Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis

Pulmonary fibrosis is the consequence of a variety of diseases with no satisfying treatment option. Therapy-induced fibrosis also limits the efficacy of chemotherapy and radiotherapy in numerous cancers. Here, we studied the potential of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors (RTKIs) to attenuate radiation-induced pulmonary fibrosis. Thoraces of C57BL/6 mice were irradiated (20 Gy), and mice were treated with three distinct PDGF RTKIs (SU9518, SU11657, or Imatinib). Irradiation was found to induce severe lung fibrosis resulting in dramatically reduced mouse survival. Treatment with PDGF RTKIs markedly attenuated the development of pulmonary fibrosis in excellent correlation with clinical, histological, and computed tomography results. Importantly, RTKIs also prolonged the life span of irradiated mice. We found that radiation up-regulated expression of PDGF (A–D) isoforms leading to phosphorylation of PDGF receptor, which was strongly inhibited by RTKIs. Our findings suggest a pivotal role of PDGF signaling in the pathogenesis of pulmonary fibrosis and indicate that inhibition of fibrogenesis, rather than inflammation, is critical to antifibrotic treatment. This study points the way to a potential new approach for treating idiopathic or therapy-related forms of lung fibrosis

Intercellular Communication by Exchange of Cytoplasmic Material via Tunneling Nano-Tube Like Structures in Primary Human Renal Epithelial Cells

Transfer of cellular material via tunneling nanotubes (TNT) was recently discovered as a novel mechanism for intercellular communication. The role of intercellular exchange in communication of renal epithelium is not known. Here we report extensive spontaneous intercellular exchange of cargo vesicles and organelles between primary human proximal tubular epithelial cells (RPTEC). Cells were labeled with two different quantum dot nanocrystals (Qtracker 605 or 525) and intercellular exchange was quantified by high-throughput fluorescence imaging and FACS analysis. In co-culture, a substantial fraction of cells (67.5%) contained both dyes indicating high levels of spontaneous intercellular exchange in RPTEC. The double positive cells could be divided into three categories based on the preponderance of 605 Qtracker (46.30%), 525 Qtracker (48.3%) and approximately equal content of both Qtrackers (4.57%). The transfer of mitochondria between RPTECs was also detected using an organelle specific dye. Inhibition of TNT genesis by actin polymerization inhibitor (Latrunculin B) markedly reduced intercellular exchange (>60%) suggesting that intercellular exchange in RPTEC was in part mediated via TNT-like structures. In contrast, induction of cellular stress by Zeocin treatment increased tube-genesis in RPTEC. Our data indicates an unexpected dynamic of intercellular communication between RPTEC by exchange of cytosolic material, which may play an important role in renal physiology

Whole blood transcriptomics in cardiac surgery identifies a gene regulatory network connecting ischemia reperfusion with systemic inflammation.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass (CS/CPB) is associated with increased risk for postoperative complications causing substantial morbidity and mortality. To identify the molecular mechanisms underlying CS/CPB-induced pathophysiology we employed an integrative systems biology approach using the whole blood transcriptome as the sentinel organ. METHODOLOGY/PRINCIPAL FINDINGS: Total RNA was isolated and globin mRNA depleted from whole blood samples prospectively collected from 10 patients at time points prior (0), 2 and 24 hours following CS/CPB. Genome-wide transcriptional analysis revealed differential expression of 610 genes after CS/CPB (p<0.01). Among the 375 CS/CPB-upregulated genes, we found a gene-regulatory network consisting of 50 genes, reminiscent of activation of a coordinated genetic program triggered by CS/CPB. Intriguingly, the highly connected hub nodes of the identified network included key sensors of ischemia-reperfusion (HIF-1alpha and C/EBPbeta). Activation of this network initiated a concerted inflammatory response via upregulation of TLR-4/5, IL1R2/IL1RAP, IL6, IL18/IL18R1/IL18RAP, MMP9, HGF/HGFR, CalgranulinA/B, and coagulation factors F5/F12 among others. Differential regulation of 13 candidate genes including novel, not hitherto CS/CBP-associated genes, such as PTX3, PGK1 and Resistin, was confirmed using real-time quantitative RT-PCR. In support of the mRNA data, differential expression of MMP9, MIP1alpha and MIP1beta plasma proteins was further confirmed in 34 additional patients. CONCLUSIONS: Analysis of blood transcriptome uncovered critical signaling pathways governing the CS/CPB-induced pathophysiology. The molecular signaling underlying ischemia reperfusion and inflammatory response is highly intertwined and includes pro-inflammatory as well as cardioprotective elements. The herein identified candidate genes and pathways may provide promising prognostic biomarker and therapeutic targets

Quantifying the spontaneous intercellular exchange rates of organelles in epithelial cells.

<p>Qtracker 605 and 525 labeled cells were co-cultured for 24 h and analyzed by flow cytometry (A). FACS analysis revealed 15.8% “double +” cells. To overcome the limitation of FACS analysis to detect and discriminate double positive cells with a low number of transfered Qdots by the tubes, we employed high-throughput fluorescence image analysis using the ImageStream™ platform (B–D). By analyzing 9335 co-cultured RPTEC, spontaneous intercellular exchange was detected in 67.5% or 6305 cells (B–C). Inhibition of tube-genesis by Lat. B resulted in marked (62%) reduction of the intercellular exchange (from 67.5% to 25.9% double positive cells). Detailed analysis revealed that the intercellular exchange resulted in three categories of double positive cells: i) transfer of 525 Qtracker to 605 Qtracker labeled cells (46.30% or 2921 cells), ii) transfer of 605 Qtracker to 525 Qtracker labeled cells (48.3% or 3046 cells) or equal content of both Qtrackers (4.57% or 288 cells) (D). Thus, the transfer of few labeled Qdots organelles contributed to the majority of spontaneous intercellular exchange among RPTEC.</p

TNT-like tubes connecting epithelial cells.

<p>Representative phase contrast images of the identified TNT-like tubes connecting HMECs. These membranous structures were able to span long distances of several micrometers between the cells (A–D). The observed “gondola”-like structures, i.e., localized changes in tube diameter (white arrows in (A)), indicated potential transfer of cargos in the lumen of the tubes. Tubes with blind ends were also found (white arrow in (B)). In contrast to TNTs the identified membranous tubes often build cross roads bridging several cells with each other (C). A frequent observation was the connection of epithelial cell “islands” via these tubes (C and D). All photomicrographs x100 view. <b>Enhanced tube-genesis followed by cellular stress</b> (E and F). Treatment of RPTEC with the glycopeptide antibiotic <i>Zeocin</i> led to a marked increase in the number of tubes in a dose dependent manner. The maximum induction of tubes was reached at 400 µg/ml dose of <i>Zeocin</i>. At higher concentrations (e.g. 1000 µg/ml) the toxicity and cell death effects of the treatment dominated the phenotype resulting in a decline of the number of tubes and vital cells per optical field. Bars represent means ± <i>SD</i> from quadruplicates measurements (p<0.01 for 50–400 µg/ml <i>Zeocin</i> treatment vs. control).</p

Tracing intercellular exchange by Qdot® nanocrystals.

<p>(<b>A–C</b>)<b>,</b> RPTEC were labeled with either Qtracker 605 (red) or 525 (green) and co-cultured for 24 h. To-Pro-3 was used to stain the cell nucleus (DNA, blue). Fluorescence microscopy revealed a number of “double positive” cells suggesting spontaneous intercellular exchange of cytoplasmic material between the RPTECs. Fluorescence particles were also detected in the lumen of the tubular structures (arrows) indicating endoluminal transport. Among the specific morphological features of RPTEC tubes as compared to classical TNTs are their larger caliber, their ability to bridge very long distances (>200 µm) and the more frequent appearance of bifurcations or connections of multiple cells (A and B). Occasionally To-Pro-3 positive DNA-signal (blue staining) was detected in the lumen of the tubes (C) suggesting the possibility of the exchange of genetic material between RPTEC.</p

Intercellular exchange of Mitochondria.

<p>To detect potential exchange of other organelles such as mitochondria between RPTECs, cells were labeled with MitoTracker® reagent which specifically labels active mitochondria <i>in-vivo</i> (green). MitoTracker labeled cells were co-cultured for 24 h together with Qtracker 605 (red) labeled cells. Mitochondria could be detected in the lumen of the TNT-like tubes in RPTEC (A). The exchange of organelles between RPTEC is shown (highlighted by white arrows). Q605 labeled cells (red arrow). MitoTracker labeled cells (green arrow). Nuclear staining (blue, To-Pro3).</p