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Published ahead of print on February 14, 2003, doi:10.1165/rcmb.2002-0276OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 172-179, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0276OC

Identification of Genes Promoting Angiogenesis in Mouse Lung by Transcriptional Profiling

Sorachai Srisuma, Shyam S. Biswal, Wayne A. Mitzner, Sandra J. Gallagher, Kim H. Mai and Elizabeth M. Wagner

Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Department of Environmental Health Sciences, Bloomberg School of Public Health, and Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland

Address correspondence to: Elizabeth M. Wagner, Ph.D., Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, 5501 Hopkins Bayview Circle, Baltimore, Maryland 21224. E-mail: wagnerem{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A better understanding of the regulation of factors that promote angiogenesis may ultimately enable improved therapeutic control of this important process. In our previous studies, obstruction of the left pulmonary artery in the mouse consistently induced the formation of a new vasculature, which developed from the visceral pleura and entered the upper left lung directly within 5–6 days after ligation. No new vessels developed to the lower left lung, despite the initial ischemic stimulus being identical to that in the upper lung. Using this unique model of angiogenesis, we have determined the temporal pattern of differential gene expression from two independent regions of the same lung: one where angiogenesis is induced, and the other where angiogenesis does not occur. Microarray analysis and quantitative real-time RT-PCR were used to compare the signals from these two lung regions in the first 3 d following ischemia. The findings reveal the important roles of ELR+ CXC chemokines as proangiogenic signals. Genes involved in tissue remodeling, inflammation, and injury were also upregulated in the proangiogenic upper lung. Results also confirm that lung ischemia, rather than hypoxia, is the essential trigger for angiogenesis. These altered profiles of expression in the early stage of lung ischemia show potential roles and interactions of the most important genes involved in promoting new blood vessel formation.

Abbreviations: a disintegrin and metalloproteinase, ADAM • expressed sequence tags, ESTs • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • hypoxia-inducible factor, HIF • interleukin, IL • keratinocyte-derived chemokines, KC • lipopolysaccharide-induced CXC chemokines, LIX • lower left lung, LLL • left pulmonary artery, LPA • macrophage receptor with collagenous structure, MARCO • macrophage inflammatory protein 2, MIP-2 • right lung, RL • upper left lung, ULL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiogenesis, the process in which new blood vessels sprout and emerge from an established network, is fundamental to the repair of all injured tissues and organs. In the lung, angiogenesis of the bronchial circulation has been consistently observed following obstruction of the pulmonary vasculature (1, 2). Angiogenic vessels from the thoracic wall to the lung have also been evident following pulmonary vascular obstruction or other lung pathologies including primary lung tumors (3, 4). The pulmonary vasculature itself seems to have minimal capacity for true angiogenesis (5).

We have recently described a unique mouse model of lung angiogenesis, initiated by ligation of the left main pulmonary artery (6). This insult leads to an extensive new blood vessel growth arising from intercostal arteries adjacent to the third intercostal space, where the thoracotomy was performed for the initial vascular ligation. Functional vessels appear by the fifth to sixth day after ligation. These vessels grow into the upper left lung (ULL) and ultimately connect with the existing pulmonary vasculature. There are no new vascular connections to the lower left lung (LLL) (Figure 1). Because the same ischemic insult in the upper and lower lung regions leads to such extreme differences in vascular response, this model provides a unique opportunity to gain insight into the molecular signaling essential for angiogenesis. We have determined the temporal pattern of differential gene expression from two independent regions of the same lung, one where angiogenesis develops and the other where it does not. This experimental design thus has intrinsic physiologic controls within each animal. The ULL can be compared with the LLL where there is no angiogenesis, and both ULL and LLL can be compared with the nonischemic right lung (RL). In this initial report, we focus on the early signaling in the first 3 d that sets the stage for the promotion of new blood vessel formation. Using oligonucleotide microarray analysis, we assessed the temporal pattern of gene expression in the different lung regions. Results show an important role for the CXC family of cytokines in promoting angiogenesis following ischemia.



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  		Figure 1. Mouse model of lung angiogenesis. 5–6 d following thoracotomy in the third intercostal space and left pulmonary artery (LPA) ligation, new blood vessels develop from upper left intercostal arteries adjacent to the upper left chest wall and grow directly to the upper left lung (ULL). No angiogenesis occurs in the lower left lung (LLL) or lungs with sham-thoracotomy.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
To examine the differential gene expression of early lung ischemia within the first 3 d, which leads to different phenotypic responses (Day 5 and later), mRNA levels from the ULL and the LLL were compared with the nonischemic RL within individual animals (Figure 2). The significant genes were clustered according to similarities in expression with time (n = 2 mice for 4, 8, 24, and 72 h after left pulmonary artery [LPA] ligation). To identify the essential early signals in the ischemic lung triggering angiogenesis from the thoracic wall, genes with differential expression of ULL to LLL were categorized according to function. Real-time RT-PCR (TaqMan) was used to validate some of selected genes from microarray data at 4, 8, 24, and 72 h after LPA ligation (n = 4 mice for 24 h and n = 3 mice for all other time points).



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  		Figure 2. Study of angiogenesis in mouse lung. Schematic depicting the timeline for experimental procedure, the formation of new vasculature from the thoracic wall and the strategy for comparison and analysis of transcriptional profiles.

 
Occlusion of Left Pulmonary Artery
Male C57BL/6J (Jackson Laboratories, Bar Harbor, ME) mice, 6 wk old, were used in this study. Animal protocols for this study were reviewed and approved by the Johns Hopkins Institutions Animal Care and Use Committee and were conducted using national guidelines for the care and protection of animals.

Mice were anesthetized with 2% isoflurane in oxygen, then intubated and mechanically ventilated with this mixture at 120 breaths per minute with a tidal volume of 0.2 ml. A left thoracotomy was performed at the third intercostal space to expose the left lung. The LPA was identified, separated from the airway and ligated using 6–0 silk suture. The thoracotomy was closed by suture around the 3rd and 4th ribs. Lidocaine was applied to the thoracotomy site and the skin incision was closed using acrylamide adhesive (Future Glue; Pacer Technologies, Rancho Cucamonga, CA). The animal was removed from the ventilator, extubated and allowed to recover. No systemic analgesic was given due to the rapidity of recovery of normal behavior (within 30 min) and the early time points of data acquisition. At the 4, 8, 24, and 72 h time points after the LPA ligation, animals were anesthetized (as above) and killed by cervical dislocation. The left and right lungs were harvested. The left lungs were divided into an upper region adjacent to the third intercostal space and a lower region below the sixth intercostal space. The ULL, LLL, and RL samples from each animal were rinsed in cold phosphate-buffered saline and then immediately frozen in liquid nitrogen.

Isolation of RNA
Total RNA isolation was prepared from the ULL, LLL, and RL samples of each animal separately at the various time points using the TRIzol reagent (Invitrogen, Carlsbad, CA). Cleanup of total RNA was performed with the RNeasy Mini Kit (Qiagen, Valencia, CA).

Transcriptional Profiling by Oligonucleotide Microarray
Total RNA (6.5 µg) was used for the starting material. Total RNA obtained from each lung sample (ULL, LLL, RL) at specific time point after ischemia (n = 2 for 4, 8, 24 and 72 h) was processed independently without pooling for use on Murine Genome U74A version 2 array (Affymetrix, Santa Clara, CA) as previously described (7).

Data Analysis Using Affymetrix Software
Scanned output files were analyzed with the Affymetrix Microarray Suite 5.0. To identify differentially expressed transcripts, comparison analyses were performed using Data Mining Tool 3.0 (DMT; Affymetrix) between the following arrays obtained from each animal: (a) ULL and LLL for angiogenic response, (b) ULL and RL as well as (c) LLL and RL for ischemic response (Figure 2). For each gene, the analysis compares the differences of perfect match and mismatch intensities of each probe pair (15–20 probe pairs for a particular probe set) in the experimental array to its matching probe pair on the reference array, thereby providing the differential fold change. Wilcoxon's signed rank test, which was used to compare the direction of change among all probe pairs in the experimental sample to the reference sample, provides the P values assigning the change call as increase or decrease (P value <= 0.0025) and no change. The threshold of fold change used in this study was based on the recommendation of the company for a gene displaying differential expression >= 2-fold (Affymetrix). In addition, we selected to include the genes showing >= 2-fold expression between experimental samples and control samples in both animals per each time point.

Hierarchical Clustering
Clustering analysis was performed on the microarray data to group the genes according to the similarity of their expression patterns by using a complete linkage hierarchical clustering algorithm. These computations were performed on the fold change value using GENE CLUSTER and illustrated in a dendrogram with the TREEVIEW program (8).

Quantitative Real-Time RT-PCR (TaqMan)
We used RNA from both the same microarray experiment and from additional animals subjected to the same surgical procedures (left thoracotomy and LPA ligation) for independent validation by RT-PCR. Reverse transcription was performed by random hexamer primers and MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). Using 100 ng of cDNA as a template, quantification was performed by an ABI Prism 5700 Sequence Detector (Applied Biosystems) using the TaqMan 5' nuclease activity from the TaqMan Universal PCR Master Mix, fluorogenic probes (Applied Biosystems) and oligonucleotide primers (Invitrogen). TaqMan assays were repeated twice for each of six selected genes (Table 1) in each lung sample (n = 3 mice for 4, 8, and 72 h, and n = 4 mice for 24 h). The mRNA expression levels of all samples were normalized to the levels for the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the same sample.


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  		TABLE 1 Oligonucleotide sequences used for quantitative real time RT-PCR analysis

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential Gene Expression of Lungs in Response to Ischemia
We employed two criteria to select the genes with significantly different expression. First, using DMT with Wilcoxon's signed rank test, we analyzed all 16–20 distinct probe pairs available for the detection of several different encoding regions of one particular transcript, and recorded a statistically significant increase or decrease in the gene expression if the P value was 0.0025 or below. Second, we selected the genes with 2-fold or greater change in both animals for each time point. With these selection criteria, out of ~ 6,000 well characterized genes and nearly 6,000 expressed sequence tags (ESTs) available on the array, we found only 343 genes and 56 ESTs that were different in the comparison of either ULL versus RL or LLL versus RL. To comparatively visualize the global gene expression between ULL and LLL in response to ischemia, we performed hierarchical clustering using CLUSTERING and TREEVIEW. Figure 3 depicted the dendrogram of clusterings for the differential expression of selected genes and ESTs in ULL and LLL, relative to RL. Upregulated genes are shown in green, and downregulated genes are shown in red. The comprehensive lists of all upregulated and downregulated genes in the comparison of ULL and LLL, relative to RL are reported in the online supplement. The dendrograms show that the pattern of gene expression in the two ischemic lung regions was generally similar, except in the areas marked with letters A to D. Of the total of 343 genes and 56 ESTs, only 29 probe sets of known genes had distinct expression profiles between ischemic lung where angiogenesis will develop (ULL) and that with no angiogenesis (LLL) and can be identified into group A to D (Figures 3A–3D). These four groups were generated from the unique temporal pattern of gene expression from ULL and LLL, compared with the nonischemic RL. The expanded version of these clusters with gene names is shown in Figures 3A–3D.



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  		Figure 3. Distinct transcriptional profiles of ischemic lung. Cluster analysis of 343 genes and 56 ESTs that changed >= 2-fold in at least one time point. The data are represented as the average fold change from the comparison of ULL and LLL relative to RL, respectively, at indicated time following left pulmonary artery obstruction (n = 2 mice at each time point). Distinct clusters (A–D) of ULL and LLL response to ischemia are magnified. The color code for signal strength is shown in the box, where induced genes are indicated by shades of green and repressed genes are indicated by shades of red. Genes shown in black are not different between the two groups being compared.

 
Cluster A contained genes upregulated in both ischemic lungs since 4 h, but the level of expression was much higher in ULL than in LLL after 4 h (Figure 3A). This cluster consisted of macrophage receptor with collagenous structure (MARCO) and arginase 1.

The expression of genes in cluster B was induced higher in early time point (4–8 h) than the late time point (24–72 h) in both ULL and LLL (Figure 3B); however, the level of expression was greater in ULL than in LLL. This cluster included cytokine/chemokine (interleukin [IL]-1ß, IL-1 receptor type II, keratinocyte-derived chemokine [KC], macrophage inflammatory protein 2 [MIP-2], lipopolysaccharide-induced CXC chemokine [LIX]), transcription factor (SRY-box containing gene 2, myelomatosis oncogene, IL-3 regulated-nuclear factor), thrombospondin 1, and cyclooxygenase 2.

The pattern in cluster C depicted the upregulation of genes in ULL, relative to no change or decrease in gene expression in LLL (Figure 3C). Genes associated with antioxidant (lactotransferrin, lipocalin 1, or von Ebnor salivary gland protein), mucin secretion–related protein (GOB-4, GOB-5, trefoil factor 2), intracellular calcium kinetics (calgranulin A, B), transcription factor (cardiac ankyrin repeat protein [CARP]), and immune function (regenerating islet–derived 3 {gamma}) have been identified in this cluster.

Cluster D contained genes repressed in LLL, but without any significant change in ULL (Figure 3D). Such genes encoded cytoskeletal protein (actin, myosin), calcium kinetics (parvalbumin, Ca-ATPase), lipid metabolism (adipsin, adiponectin), and enzyme (carbonic anhydrase 3).

The different pattern of gene expression in Cluster A, B, and C led us to postulate that the expression of these genes represented the strong inflammatory response in ULL upon ischemic exposure compared with LLL.

Transcriptional Profile of Angiogenic Response
To identify the genes responsible for angiogenesis and corroborate the differential gene expression data, we performed further analysis by using LLL as the reference array to ULL, with same criteria to cancel out the signal background of any common response that occurs due to ischemia alone. Figure 4 represents the list of genes differentially expressed within ischemic lungs and functionally classified at each time point after LPA ligation. This comparison further reduces the number of genes with a significant change (from 29 genes in Figure 3) to 25 known genes. Each of these genes showed an increased expression in the proangiogenic ULL relative to LLL. This strategy also helps to identify genes more specifically related to lung angiogenesis, and includes important genes such as IL-6, hepcidin antimicrobial peptide, a disintegrin and metalloproteinase (ADAM) 8, and acidic nuclear phosphoprotein 32 (pp32), which were not included in the comparative analysis of dendrograms (Figure 3).



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  		Figure 4. Classification of significant genes identified by differential expression comparing ULL to LLL at different time points. Data presented as the average fold change from the comparison of proangiogenic ULL to LLL (n = 2 mice at each time point). Green bars represent the genes significantly upregulated at least 2-fold.

 
Most of these 25 known genes with altered expression in the proangiogenic lung can be grouped into a few broad classifications, including genes related to inflammation, immune responses, mucin secretion, and transcription factors. The inflammation-related genes include chemokines, cytokines, and immunity-associated genes. Within 4 h after LPA obstruction, IL-1ß, IL-6, MIP-2, LIX, and KC in ULL were increased by 2- to 6-fold above that observed in LLL. This upregulation was maintained up to 72 h for MIP-2 and LIX. ADAM 8, the gene associated with tissue remodeling, was upregulated in ULL since 4 h. A total of 5 genes associated with the immune response were detected. One of them (MARCO) is present on macrophages. The others consisting of lactotransferrin, von Ebnor salivary gland protein or lipocalin 1, regenerating islet–derived gene 3 {gamma}, hepcidin were induced to a higher level during 4–72 h after ischemia. Genes related to mucin secretion and production (GOB-4, -5, and trefoil factor 2 [TFF2]) were upregulated in ULL. For the transcription factors, CARP was strongly upregulated in ULL at all time points, whereas the upregulation of pp32 was detected in ULL only at 4 h.

The other groups of highly expressed genes from the comparison of ULL versus LLL were lipid metabolism molecules, calcium kinetics, and cytoskeleton (Figures 4F, 4G, and 4H). However, these genes were not induced in ULL relative to control RL from the analysis of Figure 3D. Repressed expression of these genes in the LLL relative to the RL caused them to appear upregulated in ULL when compared with the LLL.

Validation of Microarray Data
Verification of differential gene expression was examined on a small set of known genes by using quantitative real time RT-PCR (TaqMan) (n = 3–4 mice for each time point). The levels of gene expression from RT-PCR were normalized to that of the gene for the enzyme GAPDH and expressed as the fold change in ULL relative to the LLL samples. GAPDH is one of the housekeeping genes and was not found to change in any comparisons from microarray data. Six known genes [MIP-2, KC, LIX, ADAM 8, CARP and vascular endothelial growth factor A (VEGF-A)] were selected for RT-PCR. Most of the selective genes had different patterns of expression and have distinct functional groups. MIP-2, KC and LIX are CXC chemokines consistently and highly induced in ULL. Two other important genes, ADAM 8 and CARP, were also upregulated in ULL and are in differently functional categories. VEGF-A was also chosen for validation given its prominent role in angiogenesis in other tissues although it did not show a significant difference between ULL and LLL or between ischemic left lung and nonischemic RL. Gene expression levels determined by microarray were compared with those obtained by RT-PCR, using the same lung samples employed in the microarray experiment with additional, identically treated, animals for the second level of validation. Figure 5 compares the temporal expression of these six genes with the two methods of measurement. This figure shows highly concordant quantitative measurements with the direction and magnitude of differential expression of all six genes.



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  		Figure 5. Comparison of gene expression levels determined by quantitative real time RT-PCR (TaqMan) and microarray analysis. Data representing the relative fold change of mRNA of indicated genes (A–F) in ULL by RT-PCR (shaded bars) after normalization with mRNA GAPDH is plotted relative to the level of LLL and compared with those from the microarray hybridizations (open bars). n = 3 mice for 4, 8, 72 h, and n = 4 mice for 24 h. Data are represented as mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Understanding the angiogenic process is important both for therapeutic promotion of new vessel growth in ischemic tissues as well as retardation of the process in tumors and other angiogenesis-related diseases. We used a unique angiogenesis mouse lung model coupled with oligonucleotide microarray analysis to study expression of those genes responsible for the initiation of angiogenesis. In this model, we have previously documented that new systemic vessels that perfuse the lung derive from intercostal arteries adjacent to the upper lung region (6). The reasons for this selective localization of new blood vessels are related to events that occurred at the time of pulmonary vascular ligation. To ligate the LPA, it was necessary to perform a thoracotomy through the third intercostal space. Although the ULL and LLL are both subject to the same degree of ischemia, healing of this adjacent thoracic wound is therefore the primary difference between the physical environments of the ULL and LLL. This healing process in the intercostal space provides an opportunistic potential source of new blood vessels. Apparently, there is local signaling both from the lung to the thoracic wall to "ask" for assistance, and from the chest wall to the lung to "tell" the lung that there is a potential source of blood nearby. In the first few days before functional new vessels form, these protein signals must diffuse across the pleural space. In the present study, we have chosen to focus on the early signaling in the lung, in an effort to determine those proangiogenic factors that allow the formation of functional blood vessels in the ischemic lung. Comparing specific gene expression in ischemic upper and lower lungs thus provided an internal control that allowed us to eliminate the genes not directly relevant to the angiogenic process. This approach was rewarded with our findings in which relatively few known genes—25—were significantly altered in the proangiogenic lung, and it will greatly accelerate the search for the most critical components underlying angiogenesis.

Several strategies were applied in our microarray study at the four different time points. First, we minimized and avoided biological variability across the samples by comparing samples obtained from different lung regions within the same individual animal (n = 2) at each time point. Next, we used stringent criteria for evaluation. The direction of gene change was strictly determined with high specificity by calculating the difference in intensity for all 16–20 probe pairs for each transcript on the experimental array relative to that from control tissues in the same animal. This intra-animal comparison provides much greater statistical power. Because of potential noise in the magnitude of fold change, genes were selected for consideration based on whether the statistically significant change in their relative expression was at least 2-fold in two animals at one time point. Following the identification of regulated genes, a quantitative real-time RT-PCR (TaqMan) was used to establish that selected genes detected by microarray could be validated in the same sample by an independent method. In addition, this procedure confirmed the validity of changes in selected genes across animals in the same setting by examining the gene expression levels in lung RNA from multiple similarly treated animals (n = 4 mice for 24 h, and n = 3 mice for all other time points).

Several studies have shown early increased levels of VEGF-A in animals and patients after hindlimb (9), myocardial (10), cerebral (11), and retinal (12) ischemia. For reasons not yet clear, the gene expression in ischemic mouse lungs of established angiogenic stimulators, such as VEGF-A (13), was neither altered in ULL relative to LLL, nor increased in the ischemic left lungs compared with nonischemic RL (see online supplement). This observed lack of VEGF-A induction is not likely a technical artifact. First, we noted that there are 16 sets of 25-mer probes on the array to detect VEGF-A transcript with high sensitivity. Second, other members in the same family whose probes are available on the array including VEGF-B, -C, -D, and angiopoietin-1 were also not induced. Third, we confirmed by real-time RT-PCR that VEGF-A expression was not upregulated at any time points in ischemic lungs. This observation suggests that VEGF-A is not, at least at transcriptional level, an important signaling factor in ischemic lungs. In ischemic organs other than lung, however, the main angiogenic factor is thought to be VEGF-A, and the stimulus for its expression is tissue hypoxia via hypoxia-inducible factor (HIF)-1{alpha} (14) and HIF-2{alpha} (15). This may be an important difference, because in the lung, ischemia does not result in hypoxia. Rather, a ventilated lung is actually better oxygenated following removal of perfusion with deoxygenated pulmonary arterial blood. Consistent with this suggestion, we found consistent downregulation of HIF-2{alpha} (see online supplement). The fact that lung angiogenesis develops in the absence of the HIF-1{alpha}, HIF-2{alpha}, and VEGF-A opens the provocative possibility that perhaps neither of these molecules are essential for angiogenesis in other organs.

Our strategy of comparative analysis mainly demonstrated upregulation of genes related to inflammation following ischemic injury. There were three chemokines, MIP-2, LIX, and KC, highly expressed in ULL relative to LLL at most time points. These three chemokines belong to the CXC chemokine subfamily ({alpha}-chemokine) by the presence of a single nonconserved amino acid separating two N-terminal cysteines. They also have a unique domain of three-amino-acid motif (Glu-Leu-Arg, ELR) preceding the first cysteine amino acid (ELR+ CXC) that differentiates them from the other CXC chemokines (ELR- CXC) (16). The ELR+ CXC chemokines share common properties of neutrophil recruitment and being able to directly promote angiogenesis in the absence of preceding inflammation (17). The ELR motif of these chemokines mediates their angiogenic activity by inducing endothelial cell chemotaxis and proliferation via the CXC chemokine receptor 2 (CXCR2) activation (18), whereas members of the CXC subfamily without this motif (ELR- CXC) are potent inhibitors of angiogenesis (19). Functional homologs of MIP-2, KC, and LIX in human are CXC chemokines IL-8, growth-related oncogene (GRO)-{alpha}/ß, and epithelial neutrophil-activating peptide 78 (ENA-78), respectively. IL-8, GRO{alpha}, and ENA-78 were found to be highly correlated with angiogenesis in many other situations, including wound healing (20), various malignancies (21–23), and chronic inflammatory diseases (24, 25) in experimental and clinical settings. In a lung fibrosis model, depletion of ELR+ CXC or administration of ELR- CXC chemokines resulted in marked attenuation of angiogenesis, despite persistent neutrophil accumulation (26). These results support the important and direct role of ELR+ CXC chemokines on angiogenesis, distinct from neutrophil chemotactic ability. Regarding ischemia, ELR+ CXC chemokines were also shown to be related to ischemia/reperfusion injury in various organs, by directing neutrophils to the tissue sites of inflammation (27, 28). One study of canine ischemic and reperfused myocardium demonstrated the rapid upregulation of IL-8 and expression of the proliferating cell nuclear antigen (PCNA) in endothelial cells, only in the infarcted myocardium during 24–72 h following coronary artery occlusion (29). Our findings confirmed by real-time RT-PCR show that ELR+ CXC chemokines in the ischemic lung may provide essential triggers for angiogenesis in an ischemic environment, at least at the transcriptional level.

In addition to CXC chemokines, IL-1ß, and IL-6 are other cytokines that were upregulated in ULL at 4 h. IL-1ß and IL-6 are multifunctional cytokines that can regulate various immune and inflammatory responses. The study of IL-1ß overexpression in murine lung carcinoma cells has been associated with in vivo neovascularization-induced tumor growth (30). In addition, this group demonstrated that IL-1ß mediated VEGF-A and MIP-2–induced angiogenesis, and that neutralization with CXCR2 antibody reduced tumor growth by suppression of tumor angiogenesis. The involvement of IL-6 in angiogenesis was demonstrated by elevation of IL-6 mRNA in vivo in the formation of vascular system accompanying the development of ovarian follicles and the formation of a capillary network in maternal deciduas following embryonic implantation (31). In addition, IL-6 activity in wound fluid and serum reaches a maximum within 12 h of wound healing, suggesting its role in active angiogenesis during wound healing (32).

Not surprisingly, our data also indicated a role for genes involved in tissue remodeling. The protease enzyme, ADAM 8 (CD156) is highly expressed in ULL during 4–72 h. ADAM 8, reported as a macrophage-specific cDNA, is capable of collagen digestion, extracellular proteolysis, competitively binding with integrin (33), and regulation of leukocyte recruitment and infiltration by shedding/inducing the various transmembrane adhesion molecules (34). Although the specific role of ADAM 8 on angiogenesis needs further study, extracellular proteolysis is clearly essential for tissue remodeling and the sprouting of new blood vessels during angiogenesis.

MARCO, a gene strongly expressed in the ULL, has purported roles in phagocytosis of pathogens and waste products (35). MARCO is a scavenger receptor expressed specifically on monocytes and macrophages, and is highly induced in activated macrophages (36). In addition, alveolar macrophages are reported to be capable of secreting MIP-2 and IL-6 (37), the highly induced cytokines in ULL. Taken collectively, these results not only suggest the presence of macrophages, but also that they may play an important role in promoting angiogenesis in the ischemic lung.

Genes associated with mucin secretion also displayed an elevated expression in ULL. They consisted of GOB-4, GOB-5, and TFF2. GOB-5 was found to be an important regulator of mucus overproduction and goblet cell metaplasia in the lung following allergen exposure (38). Overexpression of GOB-5 and other mucus-related genes may be an initial reaction to lubricate airway epithelium after lung ischemia and possibly facilitate the formation of new blood vessels. TFF2 was reported to enhance migration of respiratory epithelium triggering rapid repair processes in airway diseases (39).

CARP is one of the transcription factors highly overexpressed in ULL, with no induction in LLL. It was first identified as a nuclear protein and expressed abundantly in heart and skeletal muscle. Recently, CARP expression was detected in endothelium (40) and vascular smooth muscle cells (41) following vascular injury. pp32, the other nuclear protein highly induced in ULL at 4 h, has been shown to be expressed in tissue with self-renewal and associated with the suppression of cellular transformation (42). The functional significance of these transcription factors related to lung angiogenesis is presently unknown.

Among the genes that were significantly upregulated, ELR+ CXC chemokines have been recognized as important proangiogenic mediators. High induction of these chemokines in the ULL early after ischemia potentially generates the proliferative and migratory signals for dividing endothelial cells from intercostal vessels at the thoracic wall. Our findings are consistent with several models of lung disease, demonstrating an important role for the proangiogenic ELR+ CXC chemokines (23, 24, 26). Future studies using neutralizing antibodies to these chemokines as well as their receptor CXCR2 will provide new information regarding the mechanisms of lung angiogenesis.

In summary, we took advantage of a new experimental model in the mouse that completely isolates the angiogenic process from the direct effects of ischemia. The model also leads to lung angiogenesis that mimics the vascular source of many lung pathologies and allows investigation of the temporal and spatial factors that can promote or inhibit angiogenesis. This study revealed the expression patterns of genes relevant to proangiogenic signals and conditions in response to ischemia in the lung. The most notable changes were increases in the expression of genes involved in inflammation and tissue remodeling. Results from this unique animal model, which provides nonhypoxic lung ischemia, suggest that hypoxia is not an essential trigger for angiogenesis, but emphasize the important role of inflammatory cytokines in lung differentiation after ischemic insult. Thus, this large-scale expression measurement has provided and identified the possible interplay of factors contributing to lung angiogenesis. The studies also reveal potential approaches for determining critical pathways and therapeutic strategies related to the control of angiogenesis.


   Acknowledgments
 
This work was supported by National Institutes of Health Grants EHS-03819 (S.B., W.M.), HL-10342 (W.M., E.M.W.). S.S. is supported by Prince Mahidol's Fellowship from Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org.

Received in original form November 26, 2002

Received in final form January 29, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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