the corneas examined, while extracts from the lungs of ADA⫹ mice only displayed a positive angiogenesis response in 1/6 (16%) corneas. Extracts from the lungs of ADA⫺/⫺ mice treated with PEG-ADA demonstrated a reversal of lung angiogenic activity. These findings demonstrate that there is angiogenic activity in the lungs of ADA⫺/⫺ mice that is dependent on adenosine elevations. Figure 2. Analysis of CXCL1 levels in the lungs of ADA⫺/⫺ mice. A) CXCL1 transcript levels were measured in wholelung RNA extracts from postnatal day 18 ADA⫹ and ADA⫺/⫺ mice using semiquantitative RT-PCR. Findings from 2 different pairs of littermates are shown. ␤-actin was used as an RNA-positive loading control for each sample. B–D) In order to acutely lower adenosine levels, day 18 ADA⫺/⫺ mice were given 5 U of PEG-ADA enzyme via i.p. injection. Tissues were harvested 72 h after injection. B) Whole lung RNA extracts analyzed using real-time quantitative RT-PCR specific for CXCL1. Data are represented as the mean percent of CXCL1 lung transcripts over ␤-actin transcripts ⫾ sem; n ⫽ 12 for ADA⫹, n ⫽ 14 for ADA⫺/⫺ and n ⫽ 9 for ADA⫺/⫺ ⫹ PEG-ADA. C) Whole lung protein extracts were analyzed using a CXCL1 specific ELISA. Data are represented as picograms CXCL1 protein over total lung protein; n ⫽ 7 for 1030

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CXCR2, the putative CXCL1 receptor, is elevated in the lungs of ADAⴚ/ⴚ mice CXCL1 exerts its angiogenic effects through its putative receptor, CXCR2 (10). To determine the status of CXCR2 in the lungs of ADA⫺/⫺ mice, total lung RNA

ADA⫹, n ⫽ 6 for ADA⫺/⫺ and n ⫽ 6 for ADA⫺/⫺ ⫹ PEG-ADA. D) BALF CXCL1 protein levels as analyzed by ELISA. Data are represented as total picograms of CXCL1 protein present in the BAL fluid; n ⫽ 8 for ADA⫹, n ⫽ 9 for ADA⫺/⫺ and n ⫽ 6 for ADA⫺/⫺ ⫹ PEG-ADA. *P ⱕ 0.05 compared to ADA⫹ mice. **P ⱕ 0.05 compared to ADA⫺/⫺ mice.

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Figure 3. Localization of CXCL1 to alveolar macrophages. A) BAL fluid was collected from the lungs of postnatal day 18 mice and cell differentials were determined. Cells examined included macrophages, lymphocytes, eosinophils, and neutrophils. Data are represented as mean total cells ⫾ sem; n ⫽ 14 for ADA⫹, n ⫽ 6 for ADA⫺/⫺. B) CXCL1 transcript levels were determined in BAL cell pellets collected from postnatal day 18 mice using real-time quantitative RT-PCR analysis. Data are represented as the mean percentage of CXCL1 transcripts/␤-actin transcripts ⫾ sem; n ⫽ 4 for ADA⫹ and n ⫽ 6 for ADA⫺/⫺. *P ⱕ 0.05 compared to ADA⫹ mice. For CXCL1 immunohistochemistry, lungs from postnatal day 18 ADA⫹ (C) and ADA⫺/⫺ (D) mice were collected and processed for immunostaining with anti-CXCL1 antibody. Arrows denote alveolar macrophages; ND, not detected.

was analyzed for CXCR2 transcripts using a specific real-time RT-PCR assay. Results demonstrated that CXCR2 transcript levels are increased in the lungs of ADA⫺/⫺ mice on day 18 (Fig. 5). Additionally, CXCR2 transcript levels decreased in the lungs of ADA⫺/⫺ mice on treatment with PEG-ADA indicating a role for adenosine in modulating transcript levels. CXCL1 signaling mediates increased angiogenesis in the lungs of ADAⴚ/ⴚ mice To determine the contribution of CXCL1 signaling to the increased angiogenic activity of ADA⫺/⫺ lung lysates, we performed the corneal micropocket assay of angiogenesis with ADA⫺/⫺ lung lysates treated with neutralizing antibodies (Fig. 6). Neutralizating antibodies to both CXCL1 and CXCR2 resulted in the inhibition of angiogenic activity in extracts from the lungs of ADA⫺/⫺ mice. These results indicate that CXCL1 signaling through the CXCR2 receptor is responsible for the increased angiogenic activity of ADA⫺/⫺ lung homogenates. CXCL1 MEDIATED ANGIOGENESIS IN ADA-DEFICIENT MICE

DISCUSSION Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage (35). Increased levels of this molecule have been found in the lungs of asthmatics (22), indicating a role in disease pathogenesis. However, the functional consequence of elevated adenosine and its impact on pulmonary inflammation and remodeling are not fully understood. In the current study, we demonstrate that in vivo elevations in adenosine lead to tracheal angiogenesis, a feature of asthma (1). We identify the CXC chemokine, CXCL1, as a mediator of angiogenesis and characterize its expression in the ADA⫺/⫺ mouse lung. In addition, we show that lowering of adenosine levels in vivo with ADA enzyme therapy not only results in a lowering of lung CXCL1 expression, but also reverses tracheal angiogenesis. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA⫺/⫺ lung lysates results in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for adenosine-mediated angiogenesis in the lung. These are the first studies to demonstrate that adenosine can 1031

Figure 4. Corneal micropocket angiogenesis assay. A) Analysis of lung angiogenic activity reveals increased activity in the lungs of ADA⫺/⫺ as compared to ADA⫹ mice, evidenced by blood vessel growth toward implanted pellets. Lowering of adenosine levels with PEG-ADA enzyme therapy in ADA⫺/⫺ mice results in the inhibition of angiogenic activity. B) Quantification of corneal micropocket angiogenesis assay results demonstrates angiogenesis in 100% of ADA⫺/⫺ mouse lung samples while ADA⫹ mouse lung samples have 16% positive results. Treatment with PEG-ADA enzyme therapy reverses ADA⫺/⫺ mouse lung angiogenesis to control levels. Data are represented as % angiogenesis positive corneas. n ⫽ 6 per group.

promote angiogenesis in an in vivo model of chronic lung disease, in association with the regulation of angiogenic ELR⫹ chemokines. Elevated levels of adenosine are found in the BAL fluid, exhaled breath condensates and blood of patients with asthma (22, 23, 36). This damage associated increase in adenosine is not a unique feature of asthma, but is a fundamental process evident in other inflammatory diseases such as rheumatoid arthritis, sepsis, ischemia-reperfusion injury, and chronic obstructive pulmonary disease (22, 23, 37). Inflammation causes an increased energy demand, which results in ATP catabolism and subsequent elevations in adenosine. Adenosine then engages G-protein coupled adenosine receptors (A1R, A2AR, A2BR, and A3R) on the cell surface and functions to regulate the inflammatory response and restore oxygen balance (38). A key mechanism by which oxygen balance is restored during injury is through the process of new blood vessel formation or angiogenesis. 1032

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Angiogenesis is defined as the growth of new blood vessels from a preexisting vascular network (39). It is a common occurrence following chronic inflammation and has been thought to promote disease pathogenesis. Angiogenesis has been described in many injury models and has recently been identified in the chronic lung disease asthma (1– 4). Tanaka et al. were able to directly visualize airway walls in asthmatic and non asthmatic patients using a high magnification bronchovideoscope (1). They demonstrated increased airway vascularity in asthmatics as compared to controls. Despite these significant findings, the molecular signals responsible for initiating the process of new blood vessel formation in lung disease are not well understood. One candidate that is elevated in human asthma and capable of inducing angiogenesis is adenosine. Chronic elevations in adenosine have been linked to angiogenesis. Numerous in vivo studies using dipyridamole, an agent that increases extracellular adenosine levels, have shown in vivo stimulation of blood vessel growth (16 –19). These findings of adenosine mediated angiogenesis are consistent with a long term role for adenosine in restoring oxygen homeostasis in injury situations. Yet, the consequences of increased adenosine concentrations on the pulmonary vasculature during injury had not been examined. This study investigated the status of tracheal angiogenesis in the adenosine-dependent pulmonary injury seen in ADA⫺/⫺ mice. The ADA⫺/⫺ mouse, in association with elevated adenosine levels, develops pulmonary disease with many features of asthma including, chronic inflammation, airway hyperresponsiveness, mucus hyper-

Figure 5. CXCR2 transcript levels. Whole lung RNA extracts were analyzed using real-time quantitative RT-PCR specific for CXCR2. Data are represented as the mean percentage of CXCR2 transcripts/␤-actin transcripts ⫾ sem n ⫽ 4 for ADA⫹, n ⫽ 8 for ADA⫺/⫺ and n ⫽ 5 for ADA⫺/⫺ ⫹ PEG-ADA. *P ⱕ 0.05 compared to ADA⫹ mice. ADA⫺/⫺ as compared to ADA⫺/⫺ ⫹ PEG-ADA yielded a P valued of 0.08.

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Figure 6. Neutralization of CXCL1 & CXCR2 inhibits ADA⫺/⫺ lung angiogenic activity. A) Corneal micropocket analysis of angiogenic activity demonstrates inhibition of angiogenic activity in the lungs of ADA⫺/⫺ mice with neutralizing antibodies to CXCL1 and CXCR2, while normal goat serum had no effect. B) Quantification of angiogenic activity in ADA⫺/⫺ mice treated with either normal goat serum (NGS) or neutralizing antibodies to CXCL1 and CXCR2. Results demonstrate inhibition of angiogenic activity in samples treated with neutralizing antibody. Data are represented as % angiogenesis positive corneas. n ⫽ 6 per group.

secretion, and subepithelial fibrosis (25, 40). Thurston et al. described quantification of the number of vessels traversing a cartilage ring as a representative index of the total number of tracheal vessels (28). Whole mount immunohistochemistry on ADA⫺/⫺ mouse tracheas using an anti-CD31 antibody demonstrated increased vessel numbers. Although our studies do not directly measure angiogenesis in the lung, increases in tracheal angiogenesis have been correlated with increases in angiogenesis in the lung (41). Moreover, analysis of lung angiogenic potential using the corneal micropocket angiogenesis assay demonstrated increased lung angiogenic activity in ADA⫺/⫺ mice as compared to controls. While it is clear that tissue levels of adenosine are elevated in the lungs of ADA⫺/⫺ mice, these data do not allow for the determination of extracellular adenosine levels that are available for the activation of adenosine receptors. However, the regulation of tissue adenosine levels in this model with ADA enzyme therapy provides evidence that in the setting of elevated lung adenosine there is increased tracheal vascularity and increased whole lung angiogenic activity. Although the mechanism of adenosine induced angiogenesis is not well understood, most studies have largely focused on its ability to induce vascular endothelial growth factor (VEGF) expression (42, 43). However, it is becoming increasingly evident that a multifaceted mechanism is involved in blood vessel development. An emerging theme in angiogenesis research is that of CXC chemokine driven angiogenesis (10). The CXC chemokine family is a heparin binding group of chemotactic cytokines that are important factors in disease pathogenesis. The presence or abCXCL1 MEDIATED ANGIOGENESIS IN ADA-DEFICIENT MICE

sence of an (Glu-Leu-Arg) ELR motif on the NH2 terminus of these molecules subdivides this group in two. Those containing this motif (ELR⫹) are inducers of angiogenesis while those lacking it (ELR-) are inhibitors of angiogenesis (10). The importance of chemokine-induced angiogenesis is highlighted by an article from Arenberg et al. They showed that neutralization of CXC chemokine signaling in a model of human non small cell lung cancer resulted in a 40% reduction in tumor size in association with a decline in tumorassociated vascular density and angiogenic activity (44). In addition, the angiogenesis associated with BOS after lung transplantation is attributed to CXC chemokine signaling and not VEGF signaling (8). It thus appears that chemokine-driven angiogenesis plays an important role in chronic lung disease. However, little is known about the primary factors responsible for inducing CXC chemokine expression. IL-8 is an ELR⫹ member of the CXC chemokine family and has also been shown to be elevated in asthma (45). Feoktistov et al. demonstrated that adenosine receptor stimulation results in the release of IL-8 from human mast cells (26). CXCL1 is the mouse functional homologue of IL-8, which is lacking in the mouse due to an ancestral deletion (46). The results of the current study demonstrate that ADA⫺/⫺ mice have elevated whole lung CXCL1 RNA and protein levels. In addition, ADA⫺/⫺ mice exhibited elevated CXCL1 protein in the BAL fluid, which is of significance since it demonstrates that the protein is not only being made but is also being secreted into the airways. Cell pellet differentials indicate that the predominant cell in the airways of ADA⫹ and ADA⫺/⫺ mice was the alveolar 1033

macrophage. However, the ADA⫺/⫺ mouse, in addition to having a marked increase in macrophage numbers, displays altered macrophage morphology. ADA⫺/⫺ mouse macrophages have a foam cell appearance, an increased cytoplasm to nuclear ratio, and also form multi-nucleated giant cells (data not shown). Analysis of BAL cell pellet message revealed the presence of CXCL1 transcripts only in pellets isolated from ADA⫺/⫺ mouse lungs. Immunohistochemistry using a CXCL1 specific antibody demonstrated protein localization in ADA⫺/⫺ alveolar macrophages and epithelial cells. These results indicate that the alveolar macrophage is an important source of CXCL1 transcripts and protein in the lungs of ADA⫺/⫺ mice. Macrophage function can significantly impact the natural history of many inflammatory disease processes (47, 48) and our results indicate that in the setting of elevated adenosine, the alveolar macrophage becomes an activated proinflammatory cell and thus represents a potential therapeutic target. It is not yet known how adenosine regulates the expression of CXCL1 in alveolar macrophages or other cells in the lung. However, note that the low-affinity A2BR, which is activated at high concentrations of adenosine, is elevated in activated macrophages (49, 50), and is responsible for the release of IL-8 from human mast cells (26), and the expression of angiogenic factors from endothelial cells (43). Furthermore, A2BR levels are elevated in the lungs of ADA⫺/⫺ mice (51), and a recent study demonstrates that antagonism of the A2BR in ADA⫺/⫺ mice leads to decreased CXCL1 production in the lungs (52). Cumulatively, these findings implicate the A2BR in the regulation of CXCL1 expression in the lungs of ADA⫺/⫺ mice. It is also important to note that the A2AR has been implicated in the regulation of angiogenesis by its regulation of both pro- and antiangiogenic molecules (21, 53). Determining the contribution of specific adenosine receptors to adenosine mediated angiogenesis in the lung are currently under investigation. We thus conclude that in association with elevated levels of adenosine and CXCL1, ADA⫺/⫺ mice develop pulmonary angiogenesis. Interestingly, lung levels of the CXCL1 receptor CXCR2 are also elevated in ADA⫺/⫺ mice. CXCR2 has been identified as the putative receptor for ELR⫹ CXC chemokine induced angiogenesis (54). The increase in whole lung CXCR2 transcripts may either reflect an increase in inflammatory cells bearing this receptor or increased expression from resident lung cells. It is generally accepted that hypoxia can stimulate the angiogenic potential of endothelium and a study from Moldobaeva et al. specifically demonstrated hypoxia induced up-regulation of CXCR2 in aortic endothelial cells (55). Our data demonstrating that PEG-ADA enzyme therapy can reduce lung CXCR2 transcripts after 72 h suggests that hypoxia may be mediating this effect since lung inflammatory cell numbers are not significantly altered after enzyme therapy (25). To ascertain if lowering adenosine levels in vivo would result in a change in the status of CXCL1 and 1034

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angiogenesis, we began ADA enzyme therapy after the establishment of disease and pathology. Day 18 mice received PEG-ADA and were examined 72 h after treatment. Measurement of CXCL1 in lung homogenates demonstrated significantly decreased levels at both the RNA and protein level. In association with decreased levels of adenosine and CXCL1, corneal micropocket analysis of lung angiogenic activity demonstrated levels similar to control. Analysis of tracheal vascularity after PEG-ADA enzyme therapy showed complete reversal of angiogenesis as ADA⫺/⫺ mice who received PEG-ADA starting on day 18 for a period of 10 d demonstrated the same level of tracheal vascularity as ADA⫹ mice. This reversal of tracheal angiogenesis is striking and indicates a potential use of ADA enzyme therapy in the treatment of pathological angiogenesis. These results demonstrate that the increases in CXCL1 and angiogenesis in ADA⫺/⫺ mice correlate with elevated adenosine. Despite these findings it was not yet clear whether the angiogenesis in ADA⫺/⫺ mouse lungs was due to CXCL1 signaling. Analysis of ADA⫺/⫺ lung angiogenic activity after neutralization of CXCL1 demonstrated an inhibition of vessel formation. Furthermore, inhibition of CXCR2 yielded similar results indicating that the angiogenic activity in ADA⫺/⫺ mouse lungs is mediated by CXCL1 signaling through the CXCR2 receptor. In summary, we have demonstrated for the first time angiogenesis in the lungs of ADA⫺/⫺ mice. These mice, in association with elevated levels of adenosine, have increased tracheal vascularity and increased lung angiogenic activity. ADA⫺/⫺ mice have increased lung RNA and protein levels of the proangiogenic chemokine CXCL1. Furthermore, the increased lung angiogenic activity is directly attributable to CXCL1 signaling through its receptor, CXCR2. Thus, the elevated levels of adenosine and ELR⫹ CXC chemokines found in the lungs of asthmatics may be responsible for the observed angiogenesis and therefore represent potential therapeutic targets. This work was supported by NIH grants AI-43572 and HL-70952 (to M.R.B.), and P50HL67665 and HL087186 (to M.P.K).

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Received for publication September 17, 2006. Accepted for publication November 14, 2006.

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