Neonatal mice genetically modified to express the elastase inhibitor elafin are protected against the adverse effects of mechanical ventilation on lung growth

Mechanical ventilation (MV) with O(2)-rich gas (MV-O(2)) offers life-saving treatment for newborn infants with respiratory failure, but it also can promote lung injury, which in neonates translates to defective alveolar formation and disordered lung elastin, a key determinant of lung growth and repair. Prior studies in preterm sheep and neonatal mice showed that MV-O(2) stimulated lung elastase activity, causing degradation and remodeling of matrix elastin. These changes yielded an inflammatory response, with TGF-β activation, scattered elastic fibers, and increased apoptosis, culminating in defective alveolar septation and arrested lung growth. To see whether sustained inhibition of elastase activity would prevent these adverse pulmonary effects of MV-O(2), we did studies comparing wild-type (WT) and mutant neonatal mice genetically modified to express in their vascular endothelium the human serine elastase inhibitor elafin (Eexp). Five-day-old WT and Eexp mice received MV with 40% O(2) (MV-O(2)) for 24-36 h. WT and Eexp controls breathed 40% O(2) without MV. MV-O(2) increased lung elastase and MMP-9 activity, resulting in elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 increased 6-fold), apoptosis (cleaved-caspase-3 increased 10-fold), and inflammation (NF-κB activation, influx of neutrophils and monocytes) in lungs of WT vs. unventilated controls. These changes were blocked or blunted during MV-O(2) of Eexp mice. Scattered lung elastin and emphysematous alveoli observed in WT mice after 36 h of MV-O(2) were attenuated in Eexp mice. Both WT and Eexp mice showed defective VEGF signaling (decreased lung VEGF-R2 protein) and loss of pulmonary microvessels after lengthy MV-O(2), suggesting that elafin's beneficial effects during MV-O(2) derived primarily from preserving matrix elastin and suppressing lung inflammation, thereby enabling alveolar formation during MV-O(2). These results suggest that degradation and remodeling of lung elastin can contribute to defective lung growth in response to MV-O(2) and might be targeted therapeutically to prevent ventilator-induced neonatal lung injury.     

Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication

In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.     

Hyperoxia-induced emphysematous changes in subacute phase of endotoxin-induced lung injury in rats

We examined the effects of prolonged hyperoxia (75% O(2)) on lung structure and collagen metabolism in the subacute phase of lung injury induced by continuous infusion of endotoxin (LPS) in a rat model. Experimental groups included control, endotoxin alone, endotoxin plus hyperoxia, and hyperoxia alone. Endotoxin-treated rats received a bolus of LPS (10 mg/kg i.v.) followed by 500 microg.kg(-1).day(-1) in continuous infusion for 10 days. The bronchoalveolar lavage (BAL) fluid/plasma albumin concentration ratio, an index of capillary permeability, and neutrophil and macrophage counts in BAL fluid were highest in the endotoxin plus hyperoxia group. On pathological examination, prolonged hyperoxia exacerbated destruction of the alveolar wall and caused most prominent emphysematous changes in the endotoxin plus hyperoxia group. Lung tissue hydroxyproline concentration was significantly decreased in the hyperoxia group and increased in the endotoxin group. The latent forms of MMP-2 and MMP-9 increased in BAL fluid of the endotoxin- and/or hyperoxia-treated groups, whereas the activities of collagenase and gelatinase, and the active form of MMP-2 were all increased in the hyperoxia-treated groups. Added to endotoxin, prolonged hyperoxia degraded collagen, the major structural component of basement membranes, and caused emphysematous changes associated with activation of collagenase and MMP-2. Our observations suggest that, in the subacute phase of endotoxin-induced lung injury, prolonged hyperoxia causes pulmonary emphysematous changes with persistent injury to the alveolar capillary barrier. Collagenase and MMP-2 activated by hyperoxia, together with MMP-9, may play prominent roles in disruption of the alveolar basement membranes and degradation of collagen lining the alveolar walls.     

Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia

Hyperoxia disrupts vascular and alveolar growth of the developing lung and contributes to the development of bronchopulmonary dysplasia (BPD). Endothelial progenitor cells (EPC) have been implicated in repair of the vasculature, but their role in lung vascular development is unknown. Since disruption of vascular growth impairs lung structure, we hypothesized that neonatal hyperoxia impairs EPC mobilization and homing to the lung, contributing to abnormalities in lung structure. Neonatal mice (1-day-old) were exposed to 80% O(2) at Denver's altitude (= 65% at sea level) or room air for 10 days. Adult mice were also exposed for comparison. Blood, lung, and bone marrow were harvested after hyperoxia. Hyperoxia decreased pulmonary vascular density by 72% in neonatal but not adult mice. In contrast to the adult, hyperoxia simplified distal lung structure neonatal mice. Moderate hyperoxia reduced EPCs (CD45-/Sca-1+/CD133+/VEGFR-2+) in the blood (55%; P < 0.03), bone marrow (48%; P < 0.01), and lungs (66%; P < 0.01) of neonatal mice. EPCs increased in bone marrow (2.5-fold; P < 0.01) and lungs (2-fold; P < 0.03) of hyperoxia-exposed adult mice. VEGF, nitric oxide (NO), and erythropoietin (Epo) contribute to mobilization and homing of EPCs. Lung VEGF, VEGF receptor-2, endothelial NO synthase, and Epo receptor expression were reduced by hyperoxia in neonatal but not adult mice. We conclude that moderate hyperoxia decreases vessel density, impairs lung structure, and reduces EPCs in the circulation, bone marrow, and lung of neonatal mice but increases EPCs in adults. This developmental difference may contribute to the increased susceptibility of the developing lung to hyperoxia and may contribute to impaired lung vascular and alveolar growth in BPD.     

Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice

Background aimsHuman amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury.MethodsNewborn mouse pups were randomized to either normoxia (inspired O₂ content (FiO₂) = 0.21, n = 60) or hyperoxia (FiO₂ = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14.ResultsHyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-β and platelet-derived growth factor-β). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia.ConclusionsIntraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.     

Retinoic acid attenuates O2-induced inhibition of lung septation

Exposure of the newborn lung to hyperoxia is associated with impaired alveolar development. In newborn rats exposed to hyperoxia and studied at day 14 of life, retinoic acid (RA) treatment improved survival and increased lung collagen but did not improve alveolar development. To determine whether RA treatment during exposure to hyperoxia results in late improvement in alveolarization, we treated newborn rats with RA and hyperoxia from day 3 to day 14 and then weaned O2 to room air by day 20, and studied the animals on day 42. O2-exposed animals had larger mean lung volumes, larger alveoli, and decreased gas-exchange tissue relative to air-exposed animals, whereas RA-treated O2-exposed animals were not statistically different from air-exposed controls. Relative to control animals, elastin staining at day 14 was decreased in hyperoxia-exposed lung independent of RA treatment, and, at day 42, elastin staining was similar in all treatment groups. At day 14, elastin gene expression was similar in all treatment groups, whereas at day 42 lung previously exposed to hyperoxia showed increased elastin signal independent of RA treatment. These results indicate that RA treatment during hyperoxia exposure promotes septal formation without evidence of effects on elastin gene expression after 4 wk of recovery.     

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.     

Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta, IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta, MIP-2, interferon-gamma-induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha. However, IL-1beta, IL-6, macrophage migration inhibitory factor, MIP-1alpha, MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.     

Protein nitration in rat lungs during hyperoxia exposure: a possible role of myeloperoxidase

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1alpha, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.     

Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung

The functional roles of neutral lipids are poorly understood in the lung. Blocking cholesteryl ester and triglyceride metabolism in lysosomal acid lipase gene knockout mice (lal-/-) resulted in a high level of neutrophil influx in the lungs as early as 2 mo of age. Bronchoalveolar macrophages appeared foamy and gradually increased in number with age progression. Affymetrix GeneChip array analysis of lung mRNA showed increased levels of proinflammatory cytokine (including IL-1beta, IL-6, and TNF-alpha) and matrix metalloproteinase (including MMP-8, MMP-9, and MMP-12) expression in lal-/- mice. With age progression, some areas of lal-/- mice developed severe abnormal cell proliferation and alveolar remodeling. In other areas, alveolar destruction (i.e., emphysema) was observed. In addition, Clara cell hypertrophy and hyperplasia developed in conducting airways. The pathophysiological phenotypes in the lal-/- mouse lungs became more severe with increasing age. The studies support the concept that neutral lipid metabolites play essential roles in pulmonary homeostasis, inflammatory responses, remodeling, and injury repair.     

Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung

Exposure of newborn mice to high inspired oxygen elicits a distinct phenotype of compromised alveolar and vascular development, although lethality during long-term exposure is lower in newborns compared to adults. As the effects of hyperoxia are mediated by excessive reactive oxygen species (ROS) generation, we hypothesized that newborn mice may exhibit enhanced expression of antioxidant defenses or attenuated ROS generation compared with adults. We measured subcellular oxidant responses to acute hyperoxia in lung slices and alveolar epithelial cells at varying time points during postnatal murine lung development. Oxidant stress was assessed using RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. In contrast to newborn resistance to oxygen-induced mortality, cells of lung slices from younger mice demonstrated exaggerated mitochondrial matrix oxidant stress compared to adults, whereas oxidant stress responses in the cytosol were absent. Cell death in lung slices from newborn mice exposed to 48 h of hyperoxia was also greater than for adults. Consistent with these findings, expression of antioxidant enzymes in newborn lungs was lower than in adults, and induction of antioxidant levels and activity during 24 h of in vivo exposure was absent. However, expression of the reactive oxygen species-generating enzyme NADPH oxidase 1 was increased with hyperoxic exposure in the young but not the adult lung. Collectively, these results suggest that the greater lethality in adult animals may be more likely attributed to processes such as inflammation than to differences in antioxidant defenses. Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses.     

SPF级新生大鼠高氧肺损伤模型的建立

目的建立一种操作简便、性能稳定的新生大鼠高氧肺损伤动物模型。方法①设计制造能自动控制氧浓度的高氧动物饲养舱。②将孕期满21 d出生的SPF级新生大鼠随机分成4组,即：高氧组Ⅰ（入高氧舱中饲养1～7 d）,高氧组Ⅱ（入高氧舱中饲养14 d）,以及相应的空气对照组Ⅰ、Ⅱ,每组14只。舱内氧浓度≥90%,每天23 h。计算肺系数,HE染色与病理学观察。结果高氧组与相应的对照组比较：高氧组大鼠体重轻,肺系数大,HE染色显示部分肺泡萎缩、肺间质及肺泡腔有水肿、出血,中性粒细胞浸润;肺泡发育明显滞后,辐射状肺泡计数明显少。结论本动物饲养舱性能稳定,操作简便;复制的新生大鼠的肺病理变化符合高氧肺损伤的改变。     

Neuraminidase-1 is required for the normal assembly of elastic fibers

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.     

Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. We have used a mouse model of allergen-induced airway remodeling to determine whether MMP-9 plays a role in airway remodeling. MMP-9-deficient and wild-type (WT) mice were repetitively challenged intranasally with ovalbumin (OVA) antigen to develop features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. OVA-challenged MMP-9-deficient mice had less peribronchial fibrosis and total lung collagen compared with OVA-challenged WT mice. There was no reduction in mucus expression, smooth muscle thickness, or airway responsiveness in OVA-challenged MMP-9-deficient compared with OVA-challenged WT mice. OVA-challenged MMP-9-deficient mice had reduced levels of bronchoalveolar lavage (BAL) regulated on activation, normal T cell expressed, and secreted (RANTES), as well as reduced numbers of BAL and peribronchial eosinophils compared with OVA-challenged WT mice. There were no significant difference in levels of BAL eotaxin, thymus- and activation-regulated chemokine (TARC), or macrophage-derived chemokine (MDC) in OVA-challenged WT compared with MMP-9-deficient mice. Overall, this study demonstrates that MMP-9 may play a role in mediating selected aspects of allergen-induced airway remodeling (i.e., modest reduction in levels of peribronchial fibrosis) but does not play a significant role in mucus expression, smooth muscle thickness, or airway responsiveness.     

Regulatory role of CD8+ T lymphocytes in bone marrow eosinophilopoiesis

Background

The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2).

Methods

Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression.

Results

TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days.

Conclusion

Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.     

Calcitonin gene-related peptide released from endothelial progenitor cells inhibits the proliferation of rat vascular smooth muscle cells induced by angiotensin II

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.     

JNK suppresses pulmonary fibroblast elastogenesis during alveolar development

Background

The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development.

Methods

Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing.

Results

By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs.

Conclusions

JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.     

Mechanisms of emphysema in autosomal dominant cutis laxa

Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-β (TGFβ) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFβ and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL.