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J Appl Physiol 101: 1556-1564, 2006. First published August 3, 2006; doi:10.1152/japplphysiol.01031.2005
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Effect of carbon dioxide on neonatal mouse lung: a genomic approach

Guangyu Li,2,* Dan Zhou,1,* Alfin G. Vicencio,2,* Julie Ryu,1 Jin Xue,1 Amjad Kanaan,1 Orit Gavrialov,1 and Gabriel G. Haddad1

1Department of Pediatrics, University of California San Diego, San Diego, California; and 2Department of Pediatrics, Section of Respiratory and Sleep Medicine, Albert Einstein College of Medicine and Children's Hospital at Montefiore, Bronx, New York

Submitted 23 August 2005 ; accepted in final form 25 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Despite the deleterious effects associated with elevated carbon dioxide (CO2) or hypercapnia, it has been hypothesized that CO2 can protect the lung from injury. However, the effects of chronic hypercapnia on the neonatal lung are unknown. Hence, we investigated the effect of chronic hypercapnia on neonatal mouse lung to identify genes that could potentially contribute to hypercapnia-mediated lung protection. Newborn mouse litters were exposed to 8% CO2, 12% CO2, or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO2-exposed mice appeared thinner than those of controls. Analyses of gene expression differences by microarrays revealed that genes from a variety of functional categories were differentially expressed following hypercapnia treatment, including those encoding growth factors, chemokines, cytokines, and endopeptidases. In particular and of major interest, the expression level of genes encoding surfactant proteins A and D, as well as chloride channel calcium-activated 3, were significantly increased, but the expression of WNT1-inducible signaling pathway protein 2 was significantly decreased. The significant changes in gene expression occurred mostly at 8% CO2, but only a few at 12% CO2. Our results lead us to conclude that 1) there are a number of gene families that may contribute to hypercapnia-mediated lung protection; 2) the upregulation of surfactant proteins A and D may play a role as anti-inflammatory or antioxidant agents; and 3) the effects of CO2 seem to depend on the level to which the lung is exposed.

hypercapnia; lung disease; gene expression; surfactant

NUMEROUS DISEASES AND CONDITIONS are associated with hypercapnia. While hypercapnia can be acute or chronic, the causes, pathophysiology, and clinical symptoms may vary. For example, chronic pulmonary diseases, including emphysema, severe asthma, end-stage cystic fibrosis, and long-standing pulmonary fibrosis can result in conditions that are characterized by acute or chronic hypercapnia. In addition, neuromuscular disease (e.g., amyotrophic lateral sclerosis), obstructive sleep apnea syndrome, and central nervous system depression can also lead to chronic or intermittent hypercapnia.

The physiological effects of acute hypercapnia in humans and animals are well known. For example, hypercapnia resulting from acute hypoventilation leads to respiratory acidosis and, depending on severity, enzymatic dysfunction that can eventually result in death. Chronic hypercapnia, on the other hand, can be tolerated as the kidneys compensate by retaining bicarbonate (HCO3–), thus buffering the acidosis associated with elevated CO2.

Despite potential adverse effects associated with hypercapnia, recent investigations have focused on the potential beneficial effects of elevated CO2. "Permissive hypercapnia," a strategy designed to minimize lung damage, caused by artificial ventilation, is being advocated in the intensive care setting (24, 32). Previously, the lung protective effect of such a strategy was thought to be purely mechanical; lower ventilator pressures were thought to minimize damage to the airways and lungs at the expense of an elevated CO2. More recently, some investigators have hypothesized that CO2 may have an independent effect in protecting the lung from injury. Indeed, laboratory experiments have suggested that CO2 may protect the lung against inflammatory (22) and hyperoxic injury after sustained artificial ventilation.

While the number of investigations regarding the effects of short-term elevation of CO2 (i.e., hours) in adult animal models continues to grow, none has addressed the potential effects of chronic hypercapnia (i.e., days and weeks) on the neonatal lung. Importantly, many premature infants, whose lungs are still in the early stages of alveolar development, are subjected to high oxygen concentrations and prolonged ventilatory support, often resulting in lung injury, abnormal alveolar formation, and ultimately a chronic lung disease known as bronchopulmonary dysplasia (BPD). In fact, BPD remains the leading complication of prematurity, affecting ~30% of extremely premature infants (37). One strategy that has demonstrated promise as a preventive measure in the development of BPD is permissive hypercapnia (9, 29, 38, 48). While past experiments have suggested that elevated CO2 may have an independent role in lung protection, the molecular pathways affected by such treatment remain largely unknown. The present study is the first attempt to systemically investigate the effect of chronically elevated CO2 on the developing mouse lung. We have utilized DNA microarray analysis to identify gene expression changes with chronic hypercapnia in the neonatal mouse lung, and we utilized additional techniques, including RT-PCR and Western blot analysis, to further investigate those changes, which may contribute to lung protection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CO2 exposure.   A computer-controlled system (OxyCycler, Reming Bioinstruments, Redfield, NY) was utilized to introduce and maintain constant levels of CO2 (either 8 or 12%) and oxygen levels of 21%, as described previously (16). CD-1 mice were utilized for all experiments. This mouse strain has been extensively evaluated in our laboratory. Thus we are familiar with gene expression changes that result from a variety of experimental conditions. CO2 concentrations of 8 and 12% were chosen to provide moderate and severe levels of hypercapnia, and exposure duration of 2 wk was chosen to encompass the period of most active alveolar formation. All litters were culled to eight pups each. At postnatal day 2, litters and their dams were placed in Plexiglas chambers with regular 12:12-h light-dark cycles. Control litters were housed in identical chambers and exposed to room air. Dams were rotated daily, and all studies were done in pathogen-free conditions. All animals were treated according to the guidelines approved by the Animal Care Committee of the Albert Einstein College of Medicine of Yeshiva University, which is accredited by the American Association of Laboratory Animal Care. The animal use protocol was approved by the University of California San Diego Institutional Animal Care and Use Committee.

Processing of lung tissue.   For the histological analysis, animals were killed, and lung tissue was processed as described previously (43, 44). Briefly, lungs were inflated through tracheotomies to 20-cm pressure with 4% paraformaldehyde (pH 7.4), and tracheas were ligated. Lungs and hearts were excised en bloc, submersed in 4% paraformaldehyde overnight, and processed for paraffin embedding and sectioning. Lung tissues were then stained with hematoxylin for histological analysis. For the microarray studies, RT-PCR, and Western blot analysis, animals were killed as described above, and lungs were excised, frozen in liquid nitrogen, and stored at –80°C until analysis.

Measurement of mean linear intercept and septal thickness.   The degree of alveolar septation was estimated by mean linear intercept (MLI) measurements, as described previously (43, 44). Briefly, six random lung sections from four separate animals per study group were viewed at x10 magnification and under a set number of horizontal lines of known length; any line that crossed a large vessel or bronchus was excluded from evaluation. Points at which an alveolar septum intercepted a line were counted separately. The total length of all lines counted was divided by the total number of intercepts per field examined. MLI, which is inversely proportional to alveolar surface area, was expressed in micrometers (µm).

Similarly, alveolar wall thickness was measured utilizing Image J software. As for MLI, six random lung sections from four separate animals per study group were viewed at x40 magnification under a set number of horizontal lines. Thickness of each septum was measured parallel to the intersecting line, and the results were expressed in micrometers (µm).

Plasma HCO3–.   Blood was withdrawn from mice after 2 wk of exposure to chronic hypercapnia, both 8 and 12% CO2, as well as age-matched controls. Mice were removed from the chamber and quickly anesthetized with isoflurane. The thoracic cavity was opened, and blood was drawn from the right ventricle with a 23-gauge needle. Throughout the entire blood sampling procedure, air was avoided. Blood was centrifuged, and the plasma fraction was stored at –20° until tests were performed. Total CO2 content of the plasma samples was measured using the Roche Cobas Integra 700 chemistry analyzer. For each group, the mean HCO3– from eight mice was calculated and compared with control.

RNA purification and cDNA probe labeling.   Total RNA from lung of each mouse was isolated using TRIzol reagent (Invitrogen, Gaithersburg, MD). Additional total RNA clean-up was carried out with RNeasy Mini Kit (Qiagen, Valencia, CA). The quality of extracted RNA samples was determined using denaturing agarose gels. Eighty micrograms of each total RNA were applied to synthesize the cDNA probe by using oligo(dT) primer and SuperScript II enzyme (Invitrogen). cDNA probes of the control mice were labeled with fluorescent cyanine 3 (Cy3)-dUTP, and the cDNA probes of mice treated with elevated CO2 were labeled with cyanine 5 (Cy5)-dUTP (Amersham Biosciences) during the reverse transcription reaction. Both Cy3- and Cy5-labeled probes were mixed and cleaned with Microcon YM 30 (Millipore, Bedford, MA) for further hybridization. "Dye-swapping" (reverse labeling) was applied to reduce systematic bias (41, 50).

Microarray hybridization.   Oligo-microarray (32K) slides (mouse genome oligo set version 3.0) containing 31,769 70mer long oligo probes were purchased from the Microarray Facility of the Albert Einstein College of Medicine (http://microarray1k.aecom.yu.edu). The number of animals studied in each group was n = 4 from two to three different litters of each treatment. The hybridization process was conducted according to the instructions of the core facility. Briefly, labeled cDNA probes were mixed with hybridization solution containing 35% formamide, 0.5% SDS, 4x sodium chloride-sodium phosphate-EDTA, and 2.5x Denhardt's solution. The probes were denatured at 94°C for 2 min and then prehybridized at 50°C for 1 h. The microarray slides were prehybridized at 50°C for at least 2 h in prehybridization solution [35% formamide, 0.5% SDS, 4x sodium chloride-sodium phosphate-EDTA, and 2.5x Denhardt's solution, 100 µg/ml salmon sperm DNA, 1 µg/µl poly(dA), 10 µg/µl tRNA, and 1 µg/µl mouse Cot1 DNA]. After prehybridization, labeled probes in the hybridization solution were applied onto slides and incubated at 50°C overnight. Slides were washed at room temperature with 0.2x saline sodium citrate/0.1% SDS for 10 min and three times with 0.2x saline sodium citrate for 10 min.

Image acquisition and data analysis.   Microarray slides were scanned with GenePix4100A laser scanner for both Cy3 and Cy5 channel, and GenePix Pro 4.1 software (Axon Instrument, Union City, CA) was utilized to quantify the intensity of fluorescent images and to normalize results by subtracting local background fluorescence. Hybridization data were considered invalid if the median intensity of the foreground was less than twice the background. The ratios of the sample intensity to the reference red (Cy5)/green (Cy3) intensity for all targets were normalized with LOWESS normalization. Significance analysis of microarrays (42) was used to identify the genes that differentiated hypercapnia treatment from control samples. Finally, the alterations in global gene expression in the context of specific biological pathways and molecular functions were determined using GenMapp 2.0 software (14). Data of microarray analyses can be retrieved under the series access number GSE3161 in the National Institute for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo).

Quantitative real-time RT-PCR.   Six genes selected from those identified as differentially expressed in microarray experiments were confirmed by quantitative real-time RT-PCR, as described previously (51). Primers were designed using Lasergene software (DNAStar) and web-based Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/). The primer set was synthesized by Invitrogen for specific genes and listed in Table 1. The same total RNA samples used in microarray experiments were treated with DNase I (Invitrogen, Gaithersburg, MD) to remove genomic DNA. First-strand cDNA was synthesized from 1 µg of total RNA using SuperScript II reverse transcriptase and oligo(dT) primer in 20 µl of volume reaction, according to the manufacturer's instruction (Invitrogen). One microliter of 1:5 diluted cDNA was used as template in 20 µl of RT-PCR reaction containing 1x SYBRgreen master mix (Applied Biosystems, Foster City, CA) and 0.5 µM of each primer. Real-time PCR amplification was performed using ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Each RT-PCR reaction was done in triplicate. The fold changes in expression level for each specific gene were calculated by the 2–{Delta}{Delta}Ct method, where Ct is threshold cycle (28), after normalization to beta-actin and/or 18S rRNA (loading controls). The final result represents the mean fold change of four individual CO2-treated samples over controls.


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  		Table 1. Real-time RT-PCR primer sequences for selected genes

 
Western blot analysis.   Frozen lung tissue was processed as described previously (43, 44). Briefly, frozen tissues were homogenized, sheared, and centrifuged, and supernatants were taken as whole tissue lysates. Protein concentration was measured using the bicinchoninic acid kit, according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein (20 µg) were separated on 10% precast NuPAGE bis-Tris SDS-PAGE gels (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride membranes (Immobilin-P; Millipore, Bedford, MA). Western blots were performed with antibodies against actin (Santa Cruz Biotechnology, Santa Cruz, CA), 1:500, and surfactant proteins A and D (Sftp-A and Sftp-D), 1:1,000 (Chemicon International, Temecula, CA). Specific bands were visualized after incubation with the respective secondary antibodies using enhanced chemiluminescense (Biosciences Amersham, Piscataway, NJ). Densitometry measurements of Western blots from each experimental group were obtained (n ≥ 5), and absolute values were normalized to actin. Results were reported in arbitrary units, comparing each value with that obtained from each respective actin measurement.

Statistical analysis.   The Wilcoxon rank sum test was used to compare the difference between hypercapnic and control animals for testing the level of plasma HCO3–. Student's t-test was used for comparison of effects of hypercapnia on target gene mRNA level (real-time RT-PCR), protein (Western blot), and lung morphology (alveolar septation and alveolar wall thickness); differences between samples were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Growth characteristics of mice in chronic hypercapnia.   CD1 mice at postnatal day 2 were exposed to 8% or 12% CO2 for 2 wk. Age-matched control mice were also raised in similar conditions, except in eucapnia. In this investigation, a dose-response relation was sought by exposing mice to either 8 or 12% CO2. The effects of elevated CO2 on growth characteristics of neonatal mice were evaluated. Our data showed that there were no significant changes observed in body weight, hematocrits, and the weight of most organs in neonatal mice exposed to either 8 or 12% CO2 for 2 wk. A significant increase in kidney weight (~10%) was observed in those mice exposed to 12% CO2 for 2 wk.

The effects of hypercapnia on plasma HCO3– concentration were evaluated at 2 wk after exposure to 8 and 12% CO2. After 2 wk at 8% CO2, plasma HCO3– was significantly increased from 18.4 ± 2.4 mmol/l (eucapnia mice, n = 8) to 22.9 ± 1.8 mmol/l (hypercapnic mice, n = 8) (P < 0.05). Similarly, after 2 wk of 12% CO2, plasma HCO3– was increased significantly to 22.6 ± 2.6 mmol/l (P < 0.05), indicating a significant increase in HCO3– reabsorption by the kidneys.

Lung structure after chronic CO2 exposure.   Overall alveolar septation was not altered with chronic CO2. After 2 wk in either 8 or 12% CO2, alveoli were well developed, as assessed by general histological evaluation (Fig. 1A). MLI measurements confirmed that there was no difference in alveolar surface area between the groups (Fig. 1B), indicating that septation (i.e., transition from the saccular stage to the alveolar stage of development) had occurred. Interestingly, CO2 appeared to affect the characteristics of individual alveoli when viewed under high magnification (Fig. 2A). Specifically, alveoli from 8 and 12% CO2 animals demonstrated thinner walls compared with those of controls. As shown in Fig. 2B, alveolar wall thickness from both CO2 groups averaged ~50% of that of control animals (P ≤ 0.002) in both 8 and 12% groups.


Figure 1
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  		Fig. 1. Alveolar septation in chronic CO2. A: exposure of neonatal mice to 8% and 12% CO2 does not alter alveolar septation. B: histological observations were quantified with mean linear intercept (MLI) counting. MLI does not differ between groups. n = 4 Animals in each group; measurement bars = 200 µm.

 

Figure 2
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  		Fig. 2. Alveolar wall thickness in chronic CO2. A: when viewed at x40 magnification, alveoli from 8 and 12% CO2 animals appeared thinner than those from control animals. B: measurements confirmed that average wall thickness from CO2-exposed animals was ~60% that of controls. There was no difference between 8% and 12% CO2 animals. n = 4 Animals in each group; measurement bars = 60 µm.

 
Overall gene expression profile in lung with CO2 exposure.   The mouse genome oligo set (version 3.0, Qiagen) is composed of 31,769 70mer probes, representing 24,878 genes and 32,829 transcripts. Changes were considered significant only when the gene expression level was altered more than 1.5-fold, and the false discover rate was <5% (q < 5%). Our results showed that a substantial number of genes have altered expression in the lung obtained from animals exposed to 8% CO2. Indeed, 365 genes were significantly upregulated, and 342 genes were significantly downregulated (Table 1). In contrast, only two genes were identified as significantly altered after 12% CO2 exposure when the same criteria were applied. These two genes were as follows: 1) potassium channel subfamily K member 1 (Kcnk1) that was downregulated by 1.52 fold (q = 0.00), and 2) pleckstrin homology domain containing family G (with RhoGef domain) member 6 (Plekhg6) that was downregulated 2.06-fold (q = 0.00).

The altered genes in mice exposed to 8% CO2 were first analyzed based on the level of expression change. We found that most of the differentially regulated genes were altered by 1.5- to 2.5-fold. Interestingly, 13 genes from this treatment group were highly upregulated with more than fivefold changes. The most upregulated gene was a gene encoding a protein that is similar to a predicted Rattus norvegicus chitinase 3-like 4 (Chi3l4), which was upregulated over 30-fold. There were also 14 highly downregulated genes (over fivefold) in lungs exposed to 8% CO2 and the most downregulated gene encoding a protein with uncharacterized function (>60-fold downregulation) (see Supplemental Table 1, available with the online version of this article).

GenMAPP and MAPPFinder were also used to identify the changes in gene families and biological processes (14). For the 8% CO2 group, out of 569 significantly up- or downregulated genes, 266 genes with Gene Ontology ID were classified into the following major functional categories: hydrolases (45 genes, P < 0.05), peptidases (19 genes, P < 0.05), receptor binding proteins (17 genes, P < 0.05), cell adhesion molecules (21 genes, P < 0.05), and immune response genes (28 genes, P < 0.05). Tables 2–5 list the categorized genes that were significantly altered with elevated CO2.


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  		Table 2. Genes related to cell adhesion regulated by CO2

 

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  		Table 5. Genes related to signal transduction regulated by CO2

 

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  		Table 4. Genes related to cell growth and/or maintenance regulated by CO2

 
Real-time RT-PCR.   To validate our microarray results, genes from various functional categories were chosen for real-time RT-PCR analysis. The same total RNA samples were used as in the microarray study. The results obtained from RT-PCR analysis (Fig. 3) generally agreed with those obtained from the microarray analysis, although fold differences varied between the two analyses.


Figure 3
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  		Fig. 3. Real-time RT-PCR validation of microarray results. Expression levels of selected genes identified by microarray analysis following 8% CO2 treatment were confirmed using real-time quantitative RT-PCR (qRT-PCR). The ratio of each gene from qRT-PCR was normalized to 18s rRNA. The values were averaged ratios from triplicate experiments. Clca3, chloride channel calcium activated 3; Sftp-A1, pulmonary surfactant-associated protein A1; Sftp-D, pulmonary surfactant-associated protein D; TGFbi, transforming growth factor beta induced protein; Wisp2, WNT1-inducible signaling pathway protein 2.

 
Protein expression level of selected genes.   Our microarray analysis and real-time RT-PCR results demonstrated increased expression of surfactant-associated protein A and surfactant-associated protein D RNAs. Because of the potential importance of these genes in various forms of lung injury, we analyzed their protein expression levels. Western blot analysis showed that Sftp-A and Sftp-D were upregulated with chronic CO2. As shown in Fig. 4, A and B, there was a dramatic increase in Sftp-A expression with 8% CO2, but a small increase with 12% CO2. The increase of Sftp-A with 8% CO2 was ~2.5-fold compared with control. Similarly, Sftp-D demonstrated a dramatic increase in protein expression with 8% CO2, and a smaller albeit significant increase with 12% CO2. Compared with control, the increased expression of Sftp-D in 8 and 12% CO2 was found to be approximately fourfold and twofold higher, respectively (Fig. 4, C and D).


Figure 4
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  		Fig. 4. Western blot results of surfactant proteins. A: exposure of neonatal mice to 8% CO2 results in increased expression of Sftp-A (representative blot). This effect is not seen with 12% CO2. B: densitometry demonstrates Sftp-A expression in 8% CO2 to be ~2.5-fold that of control animals (n > 5 animals in each group). C: exposure of neonatal mice to 8 and 12% CO2 results in increased expression of Sftp-D, with the more dramatic effect seen at 8% (representative blot). D: densitometry demonstrates Sftp-D expression in 8 and 12% CO2 to be approximately fourfold and twofold that of control animals, respectively (n > 5 animals in each group).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Permissive hypercapnia, a ventilatory strategy in which CO2 is permitted to rise, has been increasingly utilized to minimize injury sustained from high-oxygen concentrations and barotrauma. This strategy was first popularized after epidemiological studies done in premature infants demonstrated that early initiation of continuous positive airway pressure resulting in hypercapnia was associated with a more favorable respiratory outcome (4). While this effect has mostly been attributed to minimizing lung stretch caused by mechanical ventilation, more recent evidence suggests that CO2 may have an independent effect in lung protection (9, 29, 38, 48).

It is important to note, however, that elevated CO2 in the clinical setting is often accompanied by hypoxia. Because hypoxia is generally treated with supplemental O2, the diseased or underventilated lung is often exposed to both hyperoxia and hypercapnia, as demonstrated in numerous clinical conditions, including BPD, chronic obstructive lung disease, longstanding pulmonary fibrosis, and end-stage cystic fibrosis. Understanding the specific effects of CO2 in the diseased lung in human pathological conditions can, therefore, be quite challenging because of the complex physiological responses that accompany such conditions. Hence, we developed the present model to better evaluate the specific effects of hypercapnia on the lung, understanding that the difficulty in obtaining accurate physiological measurements in neonatal mice is a clear limitation of the model. For example, while our results demonstrated elevated serum HCO3– in response to hypercapnia (suggesting renal compensation of acidosis), we are unable to determine whether the observed changes were the direct result of CO2 or an indirect result of respiratory acidosis, an alteration of respiratory pattern, or a combination of physiological changes that accompany exposure to elevated CO2.

We found that the lungs of CO2-exposed animals demonstrated normal overall surface area, as assessed by MLI, but consisted of thin-walled alveoli, which are characteristic of mature lungs. Whether such changes are the result of accelerated alveolar-capillary maturation, alterations in interstitial components, or epithelial maturation is unclear from this investigation. However, past investigations have demonstrated that the process of alveolar maturation encompasses numerous events, most notably a thinning of alveolar walls, which included a transition from a double to a single capillary bed (8, 40).

In addition to characterizing the effects of chronic hypercapnia, we performed microarray analysis on the lungs of neonatal mice exposed to elevated levels of CO2 to identify genes that could contribute to lung protection. A large number of genes were found to be differentially regulated in 8% CO2 exposed lung, whereas the significant changes in gene expression in 12% CO2-treated lungs were very few. While this may appear counterintuitive, it is well known that a high level of CO2 in the blood (and cerebrospinal fluid) may induce different mechanisms than those induced by lower levels. Indeed, it is well known that a high level of CO2 may act as a depressant. In fact, our previous electrophysiological data on neurons obtained from brains exposed to 8 or 12% CO2 have shown that 8% CO2 treatment induced major excitatory effects on hippocampal neurons, but 12% did not (15).

Sftp and cell adhesion molecules.   Interestingly, exposure to hypercapnia resulted in significant increases in expression of Sftp-A and Sftp-D (Table 2). Unlike Sftp-B and Sftp-C, Sftp-A and Sftp-D are hydrophilic proteins that do not contribute significantly to the reduction of alveolar surface tension (18, 47). Rather, they are increasingly viewed as a "first line of defense" in the lung against a variety of insults (13, 17, 21, 26). Sftp-A and Sftp-D belong to a developmentally regulated protein family known as the collectins, which are characterized by a common collagenous region and COOH-terminal lectin domain (18), and are expressed predominantly in bronchial epithelial cells and type II alveolar cells. Past investigations have demonstrated their ability to bind to (and in some cases inhibit the growth of) a variety of microorganisms, including numerous bacterial species, fungi, and viruses, suggesting an antimicrobial function (20, 23, 27, 33, 34, 36, 39, 49). Indeed, null mice for Sftp-A or Sftp-D are susceptible to infection from bacteria and demonstrate delayed pulmonary clearance of Pneumocystis carnii (2, 3). In addition, Sftp-A and Sftp-D have been shown to protect epithelial cells in vitro against oxygen radicals and diminish inflammatory mediators (7, 10, 11). Coupled with these past investigations, our findings suggest that chronic hypercapnia may play a role in lung protection via upregulating the expression of Sftp-A and Sftp-D.

In addition, we found that 8% CO2 exposure leads to the activation of several important cell adhesion molecules, including procollagens (Col2a1, Col4a1, Col4a2, and Col4a3), laminins (Lamc2 and Lamb3), and fibulin (Fbln5) in Table 2. Type IV collagens are the main component of basement membrane structures. Similarly, laminins are a family of extracellular matrix glycoproteins constituting the basement membranes. Fibulin-5 is an integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and organizes elastic fiber system to prevent disease in the lung and vasculature (31).

Inflammation, immune response and cytokine activity.   Importantly, a number of different lung insults, including infection, oxidative injury, and mechanical injury, are associated with varying degrees of inflammation. Previous studies have shown that hypercapnic acidosis can mediate the suppression of inflammatory responses that may contribute to lung protection (22, 24, 25). Our microarray results also demonstrated significant downregulation in expression of numerous inflammatory mediator genes, including interferons, interleukins, chemokines, immunoglobulins, histocompatibility complexes, and tumor necrosis factor, etc. (Table 3), which may also contribute to lung protection. For example, Toll-like receptors have been shown to play an important role in the innate immune response (1), and downregulation of such genes may suppress the inflammatory response and limit lung injury. Interestingly, Sftp-D has also been shown to diminish secondary immune responses, such as IL-dependent T-lymphocyte proliferation, allergen-induced lymphocytic responses, and release of histamine (5, 6, 45). Taken together, moderate (8%), but not higher (12%), chronic hypercapnia widely suppressed the immune and inflammatory mediator gene expression, which may reduce the susceptibility of lung to injury. Furthermore, since chitinase 3-like 3 protein was shown here to be >30-fold upregulated, this may play a role in lung structure and function (52) and might be a pharmacological target.


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  		Table 3. The immune response genes regulated in response to elevated CO2

 
Cytochrome P-450 enzymes.   In our study, hypercapnia also dramatically downregulated Cyp1a1, a member of the cytochrome P-450 superfamily. Metabolism of many endogenous and exogenous compounds occurs through the cytochrome P-450 superfamily. Cyp1a1 has been shown to metabolize environmental toxins into reactive oxidant intermediates through the process of detoxification (46). In addition, studies suggest that, when Cyp1a1 levels increase during the process of detoxification, reactive oxygen species may accumulate beyond the cell's ability to repair itself (12, 30, 35). Thus, while an acute upregulation of Cyp1a1 may protect from toxins, chronic downregulation may help to limit damage by reactive oxygen species. In fact, inhibition of Cyp1a1 has been shown to attenuate the toxic effects of hyperoxia (19).

In this work, we have shown that thinning of the alveolar walls occurs after both 8 and 12% CO2 treatment. What factors play a role in this thinning is not clear at the moment. Hence, it would be difficult to link etiologically thinning of the matrix to gene expression, except to speculate that this histopathology described here could be the result of many changes in gene expression, such as metalloproteinases, growth factors, and proteases. An even harder question to resolve at this stage is the difference in gene expression in 8 vs. 12% CO2 and the lack of a clear difference in histopathology between these two treatments. However, it should be noted that, since we adopted a cutoff criterion of 1.5-fold change (which is an accepted statistical cutoff in the literature), the assumption is that lower fold change of gene expression may not be important in the phenotype, which may be debatable.

In summary, chronic exposure to a moderate level of hypercapnia (i.e., 8% CO2) induces alteration in gene expression and morphological changes in the neonatal mouse lung. While many of the changes in gene expression could potentially contribute to protection against lung injury, we believe that the present and previous investigations suggest that upregulation of Sftp-A and Sftp-D may play a role in mediating such effects.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants PO1 HD3–2573 and RO1 HL-66327 to G. G. Haddad and the Parker B. Francis Fellowship to A. G. Vicencio. We also acknowledge the support from the cDNA Microarray Core Facility of Albert Einstein College of Medicine of Yeshiva University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics, Univ. of California, San Diego, 9500 Gilman Drive, #0735, La Jolla, CA 92093–0735 (e-mail: ghaddad{at}ucsd.edu)

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.

The online version of this article contains supplemental data.

* G. Li, D. Zhou, and A. G. Vicencio contributed equally to this project. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Aderem A and Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 406: 782–787, 2000.[CrossRef][Medline]
  2. Atochina EN, Beck JM, Preston AM, Haczku A, Tomer Y, Scanlon ST, Fusaro T, Casey J, Hawgood S, Gow AJ, and Beers MF. Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species. Infect Immun 72: 6002–6011, 2004.[Abstract/Free Full Text]
  3. Atochina EN, Gow AJ, Beck JM, Haczku A, Inch A, Kadire H, Tomer Y, Davis C, Preston AM, Poulain F, Hawgood S, and Beers MF. Delayed clearance of pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of surfactant protein-D knockout mice. J Infect Dis 189: 1528–1539, 2004.[CrossRef][ISI][Medline]
  4. Avery ME, Tooley WH, Keller JB, Hurd SS, Bryan MH, Cotton RB, Epstein MF, Fitzhardinge PM, Hansen CB, and Hansen TN. Is chronic lung disease in low birth weight infants preventable? A survey of eight centers. Pediatrics 79: 26–30, 1987.[Abstract/Free Full Text]
  5. Borron P, McCormack FX, Elhalwagi BM, Chroneos ZC, Lewis JF, Zhu S, Wright JR, Shepherd VL, Possmayer F, Inchley K, and Fraher LJ. Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor. Am J Physiol Lung Cell Mol Physiol 275: L679–L686, 1998.[Abstract/Free Full Text]
  6. Borron PJ, Crouch EC, Lewis JF, Wright JR, Possmayer F, and Fraher LJ. Recombinant rat surfactant-associated protein D inhibits human T lymphocyte proliferation and IL-2 production. J Immunol 161: 4599–4603, 1998.[Abstract/Free Full Text]
  7. Bridges JP, Davis HW, Damodarasamy M, Kuroki Y, Howles G, Hui DY, and McCormack FX. Pulmonary surfactant proteins A and D are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury. J Biol Chem 275: 38848–38855, 2000.[Abstract/Free Full Text]
  8. Burri PH, Dbaly J, and Weibel ER. The postnatal growth of the rat lung. I. Morphometry. Anat Rec 178: 711–730, 1974.[CrossRef][Medline]
  9. Carlo WA, Stark AR, Wright LL, Tyson JE, Papile LA, Shankaran S, Donovan EF, Oh W, Bauer CR, Saha S, Poole WK, and Stoll B. Minimal ventilation to prevent bronchopulmonary dysplasia in extremely-low-birth-weight infants. J Pediatr 141: 370–374, 2002.[CrossRef][ISI][Medline]
  10. Clark H and Reid K. The potential of recombinant surfactant protein D therapy to reduce inflammation in neonatal chronic lung disease, cystic fibrosis, and emphysema. Arch Dis Child 88: 981–984, 2003.[Abstract/Free Full Text]
  11. Clark H and Reid KB. Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D. Immunobiology 205: 619–631, 2002.[CrossRef][ISI][Medline]
  12. Conney AH. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res 42: 4875–4917, 1982.[Free Full Text]
  13. Crouch EC. Surfactant protein-D and pulmonary host defense. Respir Res 1: 93–108, 2000.[CrossRef][Medline]
  14. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, and Conklin BR. GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nat Genet 31: 19–20, 2002.[CrossRef][ISI][Medline]
  15. Gu XQ and Haddad GG. Differential responses of mice hippocampal neurons to various levels of CO2. In: Experimental Biology and XXXV International Congress of Physiological Sciences, San Diego. 2005, p. A1215.
  16. Gu XQ, Xue J, and Haddad GG. Effect of chronically elevated CO2 on CA1 neuronal excitability. Am J Physiol Cell Physiol 287: C691–C697, 2004.[Abstract/Free Full Text]
  17. Haagsman HP, Herias V, and van Eijk M. Surfactant phospholipids and proteins in lung defence. Acta Pharmacol Sin 24: 1301–1303, 2003.[ISI][Medline]
  18. Hakansson K and Reid KB. Collectin structure: a review. Protein Sci 9: 1607–1617, 2000.[Abstract]
  19. Hazinski TA, Noisin E, Hamon I, and DeMatteo A. Sheep lung cytochrome P4501A1 (CYP1A1): cDNA cloning and transcriptional regulation by oxygen tension. J Clin Invest 96: 2083–2089, 1995.[ISI][Medline]
  20. Hickling TP, Bright H, Wing K, Gower D, Martin SL, Sim RB, and Malhotra R. A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo. Eur J Immunol 29: 3478–3484, 1999.[CrossRef][ISI][Medline]
  21. Holmskov U, Thiel S, and Jensenius JC. Collections and ficolins: humoral lectins of the innate immune defense. Annu Rev Immunol 21: 547–578, 2003.[CrossRef][ISI][Medline]
  22. Kregenow DA and Swenson ER. The lung and carbon dioxide: implications for permissive and therapeutic hypercapnia. Eur Respir J 20: 6–11, 2002.[Free Full Text]
  23. Kuan SF, Rust K, and Crouch E. Interactions of surfactant protein D with bacterial lipopolysaccharides. Surfactant protein D is an Escherichia coli-binding protein in bronchoalveolar lavage. J Clin Invest 90: 97–106, 1992.[ISI][Medline]
  24. Laffey JG, O'Croinin D, McLoughlin P, and Kavanagh BP. Permissive hypercapnia–role in protective lung ventilatory strategies. Intensive Care Med 30: 347–356, 2004.[CrossRef][ISI][Medline]
  25. Lardner A. The effects of extracellular pH on immune function. J Leukoc Biol 69: 522–530, 2001.[Abstract/Free Full Text]
  26. LeVine AM and Whitsett JA. Pulmonary collectins and innate host defense of the lung. Microbes Infect 3: 161–166, 2001.[CrossRef][ISI][Medline]
  27. LeVine AM, Whitsett JA, Hartshorn KL, Crouch EC, and Korfhagen TR. Surfactant protein D enhances clearance of influenza A virus from the lung in vivo. J Immunol 167: 5868–5873, 2001.[Abstract/Free Full Text]
  28. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2[-Delta Delta C(T)] method. Methods 25: 402–408, 2001.[CrossRef][ISI][Medline]
  29. Mariani G, Cifuentes J, and Carlo WA. Randomized trial of permissive hypercapnia in preterm infants. Pediatrics 104: 1082–1088, 1999.[Abstract/Free Full Text]
  30. Miller EC and Miller JA. Mechanisms of chemical carcinogenesis. Cancer 47: 1055–1064, 1981.[CrossRef][ISI][Medline]
  31. Nakamura T, Lozano PR, Ikeda Y, Iwanaga Y, Hinek A, Minamisawa S, Cheng CF, Kobuke K, Dalton N, Takada Y, Tashiro K, Ross J Jr, Honjo T, and Chien KR. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature 415: 171–175, 2002.[CrossRef][Medline]
  32. O'Croinin D, Ni Chonghaile M, Higgins B, and Laffey JG. Bench-to-bedside review: Permissive hypercapnia. Crit Care 9: 51–59, 2005.[CrossRef][ISI][Medline]
  33. Ofek I, Mesika A, Kalina M, Keisari Y, Podschun R, Sahly H, Chang D, McGregor D, and Crouch E. Surfactant protein D enhances phagocytosis and killing of unencapsulated phase variants of Klebsiella pneumoniae. Infect Immun 69: 24–33, 2001.[Abstract/Free Full Text]
  34. O'Riordan DM, Standing JE, Kwon KY, Chang D, Crouch EC, and Limper AH. Surfactant protein D interacts with Pneumocystis carinii and mediates organism adherence to alveolar macrophages. J Clin Invest 95: 2699–2710, 1995.[ISI][Medline]
  35. Phillips DH. Fifty years of benzo(a)pyrene. Nature 303: 468–472, 1983.[CrossRef][Medline]
  36. Schelenz S, Malhotra R, Sim RB, Holmskov U, and Bancroft GJ. Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells. Infect Immun 63: 3360–3366, 1995.[Abstract]
  37. Smith VC, Zupancic JA, McCormick MC, Croen LA, Greene J, Escobar GJ, and Richardson DK. Trends in severe bronchopulmonary dysplasia rates between 1994 and 2002. J Pediatr 146: 469–473, 2005.[CrossRef][ISI][Medline]
  38. Strand M, Ikegami M, and Jobe AH. Effects of high PCO2 on ventilated preterm lamb lungs. Pediatr Res 53: 468–472, 2003.[CrossRef][ISI][Medline]
  39. Tino MJ and Wright JR. Surfactant protein A stimulates phagocytosis of specific pulmonary pathogens by alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 270: L677–L688, 1996.[Abstract/Free Full Text]
  40. Tschanz SA, Makanya AN, Haenni B, and Burri PH. Effects of neonatal high-dose short-term glucocorticoid treatment on the lung: a morphologic and morphometric study in the rat. Pediatr Res 53: 72–80, 2003.[CrossRef][ISI][Medline]
  41. Tseng GC, Oh MK, Rohlin L, Liao JC, and Wong WH. Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. Nucleic Acids Res 29: 2549–2557, 2001.[Abstract/Free Full Text]
  42. Tusher VG, Tibshirani R, and Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98: 5116–5121, 2001.[Abstract/Free Full Text]
  43. Vicencio AG, Eickelberg O, Stankewich MC, Kashgarian M, and Haddad GG. Regulation of TGF-beta ligand and receptor expression in neonatal rat lungs exposed to chronic hypoxia. J Appl Physiol 93: 1123–1130, 2002.[Abstract/Free Full Text]
  44. Vicencio AG, Lee CG, Cho SJ, Eickelberg O, Chuu Y, Haddad GG, and Elias JA. Conditional overexpression of bioactive transforming growth factor-beta1 in neonatal mouse lung: a new model for bronchopulmonary dysplasia? Am J Respir Cell Mol Biol 31: 650–656, 2004.[Abstract/Free Full Text]
  45. Wang JY, Shieh CC, You PF, Lei HY, and Reid KB. Inhibitory effect of pulmonary surfactant proteins A and D on allergen-induced lymphocyte proliferation and histamine release in children with asthma. Am J Respir Crit Care Med 158: 510–518, 1998.[Abstract/Free Full Text]
  46. Whitlock JP Jr. Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol 39: 103–125, 1999.[CrossRef][ISI][Medline]
  47. Whitsett JA and Weaver TE. Hydrophobic surfactant proteins in lung function and disease. N Engl J Med 347: 2141–2148, 2002.[Free Full Text]
  48. Woodgate PG and Davies MW. Permissive hypercapnia for the prevention of morbidity and mortality in mechanically ventilated newborn infants. Cochrane Database Syst Rev 2: CD002061, 2001.
  49. Wu H, Kuzmenko A, Wan S, Schaffer L, Weiss A, Fisher JH, Kim KS, and McCormack FX. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability. J Clin Invest 111: 1589–1602, 2003.[CrossRef][ISI][Medline]
  50. Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, and Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15, 2002.[Abstract/Free Full Text]
  51. Zhou D, Xue J, Gavrialov O, and Haddad GG. Na+/H+ exchanger 1 deficiency alters gene expression in mouse brain. Physiol Genomics 18: 331–339, 2004.[Abstract/Free Full Text]
  52. Zhu Z, Zheng T, Homer RJ, Kim YK, Chen NY, Cohn L, Hamid Q, and Elias JA. Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation. Science 304: 1678–1682, 2004.[Abstract/Free Full Text]



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