Reversal of MicroRNA Dysregulation in an Animal Model of Pulmonary Hypertension.

BACKGROUND: Animals models have played an important role in enhancing our understanding of the pathogenesis of pulmonary arterial hypertension (PAH). Dysregulation of the profile of microRNAs (miRNAs) has been demonstrated in human tissues from PAH patients and in animal models. In this study, we measured miRNA levels in the monocrotaline (MCT) rat model of PAH and examined whether blocking a specific dysregulated miRNA not previously reported in this model, attenuated PAH. We also evaluated changes in miRNA expression in lung specimens from MCT PAH rats overexpressing human prostacyclin synthase, which has been shown to attenuate MCT PAH. METHODS: Expression levels of a panel of miRNAs were measured in MCT-PAH rats as compared to naïve (saline) control rats. Subsequently, MCT PAH rats were injected with a specific inhibitor (antagomiR) for miR-223 (A223) or a nonspecific control oligonucleotide (A-control) 4 days after MCT administration, then weekly. Three weeks later, RV systolic pressure and RV mass were measured. Total RNA, isolated from the lungs, microdissected pulmonary arteries, and right ventricle, was reverse transcribed and real-time quantitative PCR was performed. MiRNA levels were also measured in RNA isolated from paraffin sections of MCT-PAH rats overexpressing prostacyclin synthase. RESULTS: MiRs 17, 21, and 223 were consistently upregulated, whereas miRs 126, 145, 150, 204, 424, and 503 were downregulated in MCT PAH as compared to vehicle control. A223 significantly reduced levels of miR-223 in PA and lungs of MCT PAH rats as compared to levels measured in A-control or control MCT PAH rats, but A223 did not attenuate MCT PAH. Right ventricular mass and right ventricular systolic pressure in rats treated with A223 were not different from values in A-control or MCT PAH rats. In contrast, analysis of total RNA from lung specimens of MCT PAH rats overexpressing human prostacyclin synthase (hPGIS) demonstrated reversal of MCT-induced upregulation of miRs 17, 21, and 223 and an increase in levels of miR-424 and miR-503. Reduction in bone morphogenetic receptor 2 (BMPR2) messenger (m)RNA expression was not altered by A223, whereas human prostacyclin synthase overexpression restored BMPR2 mRNA to levels in MCT PAH to levels measured in naive controls. CONCLUSIONS: Inhibition of miR-223 did not attenuate MCT PAH, whereas human prostacyclin synthase overexpression restored miRNA levels in MCT PAH to levels detected in naïve rats. These data may establish a paradigm linking attenuation of PAH to restoration of BMPR2 signaling.

Pathological changes in pulmonary circulation in carbon tetrachloride (CCl4)-induced cirrhotic mice.

RATIONALE: Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease. OBJECTIVE: We investigated the effects of CCl4-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH. METHODS AND RESULTS: Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl4 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl4-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl4 did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery. CONCLUSION: Collectively, our data demonstrate that chronic CCl4 treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.

Attenuation of monocrotaline-induced pulmonary hypertension by luminal adeno-associated virus serotype 9 gene transfer of prostacyclin synthase.

Idiopathic pulmonary arterial hypertension (iPAH) is associated with high morbidity and mortality. We evaluated whether luminal delivery of the human prostacyclin synthase (hPGIS) cDNA with adeno-associated virus (AAV) vectors could attenuate PAH. AAV serotype 5 (AAV5) and AAV9 vectors containing the hPGIS cDNA under the control of a cytomegalovirus-enhanced chicken ß-actin (CB) promoter or vehicle (saline) were instilled into lungs of rats. Two days later, rats were injected with monocrotaline (MCT, 60 mg/kg) or saline. Biochemical, hemodynamic, and morphologic assessments were performed when the rats developed symptoms (3-4 weeks) or at 6 weeks. Luminal (airway) administration of AAV5 and AAV9CBhPGIS vectors (MCT-AAV5 and MCT-AAV9 rats) significantly increased plasma levels of 6-keto-PGF1(α) as compared with MCT-controls, and closely resembled levels measured in rats not treated with MCT (saline-saline). Right ventricular (RV)/left ventricular (LV)+septum (S) ratios and RV systolic pressure (RVSP) were greater in MCT-control rats than in saline-saline rats, whereas the ratios and RVSP in MCT-AAV5CBhPGIS and MCT-AAV9CBhPGIS rats were similar to saline-saline rats. Thickening of the muscular media of small pulmonary arteries of MCT-control rats was detected in histological sections, whereas the thickness of the muscular media in MCT-AAV5CBhPGIS and MCT-AAV9CBhPGIS rats was similar to saline-saline controls. In experiments with different promoters, a trend toward increased levels of PGF1(α) expression was detected in lung homogenates, but not plasma, of MCT-treated rats transduced with an AAV9-hPGIS vector containing a CB promoter. This correlated with significant reductions in the RV/LV+S ratio and RVSP in MCT-AAV9CBhPGIS rats that resembled levels in saline-saline rats. No changes in levels of PGF1(α), RV/LV+S, or RVSP were detected in rats transduced with AAV9-hPGIS vectors containing a modified CB promoter (CB7) or a distal epithelial cell-specific promoter (CC10). Thus, AAV9CBhPGIS vectors prevented development of MCT-induced PAH and associated pulmonary vascular remodeling.

Specific modulation of airway epithelial tight junctions by apical application of an occludin peptide.

Tight junctions are directly involved in regulating the passage of ions and macromolecules (gate functions) in epithelial and endothelial cells. The modulation of these gate functions to transiently regulate the paracellular permeability of large solutes and ions could increase the delivery of pharmacological agents or gene transfer vectors. To reduce the inflammatory responses caused by tight junction-regulating agents, alternative strategies directly targeting specific tight junction proteins could prove to be less toxic to airway epithelia. The apical delivery of peptides corresponding to the first extracellular loop of occludin to transiently modulate apical paracellular flux has been demonstrated in intestinal epithelia. We hypothesized that apical application of these occludin peptides could similarly modulate tight junction permeability in airway epithelia. Thus, we investigated the effects of apically applied occludin peptide on the paracellular permeability of molecular tracers and viral vectors in well differentiated human airway epithelial cells. The effects of occludin peptide on cellular toxicity, tight junction protein expression and localization, and membrane integrity were also assessed. Our data showed that apically applied occludin peptide significantly reduced transepithelial resistance in airway epithelia and altered tight junction permeability in a concentration-dependent manner. These alterations enhanced the paracellular flux of dextrans as well as gene transfer vectors. The occludin peptide redistributed occludin but did not alter the expression or distribution of ZO-1, claudin-1, or claudin-4. These data suggest that specific targeting of occludin could be a better-suited alternative strategy for tight junction modulation in airway epithelial cells compared with current agents that modulate tight junctions.

Applications of imaging techniques to studies of epithelial tight junctions.

The intercellular junctional complex, which consists of the tight junction (TJ), adherens junction, and desmosomes, mediates cell-cell adhesion in epithelia and endothelia. The TJ forms the apical-most portion of this complex in epithelia, serving as a fence to lateral diffusion of apical and basolateral membrane components and as a semi-permeable barrier or gate to the flow of ions and solutes through the paracellular pathway. The TJ consists of a series of integral membrane and cytoplasmic plaque proteins with complex interactions. Included among the TJ proteins are the claudins, which play a major role in mediating the charge and solute selectivity of the junction. Yet, the profile of claudin and associated protein expression differs among epithelia and the function and regulation of many of the TJ proteins remain unknown. This review discusses the application of techniques to discern the function, localization, and regulation of epithelial TJs based on examples from published studies.

Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO3- secretory pathway that was defective in CF: (i). ASL [HCO3-] was higher in NL than CF ASL; (ii). activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii). NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent HCO3- secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

Role of claudin interactions in airway tight junctional permeability.

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.

Safety and efficiency of modulating paracellular permeability to enhance airway epithelial gene transfer in vivo.

We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after lumenal application of vehicle, ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), sodium caprate (C10), or sodium laurate (C12). Lung toxicity was assessed after tracheobronchial instillation to murine airways and the relative ability of these agents to enhance in vivo adenoviral gene transfer was evaluated. Lumenal C12 increased LDH release in vitro, but C10 and EGTA did not. Increased levels of interleukin 8 (IL-8) were secreted from EGTA-pretreated cystic fibrosis HAE cells after apical application of Pseudomonas aeruginosa (10(8) CFU/ml), whereas IL-8 secretion from C10- and C12-pretreated cells was not different from controls. In vivo toxicity studies demonstrated no effect of EGTA, C10, or C12 on weight gain, lung edema, or bronchoalveolar lavage fluid (BALF) albumin. EGTA increased BALF cell counts, neutrophils, and murine (m) macrophage inflammatory protein 2, mKC, mIL-6, and mIL-1 beta levels. C10 had no effect on BALF cell counts or LDH, but increased murine tumor necrosis factor alpha. C12 increased BALF LDH, neutrophils, and mIL-6 levels. Histopathological analysis revealed mild focal lung inflammation more frequently in the EGTA, C10, and C12 groups than in vehicle controls, with greater intensity in the C12 group relative to the other groups. C10 and C12 also increased airway responsiveness to methacholine challenge compared with control and EGTA groups. Adenoviral gene transfer to murine trachea in vivo was enhanced more efficiently by C10 than by C12 or EGTA. Thus, the different toxicities may permit the selection of agents that enhance gene transfer with minimal adverse effects.

Acute mechanism of medium chain fatty acid-induced enhancement of airway epithelial permeability.

The localization of viral receptors to the basolateral surface of airway epithelia is an obstacle to the effectiveness of luminal viral-mediated gene transfer to the lung. The tight junction (TJ) serves as a rate-limiting barrier to the penetration of viral vectors. We have previously identified the sodium salt of the medium chain fatty acid (MCFA) capric acid (C10) as an agent that can enhance the ability of adenoviral vectors to transduce well differentiated (WD) primary human airway epithelial (HAE) cells. Previous studies have suggested that intracellular calcium (Ca(i)2+) levels may play a central role in the long-term C10-mediated increases in junctional permeability. In this study, we investigated the effects of C10 and lauric acid (C12) on Ca(i)2+ in WD primary HAE cells and determined whether these effects were necessary for the acute MCFA-induced reduction in transepithelial resistance (R(T)) and increased permeability. In addition, we characterized the effects of C10 and C12 on components localized to the TJ, including ZO-1, junctional adhesion molecule (JAM), and the claudin family of transmembrane proteins. In addition to rapidly decreasing R(T), C10 and C12 increased cellular and paracellular permeability. C10 induced a rapid, sustained increase in Ca(i)2+. However, buffering Ca(i)2+ did not block the effects of C10 on R(T). Both C10 and C12 caused reorganization of claudins-1, -4, JAM, and beta-catenin, but not ZO-1. These data suggest that C10 and C12 exert their acute effects on airway TJs via a Ca(2+)-independent mechanism of action and may alter junctional permeability via direct effects on the claudin family of TJ proteins.

Regulation of airway tight junctions by proinflammatory cytokines.

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.