Chronic hypercapnia alters lung matrix composition in mouse pups.

RATIONALE: permissive hypercapnia, a stretch-limiting ventilation strategy, often results in high Pa(CO(2)). This strategy is associated with reduced morbidity and mortality in premature infants and its benefits have been attributed to diminished barotrauma. However, little is known about the independent effect of high CO(2) levels during the lung development. METHODS: mice were exposed to 8% CO(2) or room air for 2 wk either from postnatal day 2 through 17 or as adults (approximately 2 mo of age). Lungs were excised and processed for protein, RNA, histology, and total lung volumes. RESULTS: histologic analysis demonstrated that alveolar walls of CO(2)-exposed mouse pups were thinner than those of controls and had twice the total lung volume. Molecular analysis revealed that several matrix proteins in the lung were downregulated in mouse pups exposed to hypercapnia. Interstitial collagen type I alpha1, type III alpha1, elastin and fibronectin protein, and mRNA levels were less than half of controls while collagen IV alpha 5 was unaffected. This decrease in interstitial collagen could thus account for the thinning of the interstitial matrix and the altered lung biomechanics. Matrix metalloproteinase (MMP)-8, a collagenase that has specificity for collagen types I and III, increased in hypercapnic mouse pups, suggesting increased collagen degradation. Moreover, tissue inhibitor of MMP (TIMP)-1, a potent inhibitor of MMP-8, was significantly decreased. However, unlike pups, adult mice exposed to hypercapnia demonstrated only a mild increase in total lung volumes and did not exhibit similar molecular or histologic changes. CONCLUSIONS: although permissive hypercapnia may prevent lung injury from barotrauma, our study revealed that exposure to hypercapnia may be an important factor in lung remodeling and function, especially in early life.

Differential effects of chronic intermittent and chronic constant hypoxia on postnatal growth and development.

Exposure to chronic constant or intermittent hypoxia (CCH or CIH) may have different effects on growth and development in early life. In this work, we exposed postnatal day 2 (P2) CD1 mice to CCH or CIH (11% O2) for 4 weeks and examined the effect of hypoxia on body and organ growth until P30. Regression analysis showed that weight increased in control, CCH and CIH cohorts with age with r2 values of 0.99, 0.97, and 0.94, respectively. Between days 2 and 30, slopes were 0.93+/-0.057, 0.76+/-0.108, and 0.63+/-0.061 (g/day, means+/-SEM) for control, CIH, and CCH, respectively and significantly different from each other (P<0.001). The slopes between P2 and P16 were 0.78+/-0.012, 0.46+/-0.002, and 0.47+/-0.019 for control, CCH and CIH, respectively. From P16 to 30, slopes were 1.12+/-0.033, 1.09+/-0.143, and 0.82+/-0.08 for control, CIH, and CCH, respectively with no significant difference from each other, suggesting a catch-up growth in the latter part of the hypoxic period. Slower weight gain resulted in a 12% and 23% lower body weight in CIH and CCH mice (P<0.001) by P30. Lung/body ratios were 0.010, 0.015, 0.015 for control, CIH, and CCH at P30, respectively. The decrease in liver, kidney, and brain weight were greater in CCH than CIH. Smaller liver weight was shown to be due to a reduction in cell size and cell number. Liver in CIH and CCH mice showed a 5% and 10% reduction in cell size (P<0.05) and a reduction of 28% in cell number (P<0.001) at P30. In contrast, CCH and CIH heart weight was 13% and 33% greater than control at P30 (P<0.05), respectively. This increase in the heart weight was due to an increase in the size of cardiomyocytes which showed an increase of 12% and 14% (P<0.001) for CIH and CCH, respectively as compared to control. Brain weight was 0.48 and 0.46 g for CIH and CCH, respectively (95% and 92% of normal). We concluded that (a) CIH and CCH follow different body and organ growth patterns; (b) mostly with CCH, the liver and kidneys are reduced in size in a proportionate way to body size but heart, lung, and brain are either spared or increased in size compared to body weight; and (c) the decrease in liver is secondary mostly to a decrease in cell number.

Novel functional interaction between Na+/H+ exchanger 1 and tyrosine phosphatase SHP-2.

Besides being a intracellular pH (pHi) regulator, Na+/H+ exchanger (NHE)1 has recently been postulated as a membrane scaffold that assembles protein complexes and coordinates various signaling pathways. The aim of the present study was to uncover NHE1 interactive partners and study their functional implications. NHE1 interactive partners were screened in the mouse brain with a signal transduction AntibodyArray. Ten of 400 tested proteins appeared to be potentially associated with NHE1. These partners have been shown to be involved in either cell proliferative or apoptotic pathways. The interactions between NHE1 and Src homology 2 domain-containing protein tyrosine phosphatase (SHP-2), Bin1, and heat shock protein (HSP)70 were reciprocally confirmed by coimmunoprecipitation. Moreover, in vitro binding data have shown that NHE1 COOH terminus interacts directly with SHP-2. The functional significance of the association between NHE1 and SHP-2 was further investigated by measuring pHi, cell proliferation, and cell death with the fluorescent dye BCECF, [3H]thymidine incorporation, and medium lactate dehydrogenase activity, respectively. Our results revealed that cells with SHP-2 overexpression exhibited a higher steady-state pHi and a faster, NHE1-dependent pHi recovery rate from acid load in HEPES buffer. In addition, SHP-2 overexpression diminished the HOE-642-induced inhibition of cell proliferation and protected cells from hypoxic injury, especially in the presence of HOE-642. Together, our findings demonstrate that SHP-2 not only is physically associated with NHE1 but also modulates NHE1 functions such as pHi regulation, cell proliferation, and cell death under hypoxia.

Effect of carbon dioxide on neonatal mouse lung: a genomic approach.

Despite the deleterious effects associated with elevated carbon dioxide (CO(2)) or hypercapnia, it has been hypothesized that CO(2) 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% CO(2), 12% CO(2), or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO(2)-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% CO(2), but only a few at 12% CO(2). 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 CO(2) seem to depend on the level to which the lung is exposed.

Gene expression and phenotypic characterization of mouse heart after chronic constant or intermittent hypoxia.

Chronic constant hypoxia (CCH), such as in pulmonary diseases or high altitude, and chronic intermittent hypoxia (CIH), such as in sleep apnea, can lead to major changes in the heart. Molecular mechanisms underlying these cardiac alterations are not well understood. We hypothesized that changes in gene expression could help to delineate such mechanisms. The current study used a neonatal mouse model in CCH or CIH combined with cDNA microarrays to determine changes in gene expression in the CCH or CIH mouse heart. Both CCH and CIH induced substantial alterations in gene expression. In addition, a robust right ventricular hypertrophy and cardiac enlargement was found in CCH- but not in CIH-treated mouse heart. On one hand, upregulation in RNA and protein levels of eukaryotic translation initiation factor-2alpha and -4E (eIF-2alpha and eIF-4E) was found in CCH, whereas eIF-4E was downregulated in 1- and 2-wk CIH, suggesting that eIF-4E is likely to play an important role in the cardiac hypertrophy observed in CCH-treated mice. On the other hand, the specific downregulation of heart development-related genes (e.g., notch gene homolog-1, MAD homolog-4) and the upregulation of proteolysis genes (e.g., calpain-5) in the CIH heart can explain the lack of hypertrophy in CIH. Interestingly, apoptosis was enhanced in CCH but not CIH, and this was correlated with an upregulation of proapoptotic genes and downregulation of anti-apoptotic genes in CCH. In summary, our results indicate that 1) the pattern of gene response to CCH is different from that of CIH in mouse heart, and 2) the identified expression differences in certain gene groups are helpful in dissecting mechanisms responsible for phenotypes observed.

Na+/H+ exchanger 1 deficiency alters gene expression in mouse brain.

Na(+)/H(+) exchanger 1 (NHE1) is well known to function as a major regulator of intracellular pH (pH(i)). It is activated by low pH(i) and exchanges extracellular Na(+) for intracellular H(+) to maintain cellular homeostasis. Despite the fact that we now have evidence suggesting other roles for NHE1, there has been no comprehensive study investigating its role as a signaling molecule. Toward this aim, we used in this study NHE1 null mutant mice and cDNA microarrays to investigate the effects of NHE1 on global gene expression in various regions of the brain, e.g., cortex, hippocampus, brain stem-diencephalon, and cerebellum. We found that a total of 35 to 79 genes were up- or downregulated in each brain region, with the majority being downregulated. The effect of NHE1 null mutation on gene expression is region specific, and only 11 genes were changed in all brain regions studied. Further analysis of the cis-regulatory regions of downregulated genes revealed that transcription suppressors, BCL6 and E4BP4, were probable candidates that mediated the inhibitory effect of NHE1 null mutation. One of the genes, MCT-13, was not only downregulated in the NHE1 null mutant brain but also in tissue cultures treated with an NHE1 inhibitor. We conclude that 1) a relatively small number of genes were altered in the NHE1 null mouse brain; 2) the effects of NHE1 null mutation on gene expression are region specific; and 3) several genes implicated in neurodegeneration have altered expression, potentially offering a molecular explanation for the phenotype of the NHE1 null mouse.

Purification and crystallization of Escherichia coli oligoribonuclease.

Oligoribonuclease (Orn) is an essential 3'-to-5' hydrolytic exoribonuclease which degrades short oligoribonucleotides to 5' mononucleotides. Escherichia coli Orn has been crystallized under several different conditions using ammonium sulfate, sodium citrate and sodium acetate as precipitants. Both native and selenomethionine-labeled oligoribonuclease (SeMet-Orn) can be crystallized at room temperature in 1.4-1.55 M sodium citrate. The SeMet-Orn crystals diffract to 2.2 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.43, b = 72.87, c = 147.76 A, and two dimers in the asymmetric unit. When grown in the presence of manganese, a second crystal form (Mn-SeMet-Orn) was obtained containing a single dimer per asymmetric unit (P2(1)2(1)2(1); a = 63.74, b = 74.31, c = 74.19 A). Finally, a hexagonal crystal form was obtained using sodium acetate as a precipitant (a = 91.5, b = 91.5, c = 111.1 A). This crystal (Zn-ApUp-Orn) belongs to the P6(5) space group and has three oligoribonuclease molecules per asymmetric unit.