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Am J Physiol Lung Cell Mol Physiol 295: L272-L284, 2008. First published May 9, 2008; doi:10.1152/ajplung.00358.2007
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Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension

Christine M. Costello,1 Katherine Howell,1 Edwina Cahill,1 Jean McBryan,2 Melanie Konigshoff,3 Oliver Eickelberg,3 Sean Gaine,4 Finian Martin,2 and Paul McLoughlin1

1School of Medicine and Medical Science, and 2School of Biomolecular and Biomedical Science, UCD Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Dublin, Ireland; 3Department of Medicine II, University of Giessen Lung Center, Giessen, Germany; and 4Department of Respiratory Medicine, Mater Misericordiae University Hospital, University College Dublin, Dublin, Ireland

Submitted 31 August 2007 ; accepted in final form 28 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.

pulmonary endothelium; gremlin 1; CXC receptor 7; angiogenesis

SUSTAINED PULMONARY HYPOXIA is a common consequence of chronic lung diseases and leads to the development of pulmonary hypertension as a result of increased pulmonary vascular resistance (25, 39). When pulmonary hypertension occurs in this setting, it leads to increased morbidity and reduced survival (reviewed in Ref. 25). The increased pulmonary vascular resistance underlying chronic hypoxic pulmonary hypertension is accompanied by characteristic changes in the pulmonary arterioles, which include thickening of the pulmonary arterial wall and associated reduction in lumen diameter in vivo (47, 48). More recently, it has been shown that persistent vasoconstriction, mediated by the small G protein RhoA and its downstream kinases ROCK I and II, is also a major contributor to the hypoxia-induced increase in vascular resistance (29, 38, 53). These long-term structural and functional changes in the pulmonary vessels in response to hypoxia are unique to this circulation and are not observed in other organs of the body. These observations suggest that there are genes for which expression is selectively modulated by hypoxia in the lung while simultaneously remaining unaffected in other organs.

The important roles of the vascular endothelium in the local control of vascular smooth muscle tone and in modulating changes in the structure of the vessel wall are now well-recognized. Such effects are mediated through the release of specific signaling molecules such as nitric oxide, prostacyclin, and endothelin (1). The endothelial cells (ECs) can also produce extracellular matrix molecules such as laminin, fibronectin, and elastin that can contribute to the formation of the extracellular matrix of the vessel wall (5). However, ECs from different organs show considerable heterogeneity, and their roles in control of the vasculature differ from organ to organ. Ultrastructural diversity is well-recognized as endothelial structure ranges from discontinuous through fenestrated to continuous tight phenotypes (11, 14). Expression of cell surface molecules also varies considerably between ECs from different organs. For example, lung endothelium shows high basal expression of angiotensin-converting enzyme (4, 19), thrombomodulin (54), and membrane dipeptidase (13, 21, 40) compared with that of systemic organs. Moreover, these tissue-specific features are maintained in culture conditions suggesting that they are intrinsic to the mature ECs and do not depend on the organ-specific environment in which they normally reside (30). Taken together, these observations suggest that the pulmonary endothelium shows organ-specific responses to hypoxia that play an important role in the development of the vascular changes leading to chronic hypoxic pulmonary hypertension.

The aim of our study was to identify genes for which expression was altered in the pulmonary endothelium in response to hypoxia while remaining unaltered (or changing in the opposite direction) in the ECs of other organs. Using a microarray approach, we adopted a novel subtractive strategy to compare global gene responses with hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those observed in primary cardiac microvascular endothelial cells (HMVEC-C) and identified a cohort of lung-selective, hypoxia-responsive genes. We selected two exemplary clusters from among this cohort for further examination in mice exposed to environmental hypoxia (10% O2). This approach confirmed that gremlin 1 (GREM1) gene expression, a bone morphogenetic protein (BMP) antagonist, was selectively upregulated in the murine lung in response to hypoxia compared with other organs. We found expression of gremlin protein in the normoxic murine lung in vivo and demonstrated that this was markedly increased by hypoxia. Gremlin expression in lung tissue from patients with idiopathic pulmonary hypertension was also significantly elevated. Finally, we showed that gremlin inhibited BMP-stimulated human pulmonary EC wound healing in vitro. From a second exemplary cluster, we showed that chemokine (CXC motif) receptor 7 (CXCR7), a G protein-coupled receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified lung-selective hypoxia-responsive genes. This study gives new insights into the mechanisms of hypoxic pulmonary hypertension and, in particular, provides the first evidence of a potentially important role for gremlin, an endogenous inhibitor of the BMP pathways, in pulmonary hypertension.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and in vitro model of hypoxia. HMVEC-L and HMVEC-C cells were grown on sterile tissue culture dishes in EGM-2MV endothelial growth medium (cat. no. CC-3202) according to the manufacturer's instructions (Lonza, formerly Cambrex). At experiment start, medium was changed, and dishes were transferred to the hypoxia chamber (Coy Labs) and cultured in an atmosphere of 1% O2, 5% CO2, and 94% N2 for 3, 24, or 48 h. Control conditions were achieved by culture in 21% O2, 5% CO2, and 74% N2 in a cell culture incubator. At experiment end, cell medium was removed, and cells were washed with PBS and thoroughly lysed in RLT buffer (RNeasy Mini Kit, Qiagen). All cells used in these experiments were from passages 6 and 7 and were routinely checked for mycoplasma contamination using the VenorGeM PCR kit (Cambio). Six independent hypoxic time course experiments were carried out for both lung and cardiac cells.

RNA extraction. Total RNA extraction (RNeasy Mini Kit, Qiagen), with on-the-column DNase treatment, was carried out as previously described (17). RNA was determined to be free of contaminating genomic DNA by PCR amplification of GAPDH (17), quantified spectrophotometrically, and confirmed to be intact by standard gel electrophoresis. Hypoxic conditions were confirmed by RT-PCR with two well-established hypoxia-induced genes, VEGF-A and solute carrier family 2 member 1 [SLC2A1 (formerly GLUT1)]. Briefly, using standard RT-PCR conditions and a final primer concentration of 0.2 µM (Table 1), the PCR cycling profile was as follows: 2 min at 95°C, n cycles (Table 1) of 94°C for 45 s, 60°C for 30 s, 72°C for 90 s, and a final extension step of 72°C for 10 min. Samples were run on a 2% agarose-ethidium bromide gel and amplicons were visualized under UV light (GeneGenius BioImaging System, Syngene).


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  		Table 1. RT-PCR primer sequences for housekeeping and hypoxia-induced marker genes

 
GeneChip experiments. ECs were cultured and exposed to hypoxia on 6 separate occasions, and total RNA was extracted. Experimental samples (n = 3) were formed by randomly pooling RNA from 2 independent experiments. This process was repeated at each of 4 experimental time points yielding 12 separate arrays for lung and cardiac experiments. Affymetrix GeneChip Human Genome U133A chips, which allow the simultaneous analysis of 22,283 gene transcripts, were used. Total RNA (5 µg) was reverse transcribed to cDNA (One-Cycle cDNA Synthesis Kit), and clean-up of double-stranded cDNA, biotin labeling of anti-sense cRNA (integrity checked on the 2100 bioanalyzer, Agilent Technologies), and clean-up and fragmentation of biotinylated cRNA were all carried out according to the manufacturer's protocol (Affymetrix). Fragmented cRNA (15 µg) was hybridized to chips for 16 h at 45°C in a hybridization oven (Affymetrix) with rotation at 60 rpm. Immediately following hybridization, the array underwent automated washing and staining protocol on the automatic Fluidics Station 400 (Affymetrix), and chips were scanned (GeneChip Scanner 3000, Affymetrix). One 3-h hypoxic lung chip failed due to uneven hybridization and was therefore removed from all subsequent analysis.

Affymetrix CEL files were imported and normalized using algorithms of GeneSpring 6.0 software, i.e., data were normalized by applying the following data transformation criteria: 1) measurements less than 0.01 were set to 0.01; 2) per chip, normalized to 50th percentile; and 3) per gene, normalized to median. Genes that were absent in a majority of chips were eliminated from further analyses yielding a list of 10,476 genes. Using this list, a condition tree was generated by hierarchical clustering using a Pearson correlation (GeneSpring 6.0 software).

To discern those genes that were uniquely upregulated in the lung, we adopted the following stringent subtractive approach. We first selected those genes that increased at least twofold between basal normoxic expression and any hypoxic time point in the lung ECs. We next subtracted from that list those genes that increased at least 1.2-fold between normoxia and any hypoxic time point in the heart ECs. This strategy identified those genes that increased at least twofold in lung cells but decreased, remained the same, or increased marginally (<1.2-fold) in cardiac cells. A similar analysis was carried out to discern those genes that decreased twofold in lung cells but increased, remained the same, or decreased only marginally (<1.2-fold) in cardiac cells. These gene lists were combined and subjected to hierarchical clustering to identify interesting clusters for follow-on analyses (GeneSpring 6.0 software).

The entire raw data set discussed in this publication, which is compliant to the MIAME criteria, has been deposited in NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series acc. no. GSE11341.

Real-time PCR (TaqMan). Total RNA (1 µg) was reverse transcribed to cDNA using SuperScript II RNase H-Reverse Transcriptase kit (Invitrogen). Real-time PCR was performed on 384-well plates, and each sample was measured in duplicate. An initial minus RT reaction confirmed that all RNA samples were free of contaminating genomic DNA. The Eukaryotic 18S rRNA (VIC/TAMRA) predeveloped assay reagent kit was used as the endogenous control gene according to the TaqMan PCR protocol [Applied Biosystems (ABI)]. Probes and primers were either designed to nonredundant sequence using Primer Express software or ordered from ABI as Assay-On-Demand Gene Expression Assays (Table 2). Reactions were carried out on the ABI PRISM 7900 Sequence Detection System, and mRNA levels were determined using the standard curve method (ABI Prism 7700 Sequence Detection System User Bulletin no. 2).


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  		Table 2. Real-time PCR Assays-On-Demand

 
In vivo animal model of hypoxia. Chronic hypoxic pulmonary hypertension was induced by housing adult male C57BL6 mice (n = 8) in hypoxic conditions (inspired oxygen 10% for 2 days) (20). This mouse strain demonstrates pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling similar to that which is seen in other species following sustained exposure to a hypoxic environment (26, 29). Control mice (n = 8) were housed in normoxic conditions (inspired oxygen 21%) in the same room. After 48 h, mice were killed by cervical dislocation, and the thymus, lungs, heart (left ventricle), kidney, liver, and spleen were rapidly removed and immediately flash-frozen in liquid nitrogen. RNA extraction was carried out as described above with the exception that tissue samples were first crushed to a fine powder using a mortar and pestle and passed through QIAshredder columns (Qiagen) to ensure complete breakdown of tissue. All experiments were approved by the University College Dublin Animal Research Ethics Sub-Committee and carried out under license from the Department of Health.

Immunohistochemical analysis. Adult male C57BL6 mice were maintained in normoxia (n = 5) or exposed to 10% oxygen (n = 5) for 2 days as above. Mice were euthanized (60 mg/kg ip sodium pentobarbitone), the heart and lungs were removed en bloc, and lungs were fully inflated via the trachea with standard pressures (25 cmH2O) in fixative (4% paraformaldehyde) for 45 min and then maintained in fixative overnight and embedded in paraffin. Following fixation for 24 h, the vertical axis of each left lung was identified, and the lung was cut perpendicular to this axis into 4-mm-thick slices with a sharp blade beginning at a position chosen by random number within the first slice. Seven-micrometer-thick sections were cut from the surface of each slice, mounted onto polylysine slides, baked overnight at 37°C, dewaxed, and hydrated. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 5 min. Antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6.0) for 10 min in a microwave. Slides were washed in PBS, blocked in rabbit serum (10% diluted in PBS) for 30 min, and incubated in 1:10 dilution of primary goat polyclonal anti-Drm/gremlin (final concentration 10 µg/ml; R&D Systems) in PBS overnight at 4°C. Following PBS washes, endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol. Slides were next incubated in 1:150 dilution biotinylated rabbit anti-goat secondary antibody (final concentration 10 µg/ml; Vector Laboratories) for 1 h, washed in PBS, and incubated in immunoperoxidase solution (Vectastain ABC Kit, Vector Laboratories) for 1 h. Negative control slides were exclusion of primary or secondary antibody. After allowing the diaminobenzidine reaction product to develop for 20 min, sections were washed extensively in PBS and counterstained with hematoxylin (BDH Laboratory) before being examined microscopically on an Olympus BX61 microscope (x40 objective).

Quantification of gremlin staining. The extent of gremlin staining was determined by quantitative stereological techniques as previously described (8, 24) using a computer-based analysis system (CAST; VisioPharm, Hørsholm, Denmark) combined with light microscopy (Leica, Laboratory Instruments). Random fields of view were acquired (x40 objective) from all sections of each lung using a systematic random sampling strategy, digitized, and displayed on screen. A point-counting grid was digitally superimposed on these images to determine the fraction of the alveolar walls that was occupied by cells staining positively for gremlin.

Scratch assay protocol. At the start of the experiment, three horizontal lines were drawn on the back of 12-well plastic plates (Greiner Bio-One CELLSTAR). HMVEC-L cells were allowed grow into a confluent monolayer, and the medium was changed. Twenty-four hours later, a single vertical scratch was applied to each chamber using a 1- to 100-µl pipette tip (S1120-1840, Starlab). After injury, cells were allowed to settle for 2–3 h and then treated with either vehicle (4 mM HCl-0.1% BSA), BMP4 (80 ng/ml final concentration), BMP4 + gremlin (2 µg/ml final concentration), or BMP4 + condition medium. The condition medium was obtained from HMVEC-L cells grown in 1% O2 for 24 h and spun at 1,500 rpm for 3 min to remove any cellular debris. The BMP4 and gremlin were from R&D Systems. The scratch was visualized using phase-contrast (x10 objective), and the width of the wound was measured at 0 and 24 h (AxioVision 4.4 software). Six fields in each well were measured by taking images along the length of the scratch at six predefined positions (above and below each drawn line) and averaged. For each treatment, six separate well analyses were carried out.

Gremlin expression in human tissues. Lung tissue samples were obtained from five patients (mean age 39 ± 10 yr; 3 females) with idiopathic pulmonary arterial hypertension (IPAH) and six control subjects whose lungs were rejected from transplantation due to recipient incompatibility (organ donors, mean age 42 ± 13 yr; 3 females). Pulmonary hypertension was defined as a pulmonary mean arterial pressure >25 mmHg at rest or >30 mmHg on exercise. None of the IPAH patients exhibited known BMP receptor-2 (BMPR2) mutations. Total RNA and cDNA were prepared as described above. Real-time PCR was performed using fluorogenic SYBR Green and the Sequence Detection System Fast 7500 (PE Applied Biosystems). Human gremlin primers are shown in Table 3. Hypoxanthine phosphoribosyltransferase (HPRT)-1, a ubiquitously and equally expressed gene free of pseudogenes, was used as a reference gene (Table 3). PCR was performed using the primers at a final concentration of 200 nM, and relative transcript abundance was determined using the comparative CT method (ABI Prism 7700 Sequence Detection System User Bulletin no. 2).


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  		Table 3. Human real-time PCR assays

 
Statistical analyses. All statistics were carried out using Statistica 7.0 software. For normally distributed data, responses are reported as means ± SE. Comparison of means in two-group experiments was carried out using a t-test. For nonnormally distributed data, responses are reported as means ± SD, and statistical comparisons were made using Mann-Whitney U test. In experimental designs with three or more groups, statistical analysis was performed using the Kruskal-Wallis test followed by Mann-Whitney U test with the Bonferroni post hoc correction to test for differences between specific groups; values of P <0.05 were accepted as statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Global gene expression in hypoxia. We confirmed that primary human microvascular ECs in our in vitro model were hypoxic by showing that, as expected, the expression of VEGF-A and SLC2A1 (GLUT1) was upregulated as early as 3 h exposure to hypoxia and further increased with longer exposure to 1% O2 (Fig. 1A). Using Affymetrix U133A chips (~22,283 gene transcripts), we next investigated the global expression profile induced in cells in response to increasing periods of hypoxia. Hierarchical clustering of the arrays based on the genes for which expression was reliable (10,746 genes) clustered cells from the lung or cells from the heart together, forming clearly separate cell-specific subtrees (Fig. 1B). This finding supported our hypothesis that there were genes for which expression was modulated by hypoxia in the pulmonary endothelium in a manner that was specific to that cell type and was different from the pattern of gene response observed in cardiac ECs. In the lung cells, 428 genes were differentially up- or downregulated at some time point during hypoxia, whereas, in the cardiac cells, the corresponding number was 299 genes. A broad overview of the time course of these changes is shown in Fig. 1, C and D.


Figure 1
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  		Fig. 1. Hypoxia-modulated gene expression in pulmonary and cardiac microvascular endothelial cells. A: human microvascular endothelial cells from lung (HMVEC-Lung, HMVEC-L) and cardiac (HMVEC-Cardiac, HMVEC-C) cells under normoxic (0 h: 21% O2) and hypoxic conditions (1% O2) at various time points (3, 24, and 48 h). An incremental increase in the expression of 2 well-characterized hypoxia-responsive marker genes, namely VEGF-A and solute carrier family 2 member 1 [SLC2A1 (previously called GLUT1)], was measured by RT-PCR. GAPDH levels confirm equal sample loading. B: hierarchical clustering of arrays into a condition tree based on reliably expressed genes (10,476 genes). C: temporal profile of the number of genes up- or downregulated (>2-fold) revealed by comparison between experimental time points in HMVEC-L (428 genes) and HMVEC-C (299 genes; D).

 
Identification of lung endothelium-selective hypoxia-responsive genes. Our subtractive approach revealed 51 genes that were upregulated (Table 4) and 39 genes that were downregulated in the lung cells >2-fold (Table 5) but had a different pattern of expression in cardiac cells. These genes are presented in broad functional categories based on gene ontologies (Tables 4 and 5). Hierarchical clustering of these 90 differentially regulated genes identified distinct cohorts of genes with shared expression profiles over a time course of hypoxia (Fig. 2). One very striking finding from this analysis was a cluster of seven genes (outlined in yellow in Fig. 2A) that showed a basal gene expression that was greatly elevated (>3- to 10-fold) in lung cells compared with cardiac cells (Fig. 2B). Furthermore, this expression was upregulated >2-fold in response to hypoxia (at least 1 time point) only in the lung cells (Fig. 2C). In light of these characteristics, we chose to further examine this cluster in more detail. Within this cluster was the secreted BMP antagonist, GREM1, and the as yet unknown protein LIM and calponin homology domains 1 (LIMCH1), both of which were represented by two independent probes. This cluster also included VEGF165 RNA processing protein, Sam68-like phosphotyrosine protein (SLM2), the extracellular matrix protein spondin 1 (SPON1), and the potent tumor angiogenic factor ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) (12, 15, 51). The remaining two genes in this cluster represented genes of unknown specific function, namely protein kinase, Y-linked (PRKY) and macrophage expressed gene 1 (MPEG1).


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  		Table 4. Genes that increased more than 2-fold in lung cells

 

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  		Table 5. Genes that decreased more than 2-fold in lung cells

 

Figure 2
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  		Fig. 2. Hierarchical clustering of lung-selective hypoxia-responsive genes. A: hierarchical clustering of lung-selective genes identified a cluster (highlighted in yellow and termed the gremlin cluster) where basal gene expression was uniquely elevated in the lung cells (>3- to 10-fold, represented as mean fluorescence values expressed in arbitrary units; B) and was further upregulated in response to hypoxia (represented as fold change over time 0, i.e., nonhypoxic cell values; C). Each gene is represented by a single row of colored boxes, and each time point (hours) is represented by a single column. The color scale is red for high expression values and green for low expression values. GREM1, gremlin 1; SLM2, Sam68-like phosphotyrosine protein; SPON1, spondin 1; ENPP2, potent tumor angiogenic factor ectonucleotide pyrophosphatase/phosphodiesterase 2; PRKY, protein kinase, Y-linked; MPEG1, macrophage expressed gene 1; LIMCH1, LIM and calponin homology domains 1. Both GREM1 and LIMCH1 were represented by 2 independent probes.

 
In vitro transcriptional changes confirmed by real-time PCR analysis. We confirmed that basal expression of the majority of genes (6 of 7) in the gremlin cluster was significantly elevated in lung compared with cardiac ECs in each of six separate hypoxia experiments at 48 h (Fig. 3). Indeed, extremely low gene expression of GREM1, SPON1, ENPP2, and PRKY was observed in cardiac endothelium. Although mean LIMCH1 basal levels were higher in lung cells, this difference was not significant (P = 0.08). Finally, we confirmed that GREM1 expression was significantly increased in response to hypoxia (48 h) only in the pulmonary microvascular endothelium (Fig. 3). Analysis of all genes, at all experimental time points, confirmed that gremlin was the only significantly differentially regulated gene in hypoxia in the cell culture model (data not shown). Because vascular remodeling occurs most prominently in smaller vessels in the lung, we asked if GREM1 expression was different in ECs from large pulmonary vessels. We therefore examined GREM1 expression in ECs isolated from human pulmonary artery (HPAEC, Lonza, formerly Cambrex) in a separate series of experiments. We found low basal expression (similar to that measured in cardiac cells) in these cells, which was not altered with hypoxia (data not shown), suggesting that GREM1 expression is specific to the pulmonary microvascular endothelium.


Figure 3
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  		Fig. 3. Real-time PCR confirmation of gremlin cluster genes in in vitro cell experiments. Real-time PCR was used to confirm array results in unpooled RNA (n = 6) from normoxic and 48-h hypoxic conditions in in vitro experiments. Values are normalized to 18S rRNA and are presented as means ± SD. {dagger}Significant differences between normoxic lung cells and normoxic heart cells (P < 0.05); *significant differences between normoxic lung cells compared with hypoxic lung cells (P < 0.05). HML, human microvasculature lung; HMC, human microvasculature cardiac.

 
Lung-selective genes confirmed in an in vivo animal model of hypoxia. To examine whether the effects observed in the cell culture model were relevant to the in vivo situation, we examined gene expression in organs from mice exposed to 10% environmental oxygen for 2 days. In a similar manner to that seen in vitro, basal expression of the majority (5 of 7) of genes in this cluster were significantly elevated in the whole lung compared with the heart. Exceptions were SLM2, which was significantly higher in the heart (Fig. 4A), and PRKY, which remained unchanged in the two tissue types. We also observed a significant upregulation of GREM1 and SLM2 expression in lung tissue in response to the 2-day hypoxic challenge. To establish whether this finding was selective to the lung, we extended our gene expression analysis to include the kidney, liver, spleen, and thymus. Expression analysis confirmed that GREM1 (>3-fold) and SLM2 (~2-fold) were only upregulated in the lung in response to hypoxia (Fig. 4, B and C).


Figure 4
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  		Fig. 4. In vivo real-time PCR analysis of gremlin cluster genes in a hypoxic animal model. Mice (n = 8) were maintained in normoxic or hypoxic conditions (inspired oxygen 10%) for 2 days. A: real-time PCR analysis of all 7 genes in the gremlin cluster in whole lung and cardiac tissue from normoxic mice. Values are normalized to 18S rRNA, and data are presented as means ± SE. B and C: GREM1 (B) and SLM2 (C) expression in the heart, kidney, liver, lungs, spleen, and thymus of normoxic and hypoxic mice. Values are normalized to 18S rRNA and expressed as fold change relative to the normoxic control for each organ. {dagger}Significant differences between normoxic lung and normoxic heart (P < 0.05); **significant differences between normoxic compared with hypoxic lung (P < 0.001).

 
GREM1 protein is higher in hypoxic lung tissue. Immunohistochemical staining showed that gremlin protein was expressed basally in the normoxic lung (Fig. 5, A and B) and increased substantially after 2 days in hypoxia (Fig. 5, C and D). Gremlin immunoreactivity was clearly observed in the tissue surrounding the alveolar spaces. Gremlin staining was absent when primary antibody was omitted from normoxic (Fig. 5E) or hypoxic (Fig. 5F) tissue or when secondary antibody was omitted (data not shown). Images shown are representative of findings in five control animals and five hypoxic animals. Quantification of the volume of gremlin-stained tissue per unit volume of alveolar wall in the lung showed that gremlin protein was significantly higher in the hypoxic lungs (n = 5) than in normoxic lungs (n = 4) (Fig. 5G).


Figure 5
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  		Fig. 5. Immunohistochemical localization of gremlin in normoxic and hypoxic lungs. A: immunohistochemical staining (brown color) indicated that gremlin protein was expressed basally in normoxic lung tissue (A and B) and increased with exposure to hypoxia (C and D). Gremlin staining was not observed when primary antibody was omitted from normoxic (E) or hypoxic (F) tissue or when secondary antibody was omitted (data not shown). Two separate control and hypoxic animals are shown. Using stereology, the volume of cells within the alveolar wall that were gremlin-positive was expressed as a fraction of total alveolar wall volume (volume fraction) and was significantly higher in hypoxic lungs (G). Data are presented as means ± SE. *Significant differences between normoxic and hypoxic lung values (P < 0.05).

 
Gremlin inhibits BMP4-induced endothelial wound healing. Given the known role of gremlin as a potent antagonist of BMP-induced proliferation and migration, we next examined its ability to inhibit wound healing. As shown in Fig. 6A, the scratch applied to lung ECs treated with vehicle alone (Fig. 6A, panel 1) was still visible after 24 h, which was in sharp contrast to that seen in cells treated with BMP4 (80 ng/ml), where the scratch width was substantially reduced (Fig. 6A, panel 2). Conversely, blocking BMP4 activity with the addition of gremlin (2 µg/ml) resulted in a scratch width similar to control (Fig. 6A, panel 3). Furthermore, treatment of the cells with condition medium from hypoxic cells also blocked wound healing (Fig. 6A, panel 4). Figure 6B shows the mean scratch width from six separate wells. This showed BMP4 significantly induced wound healing and treatment with gremlin or hypoxic condition medium significantly blocked this effect.


Figure 6
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  		Fig. 6. Scratch wound assay. A: human pulmonary microvascular endothelial cells were grown to confluence, and a vertical wound was applied to the monolayer. Cells were either exposed to vehicle (panel 1), 80 ng/ml bone morphogenetic protein 4 (BMP4; panel 2), BMP4 and 2 µg/ml gremlin (panel 3), or BMP4 and hypoxic condition medium (CM) (panel 4) for 24 h. Representative images are shown. The arrow indicates the position of the original scratch. B: mean wound width was measured for 6 independent wounds. Results are expressed as % wound closure after 24 h. Horizontal bars indicate median values. #Significant difference from all other groups (P < 0.05).

 
Gremlin expression is higher in patients with IPAH. In patients with IPAH, expression of gremlin was significantly greater than in that observed in nondiseased lungs (Fig. 7).


Figure 7
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  		Fig. 7. In vivo real-time PCR analysis of gremlin mRNA in idiopathic pulmonary arterial hypertension (IPAH) patients. Real-time PCR analysis of gremlin in control and IPAH lung tissue samples showed a significant increase in mRNA levels in the patient group. Values are normalized to hypoxanthine phosphoribosyltransferase (HPRT) and expressed as fold change relative to the mean normoxic control value. *Significant differences between groups (P < 0.05).

 
Second cluster of genes examined in in vivo animal model of hypoxia. To demonstrate the robustness of our lung-selective gene findings, we next focused on a second smaller cluster of four genes, where hierarchical clustering showed a marked upregulation at 24- and 48-h hypoxia in lung ECs but remained virtually unchanged in the cardiac cells in vitro (Fig. 8A). This cluster was comprised of TM2D1 domain containing 1 protein [also annotated as β-amyloid-binding protein (BBP)], the proangiogenic CXCL12-binding G protein-coupled receptor CXCR7, the motor protein myosin IB (MYO1B), and a newly described mediator of angiogenesis, brain-derived neurotrophic factor (BDNF) (7, 27, 31, 43). Two genes were confirmed as hypoxia-responsive in vivo, namely CXCR7 and BDNF. However, CXCR7 was the only one of these two that was hypoxia-responsive in the lung but not in the heart, as confirmed by real-time PCR (Fig. 8B).


Figure 8
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  		Fig. 8. Examination of CXCR7 gene cluster in vivo. A: hierarchical clustering of CXCR7 genes, where basal levels of gene expression increased with hypoxia in the lung but remained unchanged in the cardiac endothelial cells. B: real-time PCR analysis of all 4 genes in the CXCR7 cluster in whole lung and cardiac tissue from normoxic and hypoxic mice showed that the expression levels of CXCR7 and brain-derived neurotrophic factor (BDNF) were significantly elevated in the hypoxic lung. Expression of BDNF was also significantly upregulated in the hypoxic heart. Values are normalized to 18S rRNA endogenous control, and data are presented as means ± SE. *Significant difference between normoxic and hypoxic tissues (P < 0.05). TM2D1/BBP, domain containing 1 protein (also annotated as β-amyloid-binding protein); MYO1B, myosin IB.

 
CXCR7 has only very recently been shown to bind the proangiogenic chemokine CXCL12. To investigate this pathway further, we examined gene expression in vivo. CXCL12 increased >2-fold in hypoxic lung tissue, with more modest changes observed in heart and spleen tissue (Fig. 9A). CXCR7 expression was markedly elevated after hypoxic stress, a response that was unique to the lung (Fig. 9B), implying that the CXCL12/CXCR7 axis may have a special role in hypoxic responses in lung.


Figure 9
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  		Fig. 9. In vivo expression profile of CXCL12 and CXCR7 in a panel of murine tissues. A and B: expression of CXCL12 (A) and CXCR7 (B) in tissues from mice maintained in normoxic (white columns) or hypoxic (black columns) conditions (inspired oxygen 10% for 48 h). CXCL12 expression increased >2-fold in hypoxic lung tissue with more modest changes observed in heart and spleen tissue. CXCR7 was significantly increased (>2-fold) only in lung tissue after hypoxic stress. Values are normalized to 18S rRNA and expressed as fold change relative to the normoxic control for each organ. Asterisks indicate significant difference between normoxic and hypoxic tissues (*P < 0.05; **P < 0.0001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Chronic lung diseases, including asthma, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, and occupational lung diseases, are frequently complicated by the development of pulmonary hypertension caused by a sustained elevation of pulmonary vascular resistance. This complication causes right ventricular failure, increases disability, and reduces life expectancy (25). One major stimulus that contributes to pulmonary hypertension is hypoxia caused by the underlying lung disease. In the present study, we detected genes for which expression was selectively altered in the lung in response to levels of hypoxia encountered in pulmonary disease.

A number of the 90 genes identified in our initial array studies were already linked to pathways known to be involved in the development of pulmonary hypertension, e.g., peroxisome proliferative-activated receptor and arginase (18, 44, 57). This observation provided support for our subtractive strategy; however, we did not pursue these well-explored pathways further.

We first focused our attention on a cluster of seven genes that showed high basal expression in the pulmonary ECs and hypoxic responsiveness (Figs. 2 and 3). In vivo five of these genes demonstrated very high basal expression values compared with the heart (Fig. 4A). In view of our recent demonstration that angiogenesis is a prominent feature of the pulmonary vascular responses to hypoxia (26, 29), it was of interest to note that the majority of the genes in this cluster had documented roles in angiogenesis, either inhibiting (51) or promoting it (9, 15). Two of the genes in the cluster demonstrated lung-selective hypoxia responses in vivo, although one of these, SLM2, was the one gene in the cluster that had significantly higher basal expression in the heart. The second of these two genes, gremlin, which showed both high basal expression in the lung and lung-selective hypoxia responses in vivo, was of particular interest because it is a soluble inhibitor of the BMP-signaling axis, and inhibition of this signaling pathway has recently been identified as playing a central role in the development of hypoxic pulmonary hypertension (22, 49, 50, 56). Inhibition of BMP signaling also plays a crucial role in the development of IPAH (23). Heterozygous nonfunctional BMPR2 mutations leading to haploinsufficiency strongly predispose to the development of IPAH, but the precise pathways linking the BMPR2 mutations to the vascular lesions observed have not been elucidated (28, 33, 35). Moreover, BMPs together with their antagonists, including gremlin, are key mediators of lung development in the embryo (2, 10, 36, 46, 52, 55).

Given that background, we were interested to further explore changes in gremlin expression in the lung in vivo. We found that gremlin protein was present under normoxic conditions and significantly more extensively expressed in the tissue surrounding the alveolar spaces in hypoxia, a pattern of increase similar to that of its mRNA (Fig. 5). The extensive staining observed in hypoxia suggests that other cells in addition to ECs may express gremlin, including epithelial cells, myofibroblasts, and macrophages (32, 37).

In systemic organs, BMPs have been linked to endothelial angiogenesis, but an action on the pulmonary endothelium has not previously been documented (6, 34, 41, 42, 45). We report for the first time that BMP4 promotes pulmonary endothelial wound-healing responses and that gremlin can block this action (Fig. 6). Since gremlin is a secreted BMP antagonist, we postulated that the hypoxia-induced upregulation of this protein in pulmonary ECs would exert a blocking effect on BMP. Our finding that conditioned medium from hypoxic ECs blocked BMP4 stimulation of endothelial wound healing supports this hypothesis. In contrast, Stabile et al. (45) recently reported that gremlin was proangiogenic and can play a BMP-independent role in the angiogenic process by directly binding the EC surface via as yet uncharacterized cell-surface heparan sulfate proteoglycans. These potentially divergent results most likely reflect the different cell types used in the two studies (immortalized cell lines or systemic cells in the latter study and pulmonary ECs in our study), but, clearly, further experiments are required to decipher the complex interactions between gremlin, BMPs, and their cognate receptors.

Since alterations in BMP signaling play a central role in the development of pulmonary arterial hypertension, we examined gremlin expression in the explanted lungs of patients who were undergoing transplantation and found that its expression was markedly increased compared with controls (Fig. 7). This suggests that the underlying pathogenetic mechanisms may involve a previously unsuspected role of this endogenous BMP antagonist. Furthermore, the finding of high basal expression of gremlin in the lung may provide an explanation for the previously unexplained observation that the pulmonary circulation is the only vascular bed that suffers in patients with BMPR2 mutations, although the mutation is somatic; further experiments are needed to test this possibility.

To confirm that our subtractive approach had revealed other lung-selective hypoxia-responsive genes, we examined a second cluster in vivo, the "CXCR7 cluster." This cluster showed initial low expression levels (when compared with the gremlin cluster) but marked hypoxic responses. Of the four genes identified using array data, two, CXCR7 and BDNF, were upregulated by hypoxia in the lung. Of these two, BDNF was also upregulated in the heart, demonstrating that it was not lung-specific, and so we did not pursue this further.

CXCR7, formerly the orphan receptor CMKOR1, has recently been identified as a receptor for the proangiogenic chemokine CXCL12 (3, 7). We examined the responses of both these genes to hypoxia in vivo in our extended panel of organs and found that CXCL12 was increased in the hypoxic lung but that this increase was not limited to the lung (Fig. 8). This is in good agreement with our original microarray data, where CXCL12 was >2-fold upregulated in lung cells but also increased in the heart (~1.7-fold) and so had not been identified as lung-selective (data not shown). However, it is worth noting that both gene array and real-time PCR showed that basal levels of CXCL12 in lung ECs were >15-fold higher than in cardiac ECs (data not shown). However, CXCR7 was upregulated in the lung alone at levels of hypoxia found in lung disease, suggesting a potential novel mechanism whereby CXCL12 could show lung-specific proangiogenic actions (Fig. 9). Further experiments are required to substantiate this hypothesis. Nonetheless, the demonstration that a further gene from a different cluster demonstrates lung-selective hypoxic response suggests that our array-based approach has identified a cohort of such pulmonary-selective genes.

In conclusion, we have shown that lung-selective changes in gene expression are caused by alveolar hypoxia typical of that found in disease in vivo. One pathway identified was selective upregulation of the endogenous BMP antagonist gremlin, which is of specific interest because of the key role that inhibition of BMP signaling is known to play in the development of pulmonary hypertension. We confirmed that a further gene, CXCR7, from a separate cluster identified in our cell model, showed lung-selective hypoxic responses in vivo. Thus our cohort of lung-selective hypoxia-responsive genes gives new insights into potential mechanisms underlying hypoxic pulmonary hypertension.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by grants from the Health Research Board and the Higher Education Authority (Programme for Research in Third Level Institutions) and an unrestricted research grant from Actelion. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


   ACKNOWLEDGMENTS
 
The expert technical assistance of Emilie Duval is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Costello (e-mail: christine.costello{at}ucd.ie)

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


   REFERENCES
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 REFERENCES
 

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