Palifermin induces alveolar maintenance programs in emphysematous mice.

Rationale Emphysema is characterized by destruction of alveoli with ensuing airspace enlargement and loss of alveoli. Induction of alveolar regeneration is still a major challenge in emphysema therapy. Objectives: To investigate whether therapeutic application of palifermin (Delta N23-KGF) is able to induce a regenerative response in distal lung parenchyma after induction of pulmonary emphysema. Methods: Mice were therapeutically treated at three occasions by oropharyngeal aspiration of 10 mg Delta N23-KGF per kg body weight after induction of emphysema by porcine pancreatic elastase. Measurements and Main Results: Airflow limitation associated with emphysema was largely reversed as assessed by noninvasive head-out body plethysmography. Porcine pancreatic elastase induced airspace enlargement and loss of alveoli were partially reversed as assessed by design-based stereology. AI Delta N23-KGF induced proliferation of epithelium, endothelium, and fibroblasts being associated with enhanced differentiation as well as increased expression of vascular endothelial growth factor, vascular endothelial growth factor receptors, transforming growth factor (TGF)-beta 1, TGF-beta 2, (phospho-) Smad2, plasminogen activator inhibitor-1, and elastin as assessed by quantitative reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemistry. Delta N23-KGF induced the expression of TGF-beta 1 in and release of active TGF-beta 1 from primary mouse alveolar epithelial type 2 (AE2) cells, murine AE2-like cells LA-4, and cocultures of LA-4 and murine lung fibroblasts (MLF), but not in MLF cultured alone. Recombinant TGF-beta 1 but not Delta N23-KGF induced elastin gene expression in MLF. Blockade of TGF-signaling by neutralizing antibody abolished these effects of Delta N23-KGF in LA-4/MLF cocultures.Conclusions: Our data demonstrate that therapeutic application of Delta N23-KGF has the potential to induce alveolar maintenance programs in emphysematous lungs and suggest that the regenerative effect on interstitial tissue is linked to AE2 cell-derived TGF-beta 1

ATRA results in irregular repair of septa and fails to inhibit proinflammatory macrophages.

ATRA is controversially discussed in emphysema therapy. We re-evaluated ATRA in the elastase-model and hypothesized that beneficial effects should be reflected by increased alveolar surface area, elastin expression, and downregulation of inflammatory mediators and matrix metalloproteinases (MMP). Emphysema was induced by porcine pancreatic elastase versus saline in Sprague-Dawley rats. On days 26-37, rats received daily intraperitoneal injections with ATRA (500 μg·kg(-1) b.w.) versus olive-oil. Lungs were removed at day 38. Rat alveolar epithelial L2 cells were incubated with/without elastase followed by ATRA- or vehicle-treatment, respectively. ATRA only partially ameliorated structural defects. Alveolar walls exhibited irregular architecture: increased arithmetic mean thickness, reduction in surface coverage by AEC type II. ATRA only partially restored reduced soluble elastin. It tended to increase the ratio of ED1(+):ED2(+) macrophages. Bronchoalveolar lavage (BAL) cells exhibited a pro-inflammatory state with high expression of IL-1β, CINC-1, TNF-α, NF-ĸB, MMP-2, -9, -12, TIMP-1, and -2 in emphysema with ATRA exerting only little effects. MMP-7 was highly induced by ATRA in healthy but not in emphysematous lungs. ATRA reduced both MMP-2 and TIMP-1 activity in BAL fluid of emphysematous lungs. ATRA-therapy may bear the risk of unwanted side-effects on alveolar septal architecture in emphysematous lungs

Establishment of a protocol for large-scale gene expression analyses of laser capture microdissected bladder tissue

PURPOSE: Lower urinary tract symptoms (LUTS) can be caused by structural and functional changes in different compartments of the bladder. To enable extensive investigations of individual regions even in small bladder biopsies, we established a combination protocol consisting of three molecular techniques: laser capture microdissection microscopy (LCM), RNA preamplification and quantitative polymerase chain reaction (qPCR). METHODS: Urinary bladders of ten mice were resected and frozen immediately or after a delay of 15 min. Cryosections were obtained and smooth muscle was isolated using the LCM technique. Then, RNA was extracted, including protocols with and without DNase digestion as well as with and without the addition of carrier RNA. Extracted RNA was either used for reverse transcriptase (RT)-PCR plus qPCR or for a combination of RNA preamplification and qPCR. RESULTS: Our data showed that with RNA preamplification, 10 μg cDNA can be regularly generated from 2.5 ng RNA. Depending on expression levels, this is sufficient for hundreds of pPCR reactions. The efficiency of preamplification, however, was gene-dependent. DNase digestion before preamplification lead to lower threshold cycles in qPCR. The use of partly degraded RNA for RNA preamplification did not change the results of the following qPCR. CONCLUSIONS: RNA preamplification strongly enlarges the spectrum of genes to be analyzed in distinct bladder compartments by qPCR. It is an easy and reliable method that can be realized with standard laboratory equipment. Our protocol may lead in near future to a better understanding of the pathomechanisms in LUTS