TGF-ß directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury.

TGF-ß is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-ß pathway is described, which rapidly promoted internalization of the αßγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-ß applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which--together with patch-clamp and flow cytometry studies--identified ENaC as the target of TGF-ß. TGF-ß rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of ßENaC, the subunit responsible for cell-surface stability of the αßγENaC complex. ENaC internalization was dependent on oxidation of ßENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove ßENaC internalization, which was inhibited by a TGF-ß neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-ß signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-ß-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

Nestin-expressing vascular wall cells drive development of pulmonary hypertension.

Nestin, a well-known marker of neuronal stem cells, was recently suggested to characterise stem cell-like progenitors in non-neuronal structures during development and tissue repair. Integrating novel morphological approaches (CLARITY), we investigate whether nestin expression defines the proliferating cell population that essentially drives vascular remodelling during development of pulmonary hypertension.The role of nestin was investigated in lungs of nestin-GFP (green fluorescent protein) mice, models of pulmonary hypertension (rat: monocrotaline, SU5416/hypoxia; mouse: hypoxia), samples from pulmonary hypertension patients and human pulmonary vascular smooth muscle cells (VSMCs).Nestin was solely found in lung vasculature and localised to proliferating VSMCs, but not bronchial smooth muscle cells. Nestin was shown to affect cell number and was significantly enhanced in lungs early during development of pulmonary hypertension, correlating well with increased VSMC proliferation, expression of phosphorylated (activated) platelet-derived growth factor receptor ß and downregulation of the smooth muscle cell differentiation marker calponin. At later time points when pulmonary hypertension became clinically evident, nestin expression and proliferation returned to control levels. Increase of nestin-positive VSMCs was also found in human pulmonary hypertension, both in vessel media and neointima.Nestin expression seems to be obligatory for VSMC proliferation, and specifies lung vascular wall cells that drive remodelling and (re-)generation. Our data promise novel diagnostic tools and therapeutic targets for pulmonary hypertension.

Structural and functional prevention of hypoxia-induced pulmonary hypertension by individualized exercise training in mice.

Pulmonary hypertension (PH) is a disease with a poor prognosis characterized by a vascular remodeling process and an increase in pulmonary vascular resistance. While a variety of reports demonstrated that exercise training exerts beneficial effects on exercise performance and quality of life in PH patients, it is not known how physical exercise affects vascular remodeling processes occurring in hypoxia-induced PH. Therefore, we investigated the effect of individualized exercise training on the development of hypoxia-induced PH in mice. Training effects were compared with pharmacological treatment with the phosphodiesterase 5 inhibitor Sildenafil or a combination of training plus Sildenafil. Trained mice who received Sildenafil showed a significantly improved walking distance (from 88.9 ± 8.1 to 146.4 ± 13.1 m) and maximum oxygen consumption (from 93.3 ± 2.9 to 105.5 ± 2.2% in combination with Sildenafil, to 102.2 ± 3.0% with placebo) compared with sedentary controls. Right ventricular systolic pressure, measured by telemetry, was at the level of healthy normoxic animals, whereas right heart hypertrophy did not benefit from training. Most interestingly, the increase in small pulmonary vessel muscularization was prevented by training. Respective counterregulatory processes were detected for the nitric oxide-soluble guanylate cyclase-phosphodiesterase system. We conclude that individualized daily exercise can prevent vascular remodeling in hypoxia-induced PH.

Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. METHODS AND RESULTS: Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38(MAPK) phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. CONCLUSION: BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH.

CFTR-dependent Cl- secretion in Xenopus laevis lung epithelium.

In our present study we used preparations from Xenopus laevis lungs to perform electrophysiological Ussing chamber measurements, unidirectional flux measurements, and employed molecular approaches to elucidate the presence and function of a cystic fibrosis transmembrane conductance regulator (CFTR) homolog in this tissue. Application of different CFTR blockers (NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid (NFA), glibenclamide, lonidamine, CFTR(inh)-172) to the apical side of the tissues was able to significantly decrease the measured short circuit current (I(SC)) indicating a Cl(-) secretion due to luminal located CFTR channels. This was further supported by a net (36)Cl(-) secretion determined by radioactive tracer flux experiments. Further, Xenopus pulmonary epithelia responded to apical chlorzoxazone exposure - a CFTR activator - and this activated current was inhibited by CFTR(inh)-172. We performed reverse transcription-PCR (RT-PCR) and Western blot analysis and with both approaches we found characteristic signals indicating the presence of a CFTR homolog in Xenopus lung. In addition, we were able to detect CFTR in apical membranes of Xenopus lung slices with immunohistological techniques. We conclude that Xenopus lung epithelium exhibits functional CFTR channels and that this tissue represents a valuable model for the investigation of ion transport properties in pulmonary epithelia.

Exposure to UV light causes increased biotinylation of histones in Jurkat cells.

Biotin in breakdown products of biotinylated carboxylases serves as substrate for biotinylation of histones by biotinidase. Here we determined whether biotinylation of histones might play a role in repair of damaged DNA and in apoptosis. Jurkat cells were exposed to UV light to induce DNA damage. Abundance of thymine dimers increased about three times in response to UV exposure, consistent with DNA damage. Biotin-containing carboxylases were degraded in response to UV exposure, as judged by Western blot analysis and carboxylase activities. Mitochondrial integrity decreased in response to UV exposure (as judged by confocal microscopy), facilitating the release of breakdown products of carboxylases from mitochondria. Biotinylation of histones increased in response to UV exposure; biotinylation of histones did not occur specifically at sites of newly repaired DNA. UV exposure triggered apoptosis, as judged by caspase-3 activity and analysis by confocal microscopy. In summary, this study provided evidence that increased biotinylation of histones in DNA-damaged cells might either be a side product of carboxylase degradation or a step during apoptosis.