Role of MicroRNAs in lung disease.

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate gene expression. They actively participate in the modulation of important cell physiological processes and are involved in the pathogenesis of lung diseases such as lung cancer, pulmonary fibrosis, asthma and chronic obstructive pulmonary disease. A better understanding of the role that miRNAs play in these diseases could lead to the development of new diagnostic and therapeutic tools. In this review, we discuss the role of some miRNAs in different lung diseases as well as the possible future of these discoveries in clinical applications.

Proteasomal inhibition after injury prevents fibrosis by modulating TGF-ß(1) signalling.

BACKGROUND: The development of organ fibrosis after injury requires activation of transforming growth factor ß(1) which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-ß(1)-mediated transcription. METHODS: Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively. RESULTS: Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-ß(1)-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response. CONCLUSIONS: Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.

Sensing, physiological effects and molecular response to elevated CO2 levels in eukaryotes.

Carbon dioxide (CO(2)) is an important gaseous molecule that maintains biosphere homeostasis and is an important cellular signalling molecule in all organisms. The transport of CO(2) through membranes has fundamental roles in most basic aspects of life in both plants and animals. There is a growing interest in understanding how CO(2) is transported into cells, how it is sensed by neurons and other cell types and in understanding the physiological and molecular consequences of elevated CO(2) levels (hypercapnia) at the cell and organism levels. Human pulmonary diseases and model organisms such as fungi, C. elegans, Drosophila and mice have been proven to be important in understanding of the mechanisms of CO(2) sensing and response.

Improving survival by increasing lung edema clearance: is airspace delivery of dopamine a solution?

In this issue of Critical Care Chamorro-Marin and coworkers provide new evidence that dopamine instilled into airspaces is beneficial in a rat model of ventilator-induced lung injury. This study is important because it is the first to explore the effects of dopamine on survival, albeit short term. The delivery of dopamine into the airspaces in vivo is also novel and builds upon previous studies describing the mechanisms by which dopamine exerts its effect by upregulating active Na+ transport in the lungs. Dopamine appears to increase active Na+ transport via activation of amiloride-sensitive sodium channels and the basolateral Na+/K+-ATPase within minutes, and it has been shown to be effective in normal lungs and several models of lung injury. This information is relevant to current clinical trials exploring the effects of alveolar fluid clearance stimulation in patients with acute lung injury.

Beta-adrenergic receptor stimulation and adenoviral overexpression of superoxide dismutase prevent the hypoxia-mediated decrease in Na,K-ATPase and alveolar fluid reabsorption.

Hypoxia has been shown to cause lung edema and impair lung edema clearance. In the present study, we exposed isolated rat lungs to pO(2) = 40 mm Hg for 60 min or rats to 8% O(2) for up to 24 h and then measured changes in alveolar fluid reabsorption (AFR) and Na,K-ATPase function. Low levels of oxygen severely impaired AFR in both ex vivo and in vivo models. The decrease in AFR was associated with a decrease in Na,K-ATPase activity and protein abundance in the basolateral membranes from peripheral lung tissue of hypoxic rats. Beta-adrenergic agonists restored AFR in rats exposed to 8% O(2) (from 0.02 +/- 0.07 ml/h to 0.59 +/- 0.03 ml/h), which was associated with parallel increases in Na,K-ATPase protein abundance in the basolateral membrane. Hypoxia is associated with increased production of reactive oxygen species. Therefore, we examined whether overexpression of SOD2, manganese superoxide dismutase, would prevent the hypoxia-mediated decrease in AFR. Spontaneously breathing rats were infected with a replication-deficient human type 5 adenovirus containing cDNA for SOD2. An otherwise identical virus that contained no cDNA was used as a control (Adnull). Hypoxic Adnull rats had decreased rates of AFR (0.12 +/- 0.1 ml/h) as compared with hypoxic AdSOD2 and normoxic control rats (0.47 +/- 0.04 ml/h and 0.49 +/- 0.02 ml/h, respectively), with parallel changes in Na,K-ATPase.

Effects of hypoxia on the alveolar epithelium.

An important role of the alveolar epithelium is to contribute to the alveolocapillary barrier, secrete surfactant to lower the surface tension, and clear edema. These are energy-requiring processes for which normal oxygenation is required. There are many clinical conditions in which alveolar epithelial cells are exposed to low oxygen concentrations and although they can adapt to hypoxia, there are alterations in cellular function that can impact clinical outcomes. Hypoxic alveolar cells maintain cellular ATP content by increasing glycolytic capacity and via the hypoxia inducible factor-1 activation of a myriad of genes including the vascular endothelial growth factor. In addition, they decrease ATP utilization by down regulating the high energy consuming Na,K-ATPase activity and protein synthesis. The alveolar epithelium is in close apposition to vascular endothelium, which facilitates efficient gas exchange and provides a physical barrier between luminal and interstitial/vascular spaces. Alveolar edema clearance is an active process requiring activity of many proteins of which the amiloride-sensitive sodium channel (ENaC) and Na,K-ATPase are important contributors. Exposure to hypoxia impairs alveolar edema clearance by mechanisms that down regulate both ENaC and the Na,K-ATPase function. Other effects of hypoxia on alveolar cell function include surfactant production, disruption of cytoskeleton integrity, and the triggering of apoptosis. In summary, hypoxia has deleterious effects on the alveolar epithelium. More research needs to be done to better understand the effects of hypoxia on alveolar epithelia cell and lung function.

Keratin 8 phosphorylation by protein kinase C delta regulates shear stress-mediated disassembly of keratin intermediate filaments in alveolar epithelial cells.

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

Hypocapnic but not metabolic alkalosis impairs alveolar fluid reabsorption.

Acid-base disturbances, such as metabolic or respiratory alkalosis, are relatively common in critically ill patients. We examined the effects of alkalosis (hypocapnic or metabolic alkalosis) on alveolar fluid reabsorption in the isolated and continuously perfused rat lung model. We found that alveolar fluid reabsorption after 1 hour was impaired by low levels of CO2 partial pressure (PCO2; 10 and 20 mm Hg) independent of pH levels (7.7 or 7.4). In addition, PCO2 higher than 30 mm Hg or metabolic alkalosis did not have an effect on this process. The hypocapnia-mediated decrease of alveolar fluid reabsorption was associated with decreased Na,K-ATPase activity and protein abundance at the basolateral membranes of distal airspaces. The effect of low PCO2 on alveolar fluid reabsorption was reversible because clearance normalized after correcting the PCO2 back to normal levels. These data suggest that hypocapnic but not metabolic alkalosis impairs alveolar fluid reabsorption. Conceivably, correction of hypocapnic alkalosis in critically ill patients may contribute to the normalization of lung ability to clear edema.

Augmentation of endogenous dopamine production increases lung liquid clearance.

We have previously reported that dopamine increased active Na+ transport in rat lungs by upregulating the alveolar epithelial Na,K-ATPase. Here we tested whether alveolar epithelial cells produce dopamine and whether increasing endogenous dopamine production by feeding rats a 4% tyrosine diet (TSD) would increase lung liquid clearance. Alveolar Type II cells express the enzyme aromatic-L-amino acid decarboxylase (AADC) and, when incubated with the dopamine precursor, 3-hydroxy-L-tyrosine (L-dopa), produce dopamine. Rats fed TSD, a precursor of L-dopa and dopamine, had increased urinary dopamine levels, which were inhibited by benserazide, an inhibitor of AADC. Rats fed TSD for 15, 24, and 48 hours had a 26, 46, and 45% increase in lung liquid clearance, respectively, as compared with controls. Also, dopaminergic D1 receptor antagonist--but not dopaminergic D2 receptor antagonist--inhibited the TSD-mediated increase in lung liquid clearance. Alveolar Type II cells isolated from the lungs of rats after they had been fed TSD for 24 hours demonstrated increased protein abundance of Na,K-ATPase alpha1 and beta1 subunits. Basolateral membranes isolated from peripheral lung tissue of tyrosine-fed rats had increased Na,K-ATPase activity and Na,K-ATPase alpha1 subunit. These data provide the first evidence that alveolar epithelial cells produce dopamine and that increasing endogenous dopamine increases lung liquid clearance.

Regulation of lung edema clearance by dopamine.

In the kidney, dopamine inhibits Na,K-ATPase, which results in natriuresis because less Na+ is reabsorbed by the proximal and distal tubules. In contrast, dopamine stimulates Na,K-ATPase activity in the alveolar epithelium, leading to increased alveolar fluid reabsorption. Importantly, dopamine increases alveolar fluid reabsorption not only in normal alveolar epithelium but also in models of lung injury. Dopamine short-term regulation of alveolar epithelial Na,K-ATPase occurs via D1 receptor activation, protein kinase C and protein phosphatase 2A pathways, leading to increased Na,K-ATPase activity by recruiting sodium pumps from the intracellular compartment to the basolateral membranes. Conversely, D2 receptor activation by long-term dopamine regulates (approximately 24 hours) alveolar epithelial Na,K-ATPase via the MAPK pathway, [figure: see text] which results in de novo synthesis of Na,K-ATPase proteins. Conceivably, by increasing Na,K-ATPase activity and promoting alveolar fluid reabsorption, dopamine can be of clinical relevance for the treatment of patients with acute hypoxemic respiratory failure due to pulmonary edema.

Pulmonary lymphatics and edema accumulation after brief lung injury.

In a past study of hyperoxia-induced lung injury, the extensive lymphatic filling could have resulted from lymphatic proliferation or simple lymphatic recruitment. This study sought to determine whether brief lung injury could produce similar changes, to show which lymphatic compartments fill with edema, and to compare their three-dimensional structure. Tracheostomized rats were ventilated at high tidal volume (12-16 ml) or low tidal volume (3-5 ml) or allowed to breathe spontaneously for 25 min. Light microscopy showed more perivascular, interlobular septal, and alveolar edema in the animals ventilated at high tidal volume (P < 0.0001). Scanning electron microscopy of lymphatic casts showed extensive filling of the perivascular lymphatics in the group ventilated at high tidal volume (P < 0.01), but lymphatic filling was greater in the nonventilated group than in the group that was ventilated at low tidal volume (P < 0.01). The three-dimensional structures of the cast interlobular and perivascular lymphatics were similar. There was little filling and no difference in pleural lymphatic casts among the three groups. More edema accumulated in the surrounding lymphatics of larger blood vessels than smaller blood vessels. Brief high-tidal-volume lung injury caused pulmonary edema similar to that caused by chronic hyperoxic lung injury, except it was largely restricted to perivascular and septal lymphatics and prelymphatic spaces.